RU2816477C2 - COMPOSITIONS AND METHODS ASSOCIATED WITH ENGINEERED Fc-CONSTRUCTS - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology, specifically to recombinant production of Fc-constructs, and can be used in medicine to reduce the immune response in treating an inflammatory, autoimmune or immune disease. Disclosed is an Fc-construct comprising a) a first polypeptide comprising first and second monomers of the Fc-domain connected by a linker; b) a second polypeptide comprising third and fourth Fc domain monomers connected by a linker; c) a third polypeptide comprising a fifth Fc domain monomer; and d) a fourth polypeptide comprising a sixth Fc domain monomer. First and fifth monomers of the Fc domain are combined to form a first Fc domain; second and fourth monomers of the Fc domain are combined to form a second Fc domain; third and sixth monomers of the Fc domain are combined to form a third Fc domain.

EFFECT: invention enables to obtain an Fc-construct with reduced binding to the Fc-receptor.

4 cl, 40 dwg, 11 tbl, 16 ex

Description

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to the filing date 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, and U.S. Provisional Patent Application No. 62/589,473, filed November 21, 2017. The contents of each of these applications are incorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

Therapeutic proteins, such as therapeutic antibodies and Fc domain-containing fusion proteins, have rapidly become a clinically important class of drugs for patients with immunological and inflammatory diseases, cancer and infections.

BRIEF DESCRIPTION OF THE INVENTION

The present invention provides biologically active therapeutic constructs containing an Fc domain. Such constructs may be characterized by a desired serum half-life and/or binding affinity and/or avidity for Fc receptors.

In general, the invention provides Fc constructs containing 2-10 Fc domains, for example, Fc constructs containing 2, 3, 4, 5, 6, 7, 8, 9 or 10 Fc domains. In some embodiments, the Fc construct contains 2-10 Fc domains, 2-5 Fc domains, 2-4 Fc domains, 2-3 Fc domains, 3-5 Fc domains, 2-8 Fc domains, or 2-6 Fc domains. In some embodiments, the Fc construct contains 2-4 Fc domains. In some embodiments, the Fc construct contains 5-10 Fc domains (eg, 5-6, 5-7, 5-8, 5-9, or 5-10 Fc domains).

In some embodiments, constructs (e.g., Fc constructs containing 2-4 Fc domains, e.g., 2, 3, or 4 Fc domains) and homogeneous pharmaceutical compositions (e.g., those containing Fc constructs containing 2-4 Fc domains, e.g. 2, 3 or 4 Fc domains) of the present invention are useful, for example, to reduce inflammation in a subject, to stimulate the clearance of autoantibodies in a subject, to suppress antigen presentation in a subject, to block an immune response, e.g. for blocking immune complex activation of an immune response in a subject, and for treating immunological and inflammatory diseases (eg, autoimmune diseases) in a subject. The Fc constructs described herein can be used to treat patients with immunological and inflammatory diseases without significant stimulation of immune cells. In some embodiments, constructs (e.g., Fc constructs containing 5-10 Fc domains, e.g., 5, 6, 7, 8, 9, or 10 Fc domains) and homogeneous pharmaceutical compositions (e.g., those containing Fc- constructs containing 5-10 Fc domains, for example 5, 6, 7, 8, 9 or 10 Fc domains) of the present invention are useful, for example, to induce activation of immune cells that provide an immune response in a subject to enhance phagocytosis of the target cells (ie, a cancer cell or an infected cell) in a subject and for treating diseases such as cancer and infections in the subject.

The properties of these structures ensure the efficient production of essentially homogeneous compositions. The degree of homogeneity of the composition affects the pharmacokinetics and in vivo performance of the composition. Such homogeneity of the composition is necessary to ensure the safety, effectiveness, uniformity and reliability of the composition. The Fc construct of the present invention may be in a composition or aggregate that is substantially homogeneous (eg, at least 85%, 90%, 95%, 98%, or 99% homogeneous).

As described in more detail herein, the invention provides substantially homogeneous compositions containing Fc constructs, all of which contain the same number of Fc domains, as well as methods for preparing such substantially homogeneous compositions.

In a first aspect, the present invention provides an Fc construct comprising a) a first polypeptide comprising i) a first Fc domain monomer; ii) a second Fc domain monomer; and iii) a linker connecting the first Fc domain monomer to a second Fc domain monomer; b) a second polypeptide comprising i) a third Fc domain monomer; ii) a fourth Fc domain monomer; and iii) a linker connecting a third Fc domain monomer to a fourth Fc domain monomer; c) the third polypeptide contains a fifth Fc domain monomer; and d) the fourth polypeptide contains a sixth Fc domain monomer; wherein the first Fc domain monomer and the fifth Fc domain monomer are joined to form a first Fc domain, the second Fc domain monomer and the fourth Fc domain monomer are joined to form a second Fc domain, and the third Fc domain monomer and the sixth Fc domain monomer are joined to form a second Fc domain. domains are connected to form a third Fc domain, and wherein each of the first and second polypeptides contains the sequence DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPPEEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDKLTKNQV SLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGGGGGGGGGGGGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPPEEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 76), and each of the third and fourth polypeptides contains the sequence DKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPPEEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVDGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPG (SEQ ID NO: 70).

In another aspect, the present invention provides an Fc construct comprising a) a first polypeptide comprising i) a first Fc domain monomer; ii) a second Fc domain monomer; and iii) a linker connecting the first Fc domain monomer to a second Fc domain monomer; b) a second polypeptide comprising i) a third Fc domain monomer; ii) a fourth Fc domain monomer; and iii) a linker connecting a third Fc domain monomer to a fourth Fc domain monomer; c) the third polypeptide contains a fifth Fc domain monomer; and d) the fourth polypeptide contains a sixth Fc domain monomer; wherein the first Fc domain monomer and the fifth Fc domain monomer are joined to form a first Fc domain, the second Fc domain monomer and the fourth Fc domain monomer are joined to form a second Fc domain, and the third Fc domain monomer and the sixth Fc domain monomer are joined to form a second Fc domain. domains are connected to form a third Fc domain, and wherein the at least one Fc domain contains an amino acid modification at position I253 (eg, one amino acid modification at position I253). In another aspect, the present invention provides an Fc construct comprising a) a first polypeptide comprising i) a first Fc domain monomer; ii) a second Fc domain monomer; and iii) a linker connecting the first Fc domain monomer to a second Fc domain monomer; b) a second polypeptide comprising i) a third Fc domain monomer; ii) a fourth Fc domain monomer; and iii) a linker connecting a third Fc domain monomer to a fourth Fc domain monomer; c) the third polypeptide contains a fifth Fc domain monomer; and d) the fourth polypeptide contains a sixth Fc domain monomer; wherein the first Fc domain monomer and the fifth Fc domain monomer are joined to form a first Fc domain, the second Fc domain monomer and the fourth Fc domain monomer are joined to form a second Fc domain, and the third Fc domain monomer and the sixth Fc domain monomer are joined to form a second Fc domain. domains are connected to form a third Fc domain, and wherein at least one Fc domain monomer contains an amino acid substitution at position I253.

In some cases, the first and second polypeptides are identical to each other, and the third and fourth polypeptides are identical to each other. In some embodiments, the first Fc domain contains an amino acid modification at position I253. In some cases, one or both of the first and fifth monomers of the Fc domain contain an amino acid substitution at position I253. In some embodiments, the second Fc domain contains an amino acid modification at position I253. In some embodiments, one or both of the second and fourth Fc domain monomers contain an amino acid substitution at position I253. In some embodiments, the third Fc domain contains an amino acid modification at position I253. In some embodiments, one or both of the third and sixth Fc domain monomers contain an amino acid substitution at position I253. In some embodiments, each amino acid modification (e.g., substitution) at position I253 is independently selected from the group consisting of I253A, I253C, I253D, I253E, I253F, I253G, I253H, I253I, I253K, I253L, I253M, I253N, I253P, I253Q, I253R, I253S, I253T, I253V, I253W and I253Y. In some embodiments, each amino acid modification (eg, substitution) at position I253 is I253A. In some embodiments, the Fc construct (eg, at least one Fc domain monomer) contains at least one amino acid modification at position R292. In some embodiments, the at least one Fc domain monomer contains an amino acid substitution at position R292. In some embodiments, the first Fc domain contains an amino acid modification at position R292. In some embodiments, the first Fc domain (eg, one or both of the first Fc domain monomer and the fifth Fc domain monomer) contains an amino acid substitution at position R292. In some embodiments, the second Fc domain contains an amino acid modification at position R292. In some embodiments, the second Fc domain (eg, one or both of the second Fc domain monomer and the fourth Fc domain monomer) contains an amino acid substitution at position R292. In some embodiments, the third Fc domain contains an amino acid modification at position R292. In some embodiments, the third Fc domain (eg, one or both of the third Fc domain monomer and the sixth Fc domain monomer) contains an amino acid substitution at position R292. In some embodiments, the first, second, and third Fc domains each contain an amino acid modification (eg, substitution) at position R292. In some cases, one or both of the first and fifth Fc domain monomers contain an amino acid substitution at position R292, one or both of the second and fourth Fc domain monomers contain an amino acid substitution at position R292, and one or more of the third and sixth Fc domain monomers contain an amino acid substitution at position R292. In some embodiments, the first, second, and third Fc domains each comprise an amino acid modification (eg, substitution) of R292P. In some embodiments, each amino acid modification (eg, substitution) at position R292 is independently selected from the group consisting of R292D, R292E, R292L, R292P, R292Q, R292R, R292T, and R292Y. In some embodiments, each amino acid modification (eg, substitution) at position R292 is R292P.

In some embodiments of all aspects, each of the Fc domains is based on a human IgG1 Fc sequence and contains modifications described herein (ie, is a variant of a human IgG Fc sequence). In some embodiments, the core IgG1 Fc sequence is SEQ ID NO: 42 and contains no more than 10 single amino acid modifications. In some embodiments, each of the Fc domains of the Fc constructs described herein is an IgG1 Fc sequence (eg, SEQ ID NO: 42) with no more than 10 single amino acid modifications. In some embodiments, the IgG1 Fc sequence is SEQ ID NO: 42 and comprises engineered cavity modifications, engineered protrusion modifications, and/or electrostatic coordination modifications to control polypeptide assembly and/or binding-related mutation modifications to modify the pharmacokinetics of the construct described in this document. Thus, no more than 10 single amino acid modifications may include modification (eg, substitution) in one or both of I253 (eg, I253A) and R292 (eg, R292P) and modification (eg, substitution) to provide: an engineered cavity and an engineered overhang and/or modification of electrostatic coordination to control polypeptide assembly. In some cases, each Fc domain monomer contains no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 single amino acid modification in addition to a substitution in one or both of I253 and R292. Modifications to provide engineered cavity, engineered protrusion, and/or electrostatic coordination modifications to control polypeptide assembly are preferably found in the CH3 domain(s) of the Fc domain.

In another aspect, the present invention provides an Fc construct comprising a) a first polypeptide comprising i) a first Fc domain monomer; ii) a second Fc domain monomer; and iii) a linker connecting the first Fc domain monomer to a second Fc domain monomer; b) a second polypeptide comprising i) a third Fc domain monomer; ii) a fourth Fc domain monomer; and iii) a linker connecting a third Fc domain monomer to a fourth Fc domain monomer; c) the third polypeptide contains a fifth Fc domain monomer; and d) the fourth polypeptide contains a sixth Fc domain monomer; wherein the first Fc domain monomer and the fifth Fc domain monomer are joined to form a first Fc domain, the second Fc domain monomer and the fourth Fc domain monomer are joined to form a second Fc domain, and the third Fc domain monomer and the sixth Fc domain monomer are joined to form a second Fc domain. domains are connected to form a third Fc domain, and wherein the at least one Fc domain contains an amino acid modification at position R292 (eg, one amino acid modification). In another aspect, the present invention provides an Fc construct comprising a) a first polypeptide comprising i) a first Fc domain monomer; ii) a second Fc domain monomer; and iii) a linker connecting the first Fc domain monomer to a second Fc domain monomer; b) a second polypeptide comprising i) a third Fc domain monomer; ii) a fourth Fc domain monomer; and iii) a linker connecting a third Fc domain monomer to a fourth Fc domain monomer; c) the third polypeptide contains a fifth Fc domain monomer; and d) the fourth polypeptide contains a sixth Fc domain monomer; wherein the first Fc domain monomer and the fifth Fc domain monomer are joined to form a first Fc domain, the second Fc domain monomer and the fourth Fc domain monomer are joined to form a second Fc domain, and the third Fc domain monomer and the sixth Fc domain monomer are joined to form a second Fc domain. domains are connected to form a third Fc domain, and wherein at least one Fc domain monomer contains an amino acid substitution at position R292.

In some embodiments, the first Fc domain contains an amino acid modification at position R292. In some embodiments, one or both of the first and fifth Fc domain monomers contain an amino acid substitution at position R292. In some embodiments, the second Fc domain contains an amino acid modification at position R292. In some embodiments, one or both of the second and fourth Fc domain monomers contain an amino acid substitution at position R292. In some embodiments, the third Fc domain contains an amino acid modification at position R292. In some embodiments, one or both of the third and sixth Fc domain monomers contain an amino acid substitution at position R292. In some embodiments, the first, second, and third Fc domains each contain an amino acid modification (eg, substitution) at position R292. In some embodiments, the first, second, and third Fc domains each contain an amino acid modification (eg, substitution) R292P (ie, each Fc monomer has an R292P modification, e.g., as compared to SEQ ID NO: 42). In some embodiments, one or both of the first and fifth Fc domain monomers contain an R292P amino acid substitution, one or both of the second and fourth Fc domain monomers contain an R292P amino acid substitution, and one or both of the third and sixth Fc domain monomers contain an amino acid substitution R292P.

In some embodiments, each amino acid modification (eg, substitution) at position R292 is independently selected from R292D, R292E, R292L, R292P, R292Q, R292R, R292T, or R292Y. In some embodiments, each amino acid modification (eg, substitution) at position R292 is R292P. In some embodiments, the first and third Fc domains each contain an amino acid modification (eg, substitution) I253A, and the first, second, and third Fc domains each contain an amino acid modification (eg, substitution) R292P. In some embodiments, one or both of the first and fifth Fc domain monomers contain an I253A amino acid substitution, one or both of the third and sixth Fc domain monomers contain an I253A amino acid substitution, one or both of the first and fifth Fc domain monomers contain an amino acid substitution R292P, one or both of the second and fourth Fc domain monomers contain the R292P amino acid substitution, and one or both of the third and sixth Fc domain monomers contain the R292P amino acid substitution. In some embodiments, the first, second, and third Fc domains each comprise an amino acid modification (eg, substitution) of I253A and R292P. In some embodiments, one or both of the first and fifth Fc domain monomers contain an I253A amino acid substitution, one or both of the second and fourth Fc domain monomers contain an I253A amino acid substitution, and one or both of the third and sixth Fc domain monomers contain an amino acid substitution I253A, one or both of the first and fifth Fc domain monomers contain the R292P amino acid substitution, one or both of the second and fourth Fc domain monomers contain the R292P amino acid substitution, and one or both of the third and sixth Fc domain monomers contain the R292P amino acid substitution.

In some embodiments, the sequence of each Fc domain is based on the sequence of the human IgG1 Fc domain with no more than ten single amino acid modifications. In some embodiments, the sequence of each Fc domain is based on SEQ ID NO: 42 with no more than ten single amino acid modifications.

In some embodiments, the first Fc domain and the third Fc domain each contain the amino acid substitutions I253A and R292P, and the second Fc domain contains the amino acid substitution R292P. In some cases, one or both of the first and fifth Fc domain monomers contain the I253A amino acid substitution, one or both of the first and fifth Fc domain monomers contain the R292P amino acid substitution, and one or both of the third and sixth Fc domain monomers contain the I253A amino acid substitution one or both of the third and sixth Fc domain monomers contain the R292P amino acid substitution, and one or both of the second and fourth Fc domain monomers contain the R292P amino acid substitution.

In some embodiments, the second Fc domain contains an amino acid substitution at position I253A. In some embodiments, one or both of the second and fourth Fc domain monomers contain the I253A amino acid substitution. In some embodiments, the first Fc domain and the third Fc domain each contain an I253A amino acid substitution. In some embodiments, one or both of the first and fifth Fc domain monomers contain the I253A amino acid substitution, and one or both of the third and sixth Fc domain monomers contain the I253A amino acid substitution. In some embodiments, the first Fc domain, the second Fc domain, and the third Fc domain each comprise an I253A amino acid substitution. In some embodiments, one or both of the first and fifth Fc domain monomers contain an I253A amino acid substitution, one or both of the second and fourth Fc domain monomers contain an I253A amino acid substitution, and one or both of the third and sixth Fc domain monomers contain an amino acid substitution I253A.

In some embodiments, the second Fc domain contains an amino acid substitution at position R292P. In some embodiments, one or both of the second and fourth Fc domain monomers contain the R292P amino acid substitution. In some embodiments, the second Fc domain contains amino acid substitutions I253A and R292P. In some embodiments, one or both of the second and fourth Fc domain monomers contain the I253A amino acid substitution, and one or both of the second and fourth Fc domain monomers contain the R292P amino acid substitution. In some embodiments, the first Fc domain and the third Fc domain each contain an I253A amino acid substitution, and the second Fc domain each contains an R292P amino acid substitution. In some embodiments, one or both of the first and fifth Fc domain monomers contain an I253A amino acid substitution, one or both of the third and sixth Fc domain monomers contain an I253A amino acid substitution, and one or both of the second and fourth Fc domain monomers contain an amino acid substitution R292P.

In some embodiments, the first Fc domain and the third Fc domain each contain an I253A amino acid substitution, and the second Fc domain each contains an I253A and R292P amino acid substitution. In some embodiments, one or both of the first and fifth Fc domain monomers contain an I253A amino acid substitution, one or both of the third and sixth Fc domain monomers contain an I253A amino acid substitution, one or both of the second and fourth Fc domain monomers contain an amino acid substitution I253A, and one or both of the second and fourth monomers of the Fc domain contain the amino acid substitution R292P. In some embodiments, the first Fc domain and the third Fc domain each contain an R292P amino acid substitution. In some embodiments, one or both of the first and fifth Fc domain monomers contain the R292P amino acid substitution, and one or both of the third and sixth Fc domain monomers contain the R292P amino acid substitution.

In some embodiments, the first Fc domain and the third Fc domain contain the amino acid substitution R292P, and the second Fc domain contains the amino acid substitution I253A. In some embodiments, one or both of the first and fifth Fc domain monomers contain an R292P amino acid substitution, one or both of the third and sixth Fc domain monomers contain an R292P amino acid substitution, and one or both of the second and fourth Fc domain monomers contain an amino acid substitution I253A. In some embodiments, the first Fc domain and the third Fc domain each comprise I253A and R292P (eg, contain the amino acid substitutions I253A and R292P). In some embodiments, one or both of the first and fifth Fc domain monomers contain an I253A amino acid substitution, one or both of the first and fifth Fc domain monomers contain an R292P amino acid substitution, one or both of the third and sixth Fc domain monomers contain an amino acid substitution I253A, and one or both of the third and sixth monomers of the Fc domain contain the amino acid substitution R292P.

In some embodiments, the first Fc domain and the third Fc domain each contain the amino acid substitutions I253A and R292P, and the second Fc domain contains the amino acid substitution I253A. In some embodiments, one or both of the first and fifth Fc domain monomers contain an I253A amino acid substitution, one or both of the first and fifth Fc domain monomers contain an R292P amino acid substitution, one or both of the third and sixth Fc domain monomers contain an amino acid substitution I253A, one or both of the third and sixth Fc domain monomers contain the R292P amino acid substitution, and one or both of the second and fourth Fc domain monomers contain the I253A amino acid substitution. In some embodiments, the first Fc domain, the second Fc domain, and the third Fc domain each comprise an R292P amino acid substitution. In some embodiments, one or both of the first and fifth Fc domain monomers contain an R292P amino acid substitution, one or both of the second and fourth Fc domain monomers contain an R292P amino acid substitution, and one or both of the third and sixth Fc domain monomers contain an amino acid substitution R292P.

In some embodiments, the first Fc domain and the third Fc domain each contain the amino acid substitution R292P, and 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 contain an R292P amino acid substitution, one or both of the third and sixth Fc domain monomers contain an R292P amino acid substitution, one or both of the second and fourth Fc domain monomers contain an amino acid substitution I253A, and one or both of the second and fourth monomers of the Fc domain contain the amino acid substitution R292P. In some embodiments, the first Fc domain, the second Fc domain, and the third Fc domain each comprise amino acid substitutions I253A and R292P. In some embodiments, one or both of the first and fifth Fc domain monomers contain the I253A amino acid substitution, one or both of the first and fifth Fc domain monomers contain the R292P amino acid substitution, and one or both of the second and fourth Fc domain monomers contain the amino acid substitution I253A, one or both of the second and fourth Fc domain monomers contain the R292P amino acid substitution, one or both of the third and sixth Fc domain monomers contain the I253A amino acid substitution, and one or both of the third and sixth Fc domain monomers contain the R292P amino acid substitution.

In some embodiments, the first Fc domain monomer and the fifth Fc domain monomer contain complementary dimerization selectivity modules that facilitate dimerization between the first Fc domain monomer and the fifth Fc domain monomer. In some embodiments, the second Fc domain monomer and the fourth Fc domain monomer comprise complementary dimerization selectivity modules that facilitate dimerization between the second Fc domain monomer and the fourth Fc domain monomer. In some embodiments, the third Fc domain monomer and the sixth Fc domain monomer contain complementary dimerization selectivity modules that facilitate dimerization between the third Fc domain monomer and the sixth Fc domain monomer. In some embodiments, the first polypeptide and the second polypeptide contain, consist of, or consist of substantially the same amino acid sequence, and wherein the third polypeptide and the fourth polypeptide contain, consist of, or consist of substantially the same amino acid sequence. In some embodiments, the second Fc domain monomer and the fourth Fc domain monomer each comprise D399K and either K409D or K409E. In some embodiments, the second Fc domain monomer and the fourth Fc domain monomer each comprise K392D and D399K. In some embodiments, the second Fc domain monomer and the fourth Fc domain monomer each comprise E357K and K370E. In some embodiments, the second Fc domain monomer and the fourth Fc domain monomer each comprise D356K and K439D. In some embodiments, the second Fc domain monomer and the fourth Fc domain monomer each comprise K392E and D399K. In some embodiments, the second Fc domain monomer and the fourth Fc domain monomer each comprise E357K and K370D. In some embodiments, the second Fc domain monomer and the fourth Fc domain monomer each comprise D356K and K439E. In some embodiments, the first Fc domain monomer and the third Fc domain monomer each contain S354C and T366W, and the fifth Fc domain monomer and the sixth Fc domain monomer each contain Y349C, T366S, L368A, and Y407V. In some embodiments, the third and fourth polypeptides each contain S354C and T366W, and the first Fc domain monomer and the third Fc domain monomer each contain Y349C, T366S, L368A, and Y407V. In some embodiments, the first Fc domain monomer and the third Fc domain monomer each contain E357K or E357R, and the fifth Fc domain monomer and the sixth Fc domain monomer each contain K370D and K370E. In some embodiments, the first Fc domain monomer and the third Fc domain monomer contain K370D or K370E, and the fifth Fc domain monomer and the sixth Fc domain monomer each contain E357K and E357R. In some embodiments, the first Fc domain monomer and the third Fc domain monomer each contain K409D or K409E, and the fifth Fc domain monomer and the sixth Fc domain monomer each contain D399K and D399R. In some embodiments, the first Fc domain monomer and the third Fc domain monomer contain D399K or D399R, and the fifth Fc domain monomer and the sixth Fc domain monomer each contain K409D and K409E.

In some embodiments, the linker (e.g., spacer) comprises a polypeptide having the sequence GGGGGGGGGGGGGGGGGGGG (SEQ ID NO: 23), GGGGS (SEQ ID NO: 1), GGSG (SEQ ID NO: 2), SGGG(SEQ ID NO: 3) , GSGS (SEQ ID NO: 4), GSGSGSGS (SEQ ID NO: 5), GSGSGSGS (SEQ ID NO: 6), GSGSGSGSGS (SEQ ID NO: 7), GSGSGSGSGSGS (SEQ ID NO: 8), GGSGGS (SEQ ID NO: 9), GGSGGSGGS (SEQ ID NO: 10), GGSGGSGGSGGS (SEQ ID NO: 11), GGSGGGSG (SEQ ID NO: 12), GGSGGGSGGGSG (SEQ ID NO: 13), GGSGGGSGGGSGGGSG (SEQ ID NO: 14), GGSGGGSGGGSGGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 80), GENLYFQSGG (SEQ ID NO: 28), SACYCELS (SEQ ID NO: 29), RSIAT (SEQ ID NO: 30), RPACKIPNDLKQKVMNH (SEQ ID NO: 31), GGSAGGSGSGSSGGSSGASGTGTAGGTGSGSGTGSG (SEQ ID NO : 32), AAAANSSIDLISVPVDSR (SEQ ID NO: 33), GGSGGGSEGGGSEGGGSEGGGSEGGGSEGGGSGGGS (SEQ ID NO: 34), GGGSGGGSGGGS (SEQ ID NO: 35), SGGGSGGGSGGGSGGGSGG (SEQ ID NO: 18), GGSGGGSGGGSGGGSGGS (SEQ ID NO: 36), GGGG (SEQ ID NO: 19), GGGGGGGG (SEQ ID NO: 20), GGGGGGGGGGGG (SEQ ID NO: 21) or GGGGGGGGGGGGGGGG (SEQ ID NO: 22). In other embodiments, one or more linkers in the Fc construct described herein is a spacer, such as an amino acid spacer of 2-200 amino acids (e.g., 2-100, 3-200, 3-150, 3-100, 3 -60, 3-50, 3-40, 3-30, 3-20, 3-10, 3-8, 3-5, 4-30, 5-30, 6-30, 8-30, 10-20 , 10-30, 12-30, 14-30, 20-30, 15-25, 15-30, 18-22 and 20-30 amino acids). In some cases, the amino acid spacer contains only glycine, only serine, or only serine and glycine. In some embodiments, the amino acid spacer contains only glycine.

In some embodiments, the first polypeptide and the second polypeptide each comprise, consist of, or consist essentially of the sequence DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMASRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV

HNAKTKPPEEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDKLTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGGGGGGGGGGGGGGGDKTHTCPP CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPPEEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSDLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 78). In some embodiments, the first polypeptide and the second polypeptide each comprise, consist of, or consist essentially of the sequence DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMASRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPPEEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDKLTKNQV SLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGGGGGGGGGGGGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPPEEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 78) with no more than 10 (9, 8, 7, 6, 5, 4, 3, 2 or 1) single amino acid modifications (eg, substitutions, eg, conservative substitutions). In some embodiments, the first polypeptide and the second polypeptide each contain, consist of, or consist essentially of the sequence of SEQ ID NO: 78 with no more than 10 (e.g., no more than 1, no more than 2, no more than 3, no more than 4 , no more than 5, no more than 6, no more than 7, no more than 8, no more than 9) single amino acid modifications in the region outside the linker (for example, outside the subsequence GGGGGGGGGGGGGGGGGGGG (SEQ ID NO: 23). Linker in SEQ ID NO: 78 may be replaced by an alternative linker. In some embodiments, each of the first polypeptide and the second polypeptide contains, consists of, or consists essentially of the sequence of SEQ ID NO: 78 with no more than 10 single amino acid modifications, provided that at position 33 of SEQ ID NO: 78 contains alanine, position 72 of SEQ ID NO: 78 contains proline, and position 319 of SEQ ID NO: 78 contains proline.

In some embodiments, the first polypeptide and the second polypeptide each comprise, consist of, or consist essentially of the sequence DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV

HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDKLTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGGGGGGGGGGGGGGGDKTHTCPP CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSDLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 49). In some embodiments, the first polypeptide and the second polypeptide each contain, consist of, or consist essentially of the sequence of SEQ ID NO: 49 with no more than 10 (e.g., no more than 9, 8, 7, 6, 5, 4, 3, 2 or 1) single amino acid modifications (eg substitutions, eg conservative substitutions). In some embodiments, the first polypeptide and the second polypeptide each comprise, consist of, or consist essentially of the sequence of SEQ ID NO: 49 with no more than 10 single amino acid modifications, provided that none of no more than 10 (e.g. no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9) single amino acid modifications are not located in the region outside the linker (for example, outside subsequence GGGGGGGGGGGGGGGGGGGG (SEQ ID NO: 23). The linker in SEQ ID NO: 49 may be replaced by an alternative linker. In some embodiments, the first polypeptide and the second polypeptide each contain, consist of, or consist essentially of the sequence of SEQ ID NO: 49 with no more than 10 single amino acid modifications, provided that position 134 SEQ ID NO: 49 contains cysteine, position 137 SEQ ID NO: 49 contains lysine, position 146 SEQ ID NO: 49 contains tryptophan, position 426 SEQ ID NO: 49 is lysine, and at position 436 of SEQ ID NO: 49 is aspartic acid.

In some embodiments, the first polypeptide and the second polypeptide each comprise, consist of, or consist essentially of the sequence DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDKLTKNQV SLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGGGGGGGGGGGGGGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMASRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE

EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 62). In some embodiments, the first polypeptide and the second polypeptide each contain, consist of, or consist essentially of the sequence of SEQ ID NO: 62 with no more than 10 (e.g., no more than 9, 8, 7, 6, 5, 4, 3, 2 or 1) single amino acid modifications (eg substitutions, eg conservative substitutions). In some embodiments, the first polypeptide and the second polypeptide each contain, consist of, or consist essentially of the sequence of SEQ ID NO: 62 with no more than 10 (e.g., no more than 1, no more than 2, no more than 3, no more than 4 , no more than 5, no more than 6, no more than 7, no more than 8, no more than 9) single amino acid modifications in a region outside the linker (for example, outside the subsequence GGGGGGGGGGGGGGGGGGGG (SEQ ID NO: 23). Linker in SEQ ID NO: 62 may be replaced by an alternative linker. In some embodiments, each of the first polypeptide and the second polypeptide contains, consists of, or consists essentially of the sequence of SEQ ID NO: 62 with no more than 10 single amino acid modifications, provided that at position 134 of SEQ ID NO: 62 is cysteine, position 137 SEQ ID NO: 62 is lysine, position 146 SEQ ID NO: 62 is tryptophan, position 280 SEQ ID NO: 62 is alanine, position 426 SEQ ID NO: 62 is lysine , and at position 436 of SEQ ID NO: 62 is aspartic acid.

In some embodiments, the first polypeptide and the second polypeptide each comprise, consist of, or consist essentially of the sequence DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMASRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDKLTKNQV SLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGGGGGGGGGGGGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 64). In some embodiments, the first polypeptide and the second polypeptide each contain, consist of, or consist essentially of the sequence of SEQ ID NO: 64 with no more than 10 (e.g., no more than 1, no more than 2, no more than 3, no more than 4 , no more than 5, no more than 6, no more than 7, no more than 8, no more than 9) single amino acid modifications (for example, substitutions, for example, conservative substitutions). In some embodiments, the first polypeptide and the second polypeptide each comprise, consist of, or consist essentially of the sequence of SEQ ID NO: 64 with no more than 10 single amino acid modifications in a region outside the linker (e.g., outside the subsequence GGGGGGGGGGGGGGGGGGGG (SEQ ID NO: 23).The linker in SEQ ID NO: 64 may be replaced by an alternative linker. In some embodiments, the first polypeptide and the second polypeptide each contain, consist of, or consist substantially of the sequence of SEQ ID NO: 64 with no more than 10 single amino acid modifications, provided that position 33 of SEQ ID NO: 64 is alanine.

In some embodiments, the first polypeptide and the second polypeptide each comprise, consist of, or consist essentially of the sequence DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMASRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDKLTKNQV SLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGGGGGGGGGGGGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMASRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 65). In some embodiments, the first polypeptide and the second polypeptide each contain, consist of, or consist essentially of the sequence of SEQ ID NO: 65 with no more than 10 (e.g., no more than 9, 8, 7, 6, 5, 4, 3, 2 or 1) single amino acid modifications (eg substitutions, eg conservative substitutions). In some embodiments, the first polypeptide and the second polypeptide each contain, consist of, or consist essentially of the sequence of SEQ ID NO: 65 with no more than 10 (e.g., no more than 1, no more than 2, no more than 3, no more than 4 , no more than 5, no more than 6, no more than 7, no more than 8, no more than 9) single amino acid modifications in the region outside the linker (for example, the subsequence GGGGGGGGGGGGGGGGGGGG (SEQ ID NO: 23). The linker in SEQ ID NO: 65 may be replaced by an alternative linker. In some embodiments, each of the first polypeptide and the second polypeptide contains, consists of, or consists essentially of the sequence of SEQ ID NO: 65 with no more than 10 single amino acid modifications, provided that at position 33 of SEQ ID NO: 65 is alanine, position 134 SEQ ID NO: 65 is cysteine, position 137 SEQ ID NO: 65 is lysine, position 146 SEQ ID NO: 65 is tryptophan, position 280 SEQ ID NO: 65 is alanine, at position 426 SEQ ID NO: 65 is lysine, and at position 436 SEQ ID NO: 65 is aspartic acid.

In some embodiments, the first polypeptide and the second polypeptide each comprise, consist of, or consist essentially of the sequence DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDKLTKNQV SLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGGGGGGGGGGGGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPPEEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 66). In some embodiments, the first polypeptide and the second polypeptide each contain, consist of, or consist essentially of the sequence of SEQ ID NO: 66 with no more than 10 (e.g., no more than 1, no more than 2, no more than 3, no more than 4 , no more than 5, no more than 6, no more than 7, no more than 8, no more than 9) single amino acid modifications (for example, substitutions, for example, conservative substitutions). In some embodiments, the first polypeptide and the second polypeptide each contain, consist of, or consist essentially of the sequence of SEQ ID NO: 66 with no more than 10 single amino acid modifications in a region outside the linker (e.g., outside the subsequence GGGGGGGGGGGGGGGGGGGG (SEQ ID NO: 23).The linker in SEQ ID NO: 66 may be replaced by an alternative linker. In some embodiments, the first polypeptide and the second polypeptide each contain, consist of, or consist substantially of the sequence of SEQ ID NO: 66 with no more than 10 single amino acid modifications, provided that position 134 SEQ ID NO: 66 contains cysteine, position 137 SEQ ID NO: 66 contains lysine, position 146 SEQ ID NO: 66 contains tryptophan, position 319 SEQ ID NO: 66 contains proline, at position 426 of SEQ ID NO: 66 is lysine, and at position 436 of SEQ ID NO: 66 is aspartic acid.

In some embodiments, the first polypeptide and the second polypeptide each comprise, consist of, or consist essentially of the sequence DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDKLTKNQV SLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGGGGGGGGGGGGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMASRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPPEEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 67). In some embodiments, the first polypeptide and the second polypeptide each contain, consist of, or consist essentially of the sequence of SEQ ID NO: 67 with no more than 10 (e.g., no more than 9, 8, 7, 6, 5, 4, 3, 2 or 1) single amino acid modifications (eg substitutions, eg conservative substitutions). In some embodiments, the first polypeptide and the second polypeptide each contain, consist of, or consist essentially of the sequence of SEQ ID NO: 67 with no more than 10 (e.g., no more than 1, no more than 2, no more than 3, no more than 4 , no more than 5, no more than 6, no more than 7, no more than 8, no more than 9) single amino acid modifications in the region outside the linker (for example, the subsequence GGGGGGGGGGGGGGGGGGGG (SEQ ID NO: 23). The linker in SEQ ID NO: 67 may be replaced by an alternative linker. In some embodiments, each of the first polypeptide and the second polypeptide contains, consists of, or consists essentially of the sequence of SEQ ID NO: 67 with no more than 10 single amino acid modifications, provided that at position 134SEQ ID NO: : 67 is cysteine, position 137 SEQ ID NO: 67 is lysine, position 146 SEQ ID NO: 67 is tryptophan, position 280 SEQ ID NO: 67 is alanine, position 318 SEQ ID NO: 67 is proline, position 426 SEQ ID NO: 67 contains lysine, and position 436 SEQ ID NO: 67 contains aspartic acid.

In some embodiments, the first polypeptide and the second polypeptide each comprise, consist of, or consist essentially of the sequence DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMASRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDKLTKNQV SLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGGGGGGGGGGGGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPPEEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 68). In some embodiments, the first polypeptide and the second polypeptide each contain, consist of, or consist essentially of the sequence of SEQ ID NO: 68 with no more than 10 (e.g., no more than 9, 8, 7, 6, 5, 4, 3, 2 or 1) single amino acid modifications (eg substitutions, eg conservative substitutions). In some embodiments, the first polypeptide and the second polypeptide each contain, consist of, or consist essentially of the sequence of SEQ ID NO: 68 with no more than 10 (e.g., no more than 1, no more than 2, no more than 3, no more than 4 , no more than 5, no more than 6, no more than 7, no more than 8, no more than 9) single amino acid modifications in the region outside the linker (for example, the subsequence GGGGGGGGGGGGGGGGGGGG (SEQ ID NO: 23). The linker in SEQ ID NO: 68 may be replaced by an alternative linker. In some embodiments, each of the first polypeptide and the second polypeptide contains, consists of, or consists essentially of the sequence of SEQ ID NO: 68 with no more than 10 single amino acid modifications, provided that at position 33SEQ ID NO : 68 is alanine, position 134 SEQ ID NO: 68 is cysteine, position 137 SEQ ID NO: 68 is lysine, position 146 SEQ ID NO: 68 is tryptophan, position 319 SEQ ID NO: 68 is proline, position 426 SEQ ID NO: 68 contains lysine, and position 436 SEQ ID NO: 68 contains aspartic acid.

In some embodiments, the first polypeptide and the second polypeptide each comprise, consist of, or consist essentially of the sequence DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMASRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV

YTLPPCRDKLTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGGGGGGGGGGGGGGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMASRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPPEEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 69). In some embodiments, the first polypeptide and the second polypeptide each contain, consist of, or consist essentially of the sequence of SEQ ID NO: 69 with no more than 10 (e.g., no more than 9, 8, 7, 6, 5, 4, 3, 2 or 1) single amino acid modifications (eg substitutions, eg conservative substitutions). In some embodiments, the first polypeptide and the second polypeptide each contain, consist of, or consist essentially of the sequence of SEQ ID NO: 69 with no more than 10 (e.g., no more than 1, no more than 2, no more than 3, no more than 4 , no more than 5, no more than 6, no more than 7, no more than 8, no more than 9) single amino acid modifications in the region outside the linker (for example, the subsequence GGGGGGGGGGGGGGGGGGGG (SEQ ID NO: 23). The linker in SEQ ID NO: 69 may be replaced by an alternative linker. In some embodiments, each of the first polypeptide and the second polypeptide contains, consists of, or consists essentially of the sequence of SEQ ID NO: 69 with no more than 10 single amino acid modifications, provided that at position 33 of SEQ ID NO: 69 is alanine, position 134 SEQ ID NO: 69 is cysteine, position 137 SEQ ID NO: 69 is lysine, position 146 SEQ ID NO: 69 is tryptophan, position 280 SEQ ID NO: 69 is alanine, position 319 of SEQ ID NO: 69 contains proline, position 426 of SEQ ID NO: 69 contains lysine, and position 436 of SEQ ID NO: 69 contains aspartic acid.

In some embodiments, the first polypeptide and the second polypeptide each comprise, consist of, or consist essentially of the sequence DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPPEEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDKLTKNQV SLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGGGGGGGGGGGGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 71). In some embodiments, the first polypeptide and the second polypeptide each contain, consist of, or consist essentially of the sequence of SEQ ID NO: 71 with no more than 10 (e.g., no more than 9, 8, 7, 6, 5, 4, 3, 2 or 1) single amino acid modifications (eg substitutions, eg conservative substitutions). In some embodiments, the first polypeptide and the second polypeptide each contain, consist of, or consist essentially of the sequence of SEQ ID NO: 71 with no more than 10 (e.g., no more than 1, no more than 2, no more than 3, no more than 4 , no more than 5, no more than 6, no more than 7, no more than 8, no more than 9) single amino acid modifications in the region outside the linker (for example, the subsequence GGGGGGGGGGGGGGGGGGGG (SEQ ID NO: 23). The linker in SEQ ID NO: 71 may be replaced by an alternative linker. In some embodiments, each of the first polypeptide and the second polypeptide contains, consists of, or consists essentially of the sequence of SEQ ID NO: 71 with no more than 10 single amino acid modifications, provided that at position 71SEQ ID NO: 71 71 contains proline, position 134 SEQ ID NO: 71 contains cysteine, position 137 SEQ ID NO: 71 contains lysine, position 146 SEQ ID NO: 71 contains tryptophan, position 426 SEQ ID NO: 71 contains lysine, and position 436 SEQ ID NO: 71 contains aspartic acid.

In some embodiments, the first polypeptide and the second polypeptide each comprise, consist of, or consist essentially of the sequence DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPPEEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDKLTKNQV SLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGGGGGGGGGGGGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMASRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 72). In some embodiments, the first polypeptide and the second polypeptide each contain, consist of, or consist essentially of the sequence of SEQ ID NO: 72 with no more than 10 (e.g., no more than 9, 8, 7, 6, 5, 4, 3, 2 or 1) single amino acid modifications (eg substitutions, eg conservative substitutions). In some embodiments, the first polypeptide and the second polypeptide each contain, consist of, or consist essentially of the sequence of SEQ ID NO: 72 with no more than 10 (e.g., no more than 1, no more than 2, no more than 3, no more than 4 , no more than 5, no more than 6, no more than 7, no more than 8, no more than 9) single amino acid modifications in the region outside the linker (for example, the subsequence GGGGGGGGGGGGGGGGGGGG (SEQ ID NO: 23). The linker in SEQ ID NO: 72 may be replaced by an alternative linker. In some embodiments, each of the first polypeptide and the second polypeptide contains, consists of, or consists essentially of the sequence of SEQ ID NO: 72 with no more than 10 single amino acid modifications, provided that at position 72SEQ ID NO: : 72 is proline, position 134 SEQ ID NO: 72 is cystine, position 137 SEQ ID NO: 72 is lysine, position 146 SEQ ID NO: 72 is tryptophan, position 426 SEQ ID NO: 72 is lysine, and at position 436 of SEQ ID NO: 72 is aspartic acid.

In some embodiments, the first polypeptide and the second polypeptide each comprise, consist of, or consist essentially of the sequence DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMASRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPPEEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDKLTKNQV SLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGGGGGGGGGGGGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 74). In some embodiments, the first polypeptide and the second polypeptide each contain, consist of, or consist essentially of the sequence of SEQ ID NO: 74 with no more than 10 (e.g., no more than 9, 8, 7, 6, 5, 4, 3, 2 or 1) single amino acid modifications (eg substitutions, eg conservative substitutions). In some embodiments, the first polypeptide and the second polypeptide each contain, consist of, or consist essentially of the sequence of SEQ ID NO: 74 with no more than 10 (e.g., no more than 1, no more than 2, no more than 3, no more than 4 , no more than 5, no more than 6, no more than 7, no more than 8, no more than 9) single amino acid modifications in the region outside the linker (for example, the subsequence GGGGGGGGGGGGGGGGGGGG (SEQ ID NO: 23). The linker in SEQ ID NO: 74 may be replaced by an alternative linker. In some embodiments, each of the first polypeptide and the second polypeptide contains, consists of, or consists essentially of the sequence of SEQ ID NO: 74 with no more than 10 single amino acid modifications, provided that at position 33 of SEQ ID NO: 74 is alanine, position 72 SEQ ID NO: 74 is proline, position 134 SEQ ID NO: 74 is cysteine, position 137 SEQ ID NO: 74 is lysine, position 146 SEQ ID NO: 74 is tryptophan, at position 426 of SEQ ID NO: 74 is lysine, and at position 436 of SEQ ID NO: 74 is aspartic acid.

In some embodiments, the first polypeptide and the second polypeptide each comprise, consist of, or consist essentially of the sequence DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMASRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPPEEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDKLTKNQV SLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGGGGGGGGGGGGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMASRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 75). In some embodiments, the first polypeptide and the second polypeptide each contain, consist of, or consist essentially of the sequence of SEQ ID NO: 75 with no more than 10 (e.g., no more than 9, 8, 7, 6, 5, 4, 3, 2 or 1) single amino acid modifications (eg substitutions, eg conservative substitutions). In some embodiments, the first polypeptide and the second polypeptide each contain, consist of, or consist essentially of the sequence of SEQ ID NO: 75 with no more than 10 (e.g., no more than 1, no more than 2, no more than 3, no more than 4 , no more than 5, no more than 6, no more than 7, no more than 8, no more than 9) single amino acid modifications in the region outside the linker (for example, the subsequence GGGGGGGGGGGGGGGGGGGG (SEQ ID NO: 23). The linker in SEQ ID NO: 75 may be replaced by an alternative linker. In some embodiments, each of the first polypeptide and the second polypeptide contains, consists of, or consists essentially of the sequence of SEQ ID NO: 75 with no more than 10 single amino acid modifications, provided that at position 33 of SEQ ID NO: 75 is alanine, position 72 SEQ ID NO: 75 is proline, position 134 SEQ ID NO: 75 is cysteine, position 137 SEQ ID NO: 75 is lysine, position 146 SEQ ID NO: 75 is tryptophan, position 280 of SEQ ID NO: 75 contains alanine, position 426 of SEQ ID NO: 75 contains lysine, and position 436 of SEQ ID NO: 75 contains aspartic acid.

In some embodiments, the first polypeptide and the second polypeptide each comprise, consist of, or consist essentially of the sequence DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPPEEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDKLTKNQV SLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGGGGGGGGGGGGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPPEEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 76). In some embodiments, the first polypeptide and the second polypeptide each contain, consist of, or consist essentially of the sequence of SEQ ID NO: 76 with no more than 10 (e.g., no more than 9, 8, 7, 6, 5, 4, 3, 2 or 1) single amino acid modifications (eg substitutions, eg conservative substitutions). In some embodiments, the first polypeptide and the second polypeptide each contain, consist of, or consist essentially of the sequence of SEQ ID NO: 76 with no more than 10 (e.g., no more than 1, no more than 2, no more than 3, no more than 4 , no more than 5, no more than 6, no more than 7, no more than 8, no more than 9) single amino acid modifications in the region outside the linker (for example, the subsequence GGGGGGGGGGGGGGGGGGGG (SEQ ID NO: 23). The linker in SEQ ID NO: 76 may be replaced by an alternative linker. In some embodiments, each of the first polypeptide and the second polypeptide contains, consists of, or consists essentially of the sequence of SEQ ID NO: 76 with no more than 10 single amino acid modifications, provided that at position 72 of SEQ ID NO: 76 is a proline and SEQ ID NO: 76 is a proline at position 319. In some embodiments, the first polypeptide and the second polypeptide each comprise, consist of, or consist essentially of the sequence DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPPEEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPCRDKLTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGGGGGGGGGGGGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMASRTPEVTCVVVDVSHEDPEVKFNW (SEQ ID NO: 77). In some embodiments, the first polypeptide and the second polypeptide each contain, consist of, or consist essentially of the sequence of SEQ ID NO: 77 with no more than 10 (e.g., no more than 9, 8, 7, 6, 5, 4, 3, 2 or 1) single amino acid modifications (eg substitutions, eg conservative substitutions). In some embodiments, the first polypeptide and the second polypeptide each contain, consist of, or consist essentially of the sequence of SEQ ID NO: 77 with no more than 10 (e.g., no more than 1, no more than 2, no more than 3, no more than 4 , no more than 5, no more than 6, no more than 7, no more than 8, no more than 9) single amino acid modifications in the region outside the linker (for example, the subsequence GGGGGGGGGGGGGGGGGGGG (SEQ ID NO: 23). The linker in SEQ ID NO: 77 may be replaced by an alternative linker. In some embodiments, each of the first polypeptide and the second polypeptide contains, consists of, or consists essentially of the sequence of SEQ ID NO: 77 with no more than 10 single amino acid modifications, provided that at position 72 of SEQ ID NO: 77 is proline, position 134 SEQ ID NO: 77 is cysteine, position 137 SEQ ID NO: 77 is lysine, position 146 SEQ ID NO: 77 is tryptophan, position 280 SEQ ID NO: 77 is alanine, position 319 SEQ ID NO: 77 contains proline, position 426 SEQ ID NO: 77 contains lysine, and position 436 SEQ ID NO: 77 contains aspartic acid.

In some embodiments, the first polypeptide and the second polypeptide each comprise, consist of, or consist essentially of the sequence DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMASRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPPEEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDKLTKNQV SLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGGGGGGGGGGGGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMASRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPPEEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 79). In some embodiments, the first polypeptide and the second polypeptide each contain, consist of, or consist essentially of the sequence of SEQ ID NO: 79 with no more than 10 (e.g., no more than 9, 8, 7, 6, 5, 4, 3, 2 or 1) single amino acid modifications (eg substitutions, eg conservative substitutions). In some embodiments, the first polypeptide and the second polypeptide each contain, consist of, or consist essentially of the sequence of SEQ ID NO: 79 with no more than 10 (e.g., no more than 1, no more than 2, no more than 3, no more than 4 , no more than 5, no more than 6, no more than 7, no more than 8, no more than 9) single amino acid modifications in the region outside the linker (for example, the subsequence GGGGGGGGGGGGGGGGGGGG (SEQ ID NO: 23). The linker in SEQ ID NO: 79 may be replaced by an alternative linker. In some embodiments, each of the first polypeptide and the second polypeptide contains, consists of, or consists essentially of the sequence of SEQ ID NO: 79 with no more than 10 single amino acid modifications, provided that at position 33 of SEQ ID NO: 79 contains alanine, position 72 of SEQ ID NO: 79 contains proline, position 280 of SEQ ID NO: 79 contains alanine, and position 319 of SEQ ID NO: 79 contains proline.

In some embodiments, each of the third polypeptide and the fourth polypeptide contains, consists of, or consists essentially of the sequence

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMASRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPPEEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVDGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 73). In some embodiments, the third polypeptide and the fourth polypeptide each contain, consist of, or consist essentially of the sequence of SEQ ID NO: 73 with no more than 10 (e.g., no more than 9, 8, 7, 6, 5, 4, 3, 2 or 1) single amino acid modifications (eg substitutions, eg conservative substitutions). In some embodiments, each of the third polypeptide and the fourth polypeptide contains, consists of, or consists substantially of the sequence of SEQ ID NO: 73 with no more than 10 single amino acid modifications, provided that at position 33 of SEQ ID NO: 73 is an alanine, and at position 72 of SEQ ID NO: 73 is proline.

In some embodiments, each of the third polypeptide and the fourth polypeptide contains, consists of, or consists essentially of the sequence

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVDGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 61). In some embodiments, the third polypeptide and the fourth polypeptide each contain, consist of, or consist essentially of the sequence of SEQ ID NO: 61 with no more than 10 (e.g., no more than 9, 8, 7, 6, 5, 4, 3, 2 or 1) single amino acid modifications (eg substitutions, eg conservative substitutions). In some embodiments, each of the third polypeptide and the fourth polypeptide contains, consists of, or consists substantially of the sequence of SEQ ID NO: 61 with no more than 10 single amino acid modifications, provided that at position 129 of SEQ ID NO: 61 is a cysteine, position 146 SEQ ID NO: 61 contains serine, position 148 SEQ ID NO: 61 contains alanine, position 150 SEQ ID NO: 61 contains aspartic acid, and position 187 SEQ ID NO: 61 contains valine.

In some embodiments, each of the third polypeptide and the fourth polypeptide contains, consists of, or consists essentially of the sequence

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMASRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVDGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 63). In some embodiments, the third polypeptide and the fourth polypeptide each contain, consist of, or consist essentially of the sequence of SEQ ID NO: 63 with no more than 10 (e.g., no more than 9, 8, 7, 6, 5, 4, 3, 2 or 1) single amino acid modifications (eg substitutions, eg conservative substitutions). In some embodiments, the third polypeptide and the fourth polypeptide each contain, consist of, or consist essentially of the sequence of SEQ ID NO: 63 with no more than 10 single amino acid modifications, provided that position 33 of SEQ ID NO: 63 is an alanine.

In some embodiments, each of the third polypeptide and the fourth polypeptide contains, consists of, or consists essentially of the sequence

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPPEEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVDGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 70). In some embodiments, the third polypeptide and the fourth polypeptide each contain, consist of, or consist essentially of the sequence of SEQ ID NO: 70 with no more than 10 (e.g., no more than 9, 8, 7, 6, 5, 4, 3, 2 or 1) single amino acid modifications (eg substitutions, eg conservative substitutions). In some embodiments, the third polypeptide and the fourth polypeptide each contain, consist of, or consist essentially of the sequence of SEQ ID NO: 70 with no more than 10 single amino acid modifications, provided that at position 72 of SEQ ID NO: 70 is a proline.

In some embodiments, each of the first and second polypeptides contains, consists of, or consists essentially of the sequence

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMASRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPPEEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDKLTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGGGGGGGGGGGGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPPEEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP SRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 78), and each of the third and fourth polypeptides contains, consists of, or consists essentially of the sequence

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMASRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPPEEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVDGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 73). In some embodiments, the first and second polypeptides each contain, consist of, or consist essentially of the sequence of SEQ ID NO: 78 with no more than 10 (e.g., no more than 9, 8, 7, 6, 5, 4, 3 , 2 or 1) single amino acid modifications (e.g., substitutions, e.g., conservative substitutions), and each of the third and fourth polypeptides contains, consists of, or consists essentially of the sequence SEQ ID NO: 73 with no more than 10 (9, 8 , 7, 6, 5, 4, 3, 2 or 1) single amino acid modifications (eg, substitutions, eg conservative substitutions). In some embodiments, each of the first and second polypeptides contains, consists of, or consists substantially of the sequence of SEQ ID NO: 78 with no more than 10 single amino acid modifications, provided that none of more than 10 (e.g., more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9) single amino acid modifications not located in the region outside the linker (for example, subsequences GGGGGGGGGGGGGGGGGGGG (SEQ ID NO: 23) The linker in SEQ ID NO: 78 may be replaced by an alternative linker.In some embodiments, the first and second polypeptides each contain, consist of, or consist essentially of the sequence of SEQ ID NO: 78 with no more than than 10 single amino acid modifications, provided that position 33 of SEQ ID NO: 78 is alanine, position 72 of SEQ ID NO: 78 is proline, and position 319 of SEQ ID NO: 78 is proline, and each of the third and fourth polypeptides contains, consists of, or consists essentially of the sequence of SEQ ID NO: 73 with no more than 10 single amino acid modifications, provided that at position 33 of SEQ ID NO: 73 there is an alanine and at position 72 of SEQ ID NO: 73 there is proline.

In some embodiments, each of the first and second polypeptides contains, consists of, or consists essentially of the sequence

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDKLTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGGGGGGGGGGGGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP SRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 49), and each of the third and fourth polypeptides contains, consists of, or consists essentially of the sequence

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVDGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 61). In some embodiments, each of the first and second polypeptides contains, consists of, or consists substantially of the sequence of SEQ ID NO: 49 with no more than 10 (9, 8, 7, 6, 5, 4, 3, 2, or 1) single amino acid modifications (e.g., substitutions, e.g., conservative substitutions), and each of the third and fourth polypeptides contains, consists of, or consists essentially of the sequence SEQ ID NO: 61 with no more than 10 (9, 8, 7, 6, 5, 4, 3, 2 or 1) single amino acid modifications (eg substitutions, eg conservative substitutions). In some embodiments, each of the first and second polypeptides contains, consists of, or consists substantially of the sequence of SEQ ID NO: 49 with no more than (e.g., no more than 1, no more than 2, no more than 3, no more than 4, no more than more than 5, no more than 6, no more than 7, no more than 8, no more than 9) single amino acid modifications in the region outside the linker (for example, subsequence GGGGGGGGGGGGGGGGGGGG (SEQ ID NO: 23); the linker in SEQ ID NO: 76 can be replaced alternative linker) and each of the third and fourth polypeptides contains, consists of, or consists essentially of the sequence of SEQ ID NO: 61 with no more than 10 single amino acid modifications. In some embodiments, each of the first and second polypeptides contains, consists of, or consists substantially of the sequence of SEQ ID NO: 49 with no more than 10 single amino acid modifications, provided that at position 134 of SEQ ID NO: 49 is a cysteine, at position 137 SEQ ID NO: 49 there is lysine, at position 146 SEQ ID NO: 49 there is tryptophan, at position 426 SEQ ID NO: 49 there is lysine, and at position 436 SEQ ID NO: 49 there is aspartic acid, and each of the third and a fourth polypeptide contains, consists of, or consists essentially of the sequence of SEQ ID NO: 61 with no more than 10 single amino acid modifications, provided that at position 129 of SEQ ID NO: 61 there is a cysteine, at 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 SEQ ID NO: 61 is valine.

In some embodiments, each of the first and second polypeptides contains, consists of, or consists essentially of the sequence

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDKLTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGGGGGGGGGGGGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMASRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP SRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 62), and each of the third and fourth polypeptides contains, consists of, or consists essentially of the sequence

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVDGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 61). In some embodiments, each of the first and second polypeptides contains, consists of, or consists substantially of the sequence of SEQ ID NO: 62 with no more than 10 (9, 8, 7, 6, 5, 4, 3, 2, or 1) single amino acid modifications (e.g., substitutions, e.g., conservative substitutions), and each of the third and fourth polypeptides contains, consists of, or consists essentially of the sequence SEQ ID NO: 61 with no more than 10 (9, 8, 7, 6, 5, 4, 3, 2 or 1) single amino acid modifications (eg substitutions, eg conservative substitutions). In some embodiments, each of the first and second polypeptides contains, consists of, or consists substantially of the sequence of SEQ ID NO: 62 with no more than 10 (e.g., no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9) single amino acid modifications in the region outside the linker (e.g., subsequence GGGGGGGGGGGGGGGGGGGG (SEQ ID NO: 23); the linker in SEQ ID NO: 62 may be replaced by an alternative linker) and each of the third and fourth polypeptides contains, consists of, or consists essentially of the sequence of SEQ ID NO: 61 with no more than 10 single amino acid modifications. In some embodiments, each of the first and second polypeptides contains, consists of, or consists substantially of the sequence of SEQ ID NO: 62 with no more than 10 single amino acid modifications, provided that at position 134 of SEQ ID NO: 62 is a cysteine, position 137 SEQ ID NO: 62 contains lysine, position 146 SEQ ID NO: 62 contains tryptophan, position 280 SEQ ID NO: 62 contains alanine, position 426 SEQ ID NO: 62 contains lysine, and position 436 SEQ ID NO: 62 is aspartic acid, and each of the third and fourth polypeptides contains, consists of, or consists essentially of the sequence of SEQ ID NO: 61 with no more than 10 single amino acid modifications, provided that at position 129 of SEQ ID NO: 61 is cysteine, position 146 SEQ ID NO: 61 is serine, position 148 SEQ ID NO: 61 is alanine, position 150 SEQ ID NO: 61 is aspartic acid, and position 187 SEQ ID NO: 61 is valine.

In some embodiments, each of the first and second polypeptides contains, consists of, or consists essentially of the sequence

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMASRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDKLTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGGGGGGGGGGGGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP SRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 64), and each of the third and fourth polypeptides contains, consists of, or consists essentially of the sequence

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMASRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVDGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 63). In some embodiments, each of the first and second polypeptides contains, consists of, or consists substantially of the sequence of SEQ ID NO: 64 with no more than 10 (9, 8, 7, 6, 5, 4, 3, 2, or 1) single amino acid modifications (e.g., substitutions, e.g., conservative substitutions), and each of the third and fourth polypeptides contains, consists of, or consists substantially of the sequence SEQ ID NO: 63 with no more than 10 (9, 8, 7, 6, 5, 4, 3, 2 or 1) single amino acid modifications (eg substitutions, eg conservative substitutions). In some embodiments, each of the first and second polypeptides contains, consists of, or consists substantially of the sequence of SEQ ID NO: 64 of no more than 10 (e.g., no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9) single amino acid modifications in the region outside the linker (for example, subsequence GGGGGGGGGGGGGGGGGGGG (SEQ ID NO: 23); the linker in SEQ ID NO: 64 can be replaced alternative linker) and each of the third and fourth polypeptides contains, consists of, or consists essentially of the sequence of SEQ ID NO: 63 with no more than 10 single amino acid modifications. In some embodiments, each of the first and second polypeptides contains, consists of, or consists substantially of the sequence of SEQ ID NO: 64 with no more than 10 single amino acid modifications, provided that at position 33 of SEQ ID NO: 64 is an alanine, and each of the third and fourth polypeptides contains, consists of, or consists substantially of the sequence of SEQ ID NO: 63 with no more than 10 single amino acid modifications, provided that at position 33 of SEQ ID NO: 63 is an alanine.

In some embodiments, each of the first and second polypeptides contains, consists of, or consists essentially of the sequence

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMASRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDKLTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGGGGGGGGGGGGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMASRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP SRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 65), and each of the third and fourth polypeptides contains, consists of, or consists essentially of the sequence

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMASRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVDGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 63). In some embodiments, each of the first and second polypeptides contains, consists of, or consists substantially of the sequence of SEQ ID NO: 65 with no more than 10 (9, 8, 7, 6, 5, 4, 3, 2, or 1) single amino acid modifications (e.g., substitutions, e.g., conservative substitutions), and each of the third and fourth polypeptides contains, consists of, or consists substantially of the sequence SEQ ID NO: 63 with no more than 10 (9, 8, 7, 6, 5, 4, 3, 2 or 1) single amino acid modifications (eg substitutions, eg conservative substitutions). In some embodiments, each of the first and second polypeptides contains, consists of, or consists substantially of the sequence of SEQ ID NO: 65 with no more than 10 (e.g., no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9) single amino acid modifications in the region outside the linker (e.g., subsequence GGGGGGGGGGGGGGGGGGGG (SEQ ID NO: 23); the linker in SEQ ID NO: 65 may be replaced by an alternative linker) and each of the third and fourth polypeptides contains, consists of, or consists essentially of the sequence of SEQ ID NO: 63 with no more than 10 single amino acid modifications. In some embodiments, each of the first and second polypeptides contains, consists of, or consists substantially of the sequence of SEQ ID NO: 65 with no more than 10 single amino acid modifications, provided that at position 33 of SEQ ID NO: 65 is an alanine, at position 134 SEQ ID NO: 65 there is cysteine, at position 137 SEQ ID NO: 65 there is lysine, at position 146 SEQ ID NO: 65 there is tryptophan, at position 280 SEQ ID NO: 65 there is alanine, at position 426 SEQ ID NO : 65 is lysine, and at position 436 of SEQ ID NO: 65 is aspartic acid, and each of the third and fourth polypeptides contains, consists of, or consists substantially of the sequence of SEQ ID NO: 63 with no more than 10 single amino acid modifications, provided that position 33 of SEQ ID NO: 63 is alanine.

In some embodiments, each of the first and second polypeptides contains, consists of, or consists essentially of the sequence

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDKLTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGGGGGGGGGGGGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPPEEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP SRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 66), and each of the third and fourth polypeptides contains, consists of, or consists essentially of the sequence DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY (SEQ ID NO: 61). In some embodiments, each of the first and second polypeptides contains, consists of, or consists substantially of the sequence of SEQ ID NO: 66 with no more than 10 (9, 8, 7, 6, 5, 4, 3, 2, or 1) single amino acid modifications (e.g., substitutions, e.g., conservative substitutions), and each of the third and fourth polypeptides contains, consists of, or consists essentially of the sequence SEQ ID NO: 61 with no more than 10 (9, 8, 7, 6, 5, 4, 3, 2 or 1) single amino acid modifications (eg substitutions, eg conservative substitutions). In some embodiments, each of the first and second polypeptides contains, consists of, or consists substantially of the sequence of SEQ ID NO: 66 with no more than 10 (e.g., no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9) single amino acid modifications in the region outside the linker (e.g., subsequence GGGGGGGGGGGGGGGGGGGG (SEQ ID NO: 23); the linker in SEQ ID NO: 66 may be replaced by an alternative linker) and each of the third and fourth polypeptides contains, consists of, or consists essentially of the sequence of SEQ ID NO: 61 with no more than 10 single amino acid modifications. In some embodiments, each of the first and second polypeptides contains, consists of, or consists substantially of the sequence of SEQ ID NO: 66 with no more than 10 single amino acid modifications, provided that at position 134 of SEQ ID NO: 66 is a cysteine, position 137 SEQ ID NO: 66 contains lysine, position 146 SEQ ID NO: 66 contains tryptophan, position 319 SEQ ID NO: 66 contains proline, position 426 SEQ ID NO: 66 contains lysine, and position 436 SEQ ID NO: 66 is aspartic acid, and each of the third and fourth polypeptides contains, consists of, or consists essentially of the sequence of SEQ ID NO: 61 with no more than 10 single amino acid modifications, provided that at position 129 of SEQ ID NO: 61 is cysteine, position 146 SEQ ID NO: 61 is serine, position 148 SEQ ID NO: 61 is alanine, position 150 SEQ ID NO: 61 is aspartic acid, and position 187 SEQ ID NO: 61 is valine.

In some embodiments, each of the first and second polypeptides contains, consists of, or consists essentially of the sequence

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDKLTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGGGGGGGGGGGGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMASRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPPEEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP SRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 67), and each of the third and fourth polypeptides contains, consists of, or consists essentially of the sequence

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVDGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 61). In some embodiments, each of the first and second polypeptides contains, consists of, or consists substantially of the sequence of SEQ ID NO: 67 with no more than 10 (9, 8, 7, 6, 5, 4, 3, 2, or 1) single amino acid modifications (e.g., substitutions, e.g., conservative substitutions), and each of the third and fourth polypeptides contains, consists of, or consists essentially of the sequence SEQ ID NO: 61 with no more than 10 (9, 8, 7, 6, 5, 4, 3, 2 or 1) single amino acid modifications (eg substitutions, eg conservative substitutions). In some embodiments, each of the first and second polypeptides contains, consists of, or consists substantially of the sequence of SEQ ID NO: 67 with no more than 10 (e.g., no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9) single amino acid modifications in the region outside the linker (for example, the subsequence GGGGGGGGGGGGGGGGGGGG (SEQ ID NO: 23; the linker in SEQ ID NO: 67 can be replaced alternative linker) and each of the third and fourth polypeptides contains, consists of, or consists essentially of the sequence of SEQ ID NO: 61 with no more than 10 single amino acid modifications. In some embodiments, each of the first and second polypeptides contains, consists of, or consists essentially of the sequence under SEQ ID NO: 67 with no more than 10 single amino acid modifications, provided that at position 134 SEQ ID NO: 67 there is a cysteine, at position 137 SEQ ID NO: 67 there is a lysine, at position 146 SEQ ID NO: 67 contains tryptophan, position 280 SEQ ID NO: 67 contains alanine, position 318 SEQ ID NO: 67 contains proline, position 426 SEQ ID NO: 67 contains lysine, and position 436 SEQ ID NO: 67 contains aspartic acid, and each of the third and fourth polypeptides contains, consists of, or consists essentially of the sequence of SEQ ID NO: 61 with no more than 10 single amino acid modifications, provided that at position 129 of SEQ ID NO: 61 is a cysteine, position 146 SEQ ID NO: 61 contains serine, position 148 SEQ ID NO: 61 contains alanine, position 150 SEQ ID NO: 61 contains aspartic acid, and position 187 SEQ ID NO: 61 contains valine.

In some embodiments, each of the first and second polypeptides contains, consists of, or consists essentially of the sequence

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMASRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDKLTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGGGGGGGGGGGGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPPEEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP SRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 68), and each of the third and fourth polypeptides contains, consists of, or consists essentially of the sequence

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMASRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVDGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 63). In some embodiments, each of the first and second polypeptides contains, consists of, or consists substantially of the sequence of SEQ ID NO: 68 with no more than 10 (9, 8, 7, 6, 5, 4, 3, 2, or 1) single amino acid modifications (e.g., substitutions, e.g., conservative substitutions), and each of the third and fourth polypeptides contains, consists of, or consists substantially of the sequence SEQ ID NO: 63 with no more than 10 (9, 8, 7, 6, 5, 4, 3, 2 or 1) single amino acid modifications (eg substitutions, eg conservative substitutions). In some embodiments, each of the first and second polypeptides contains, consists of, or consists substantially of the sequence of SEQ ID NO: 68 with no more than 10 (e.g., no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9) single amino acid modifications in the region outside the linker (e.g., subsequence GGGGGGGGGGGGGGGGGGGG (SEQ ID NO: 23); the linker in SEQ ID NO: 68 may be replaced by an alternative linker) and each of the third and fourth polypeptides contains, consists of, or consists essentially of the sequence of SEQ ID NO: 63 with no more than 10 single amino acid modifications. In some embodiments, each of the first and second polypeptides contains, consists of, or consists substantially of the sequence of SEQ ID NO: 68 with no more than 10 single amino acid modifications, provided that at position 33 of SEQ ID NO: 68 is an alanine, at position 134 SEQ ID NO: 68 there is cysteine, at position 137 SEQ ID NO: 68 there is lysine, at position 146 SEQ ID NO: 68 there is tryptophan, at position 319 SEQ ID NO: 68 there is proline, at position 426 SEQ ID NO : 68 is lysine, and at position 436 of SEQ ID NO: 68 is aspartic acid, and each of the third and fourth polypeptides contains, consists of, or consists substantially of the sequence of SEQ ID NO: 63 with no more than 10 single amino acid modifications, provided that position 33 of SEQ ID NO: 63 is alanine.

In some embodiments, each of the first and second polypeptides contains, consists of, or consists essentially of the sequence

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMASRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDKLTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGGGGGGGGGGGGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMASRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPPEEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP SRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 69), and each of the third and fourth polypeptides contains, consists of, or consists essentially of the sequence

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMASRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVDGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 63). In some embodiments, each of the first and second polypeptides contains, consists of, or consists substantially of the sequence of SEQ ID NO: 69 with no more than 10 (9, 8, 7, 6, 5, 4, 3, 2, or 1) single amino acid modifications (e.g., substitutions, e.g., conservative substitutions), and each of the third and fourth polypeptides contains, consists of, or consists substantially of the sequence SEQ ID NO: 63 with no more than 10 (9, 8, 7, 6, 5, 4, 3, 2 or 1) single amino acid modifications (eg substitutions, eg conservative substitutions). In some embodiments, each of the first and second polypeptides contains, consists of, or consists substantially of the sequence of SEQ ID NO: 69 with no more than 10 (e.g., no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9) single amino acid modifications in the region outside the linker (e.g., subsequence GGGGGGGGGGGGGGGGGGGG (SEQ ID NO: 23); the linker in SEQ ID NO: 69 may be replaced by an alternative linker) and each of the third and fourth polypeptides contains, consists of, or consists essentially of the sequence of SEQ ID NO: 63 with no more than 10 single amino acid modifications. In some embodiments, each of the first and second polypeptides contains, consists of, or consists substantially of the sequence of SEQ ID NO: 69 with no more than 10 single amino acid modifications, provided that at position 33 of SEQ ID NO: 69 is an alanine, position 134 SEQ ID NO: 69 contains cysteine, position 137 SEQ ID NO: 69 contains lysine, position 146 SEQ ID NO: 69 contains tryptophan, position 280 SEQ ID NO: 69 contains alanine, position 319 SEQ ID NO : 69 is proline, position 426 of SEQ ID NO: 69 is lysine, and position 436 of SEQ ID NO: 69 is aspartic acid, and the third and fourth polypeptides each contain, consist of, or consist essentially of the sequence of SEQ ID NO: : 63 with no more than 10 single amino acid modifications, provided that position 33 of SEQ ID NO: 63 is an alanine.

In some embodiments, each of the first and second polypeptides contains, consists of, or consists essentially of the sequence

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPPEEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDKLTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGGGGGGGGGGGGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP SRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 71), and each of the third and fourth polypeptides contains, consists of, or consists essentially of the sequence

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPPEEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVDGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 70). In some embodiments, each of the first and second polypeptides contains, consists of, or consists substantially of the sequence of SEQ ID NO: 71 with no more than 10 (9, 8, 7, 6, 5, 4, 3, 2, or 1) single amino acid modifications (e.g., substitutions, e.g., conservative substitutions), and each of the third and fourth polypeptides contains, consists of, or consists substantially of the sequence SEQ ID NO: 70 with no more than 10 (9, 8, 7, 6, 5, 4, 3, 2 or 1) single amino acid modifications (eg substitutions, eg conservative substitutions). In some embodiments, each of the first and second polypeptides contains, consists of, or consists substantially of the sequence of SEQ ID NO: 71 with no more than 10 (e.g., no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9) single amino acid modifications in the region outside the linker (e.g., subsequence GGGGGGGGGGGGGGGGGGGG (SEQ ID NO: 23); the linker in SEQ ID NO: 71 may be replaced by an alternative linker) and each of the third and fourth polypeptides contains, consists of, or consists essentially of the sequence of SEQ ID NO: 70 with no more than 10 single amino acid modifications. In some embodiments, each of the first and second polypeptides contains, consists of, or consists substantially of the sequence of SEQ ID NO: 71 with no more than 10 single amino acid modifications, provided that at position 71 of SEQ ID NO: 71 is a proline, at position 134 SEQ ID NO: 71 there is cysteine, at position 137 SEQ ID NO: 71 there is lysine, at position 146 SEQ ID NO: 71 there is tryptophan, at position 426 SEQ ID NO: 71 there is lysine, and at position 436 SEQ ID NO : 71 is aspartic acid, and each of the third and fourth polypeptides contains, consists of, or consists essentially of the sequence of SEQ ID NO: 70 with no more than 10 single amino acid modifications, provided that at position 72 SEQ ID NO: 70 there is proline.

In some embodiments, each of the first and second polypeptides contains, consists of, or consists essentially of the sequence

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPPEEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDKLTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGGGGGGGGGGGGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMASRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP SRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 72), and each of the third and fourth polypeptides contains, consists of, or consists essentially of the sequence

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPPEEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVDGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 70). In some embodiments, the first and second polypeptides each contain, consist of, or consist essentially of the sequence of SEQ ID NO: 72 with no more than 10 (9, 8, 7, 6, 5, 4, 3, 2, or 1) single amino acid modifications (e.g., substitutions, e.g., conservative substitutions), and each of the third and fourth polypeptides contains, consists of, or consists substantially of the sequence SEQ ID NO: 70 with no more than 10 (9, 8, 7, 6, 5, 4, 3, 2 or 1) single amino acid modifications (eg substitutions, eg conservative substitutions). In some embodiments, each of the first and second polypeptides contains, consists of, or consists substantially of the sequence of SEQ ID NO: 72 with no more than 10 (e.g., no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9) single amino acid modifications in the region outside the linker (e.g., subsequence GGGGGGGGGGGGGGGGGGGG (SEQ ID NO: 23); the linker in SEQ ID NO: 72 may be replaced by an alternative linker) and each of the third and fourth polypeptides contains, consists of, or consists essentially of the sequence of SEQ ID NO: 70 with no more than 10 single amino acid modifications. In some embodiments, each of the first and second polypeptides contains, consists of, or consists substantially of the sequence of SEQ ID NO: 72 with no more than 10 single amino acid modifications, provided that at position 72 of SEQ ID NO: 72 is a proline, position 134 SEQ ID NO: 72 contains cystine, position 137 SEQ ID NO: 72 contains lysine, position 146 SEQ ID NO: 72 contains tryptophan, position 426 SEQ ID NO: 72 contains lysine, and position 436 SEQ ID NO: 72 is aspartic acid, and each of the third and fourth polypeptides contains, consists of, or consists essentially of the sequence of SEQ ID NO: 70 with no more than 10 single amino acid modifications, and each of the third and fourth polypeptides contains, consists of or consists essentially of the sequence of SEQ ID NO: 70 with no more than 10 single amino acid modifications, provided that at position 72 of SEQ ID NO: 70 is a proline.

In some embodiments, each of the first and second polypeptides contains, consists of, or consists essentially of the sequence

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMASRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPPEEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDKLTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGGGGGGGGGGGGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP SRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 74), and each of the third and fourth polypeptides contains, consists of, or consists essentially of the sequence

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMASRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPPEEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVDGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 73). In some embodiments, each of the first and second polypeptides contains, consists of, or consists substantially of the sequence of SEQ ID NO: 74 with no more than 10 (9, 8, 7, 6, 5, 4, 3, 2, or 1) single amino acid modifications (e.g., substitutions, e.g., conservative substitutions), and each of the third and fourth polypeptides contains, consists of, or consists essentially of the sequence SEQ ID NO: 73 with no more than 10 (9, 8, 7, 6, 5, 4, 3, 2 or 1) single amino acid modifications (eg substitutions, eg conservative substitutions). In some embodiments, each of the first and second polypeptides contains, consists of, or consists substantially of the sequence of SEQ ID NO: 74 with no more than 10 (e.g., no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9) single amino acid modifications in the region outside the linker (e.g., subsequence GGGGGGGGGGGGGGGGGGGG (SEQ ID NO: 23); the linker in SEQ ID NO:74 may be replaced by an alternative linker) and each of the third and fourth polypeptides contains, consists of, or consists essentially of the sequence of SEQ ID NO: 73 with no more than 10 single amino acid modifications. In some embodiments, each of the first and second polypeptides contains, consists of, or consists substantially of the sequence of SEQ ID NO: 74 with no more than 10 single amino acid modifications, provided that at position 33 of SEQ ID NO: 74 is an alanine, position 72 SEQ ID NO: 74 contains proline, position 134 SEQ ID NO: 74 contains cysteine, position 137 SEQ ID NO: 74 contains lysine, position 146 SEQ ID NO: 74 contains tryptophan, position 426 SEQ ID NO : 74 is lysine, and at position 436 of SEQ ID NO: 74 is aspartic acid, and each of the third and fourth polypeptides contains, consists of, or consists substantially of the sequence of SEQ ID NO: 73 with no more than 10 single amino acid modifications, provided that position 33 of SEQ ID NO: 73 is alanine and position 72 of SEQ ID NO: 73 is proline.

In some embodiments, each of the first and second polypeptides contains, consists of, or consists essentially of the sequence

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMASRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPPEEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDKLTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGGGGGGGGGGGGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMASRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP SRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 75), and each of the third and fourth polypeptides contains, consists of, or consists essentially of the sequence

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMASRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPPEEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVDGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 73). In some embodiments, each of the first and second polypeptides contains, consists of, or consists substantially of the sequence of SEQ ID NO: 75 with no more than 10 (9, 8, 7, 6, 5, 4, 3, 2, or 1) single amino acid modifications (e.g., substitutions, e.g., conservative substitutions), and each of the third and fourth polypeptides contains, consists of, or consists essentially of the sequence SEQ ID NO: 73 with no more than 10 (9, 8, 7, 6, 5, 4, 3, 2 or 1) single amino acid modifications (eg substitutions, eg conservative substitutions). In some embodiments, each of the first and second polypeptides contains, consists of, or consists substantially of the sequence of SEQ ID NO: 75 with no more than 10 (e.g., no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9) single amino acid modifications in the region outside the linker (e.g., subsequence GGGGGGGGGGGGGGGGGGGG (SEQ ID NO: 23); the linker in SEQ ID NO: 75 may be replaced by an alternative linker) and each of the third and fourth polypeptides contains, consists of, or consists essentially of the sequence of SEQ ID NO: 73 with no more than 10 single amino acid modifications. In some embodiments, each of the first and second polypeptides contains, consists of, or consists substantially of the sequence of SEQ ID NO: 75 with no more than 10 single amino acid modifications, provided that at position 33 of SEQ ID NO: 75 is an alanine, at position 72 SEQ ID NO: 75 there is proline, at position 134 SEQ ID NO: 75 there is cysteine, at position 137 SEQ ID NO: 75 there is lysine, at position 146 SEQ ID NO: 75 there is tryptophan, at position 280 SEQ ID NO : 75 is alanine, position 426 of SEQ ID NO: 75 is lysine, and position 436 of SEQ ID NO: 75 is aspartic acid, and each of the third and fourth polypeptides contains, consists of, or consists essentially of the sequence of SEQ ID NO: : 73 with no more than 10 single amino acid modifications, provided that position 33 of SEQ ID NO: 73 is alanine and position 72 of SEQ ID NO: 73 is proline.

In some embodiments, each of the first and second polypeptides contains, consists of, or consists essentially of the sequence

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPPEEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDKLTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGGGGGGGGGGGGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPPEEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP SRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 76), and each of the third and fourth polypeptides contains, consists of, or consists essentially of the sequence

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPPEEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVDGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 70). In some embodiments, each of the first and second polypeptides contains, consists of, or consists substantially of the sequence of SEQ ID NO: 76 with no more than 10 (9, 8, 7, 6, 5, 4, 3, 2, or 1) single amino acid modifications (e.g., substitutions, e.g., conservative substitutions), and each of the third and fourth polypeptides contains, consists of, or consists substantially of the sequence SEQ ID NO: 70 with no more than 10 (9, 8, 7, 6, 5, 4, 3, 2 or 1) single amino acid modifications (eg substitutions, eg conservative substitutions). In some embodiments, each of the first and second polypeptides contains, consists of, or consists substantially of the sequence of SEQ ID NO: 76 with no more than 10 (e.g., no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9) single amino acid modifications in the region outside the linker (e.g., subsequence GGGGGGGGGGGGGGGGGGGG (SEQ ID NO: 23); the linker in SEQ ID NO: 76 may be replaced by an alternative linker) and each of the third and fourth polypeptides contains, consists of, or consists essentially of the sequence of SEQ ID NO: 70 with no more than 10 single amino acid modifications. In some embodiments, each of the first and second polypeptides contains, consists of, or consists substantially of the sequence of SEQ ID NO: 76 with no more than 10 single amino acid modifications, provided that at position 72 of SEQ ID NO: 76 is a proline and at position 319 of SEQ ID NO: 76 is a proline, and each of the third and fourth polypeptides contains, consists of, or consists substantially of the sequence of SEQ ID NO: 70 with no more than 10 single amino acid modifications, provided that at position 72 SEQ ID NO: 70 contains proline.

In some embodiments, each of the first and second polypeptides contains, consists of, or consists essentially of the sequence

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPPEEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDKLTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGGGGGGGGGGGGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPPEEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP SRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 76), and each of the third and fourth polypeptides contains, consists of, or consists essentially of the sequence

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPPEEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVDGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 70).

In some embodiments, each of the first and second polypeptides contains, consists of, or consists essentially of the sequence

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPPEEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDKLTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGGGGGGGGGGGGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMASRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPPEEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP SRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 77), and each of the third and fourth polypeptides contains, consists of, or consists essentially of the sequence

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPPEEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVDGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 70). In some embodiments, the first and second polypeptides each contain, consist of, or consist essentially of the sequence of SEQ ID NO: 77 with no more than 10 (9, 8, 7, 6, 5, 4, 3, 2, or 1) single amino acid modifications (e.g., substitutions, e.g., conservative substitutions), and each of the third and fourth polypeptides contains, consists of, or consists substantially of the sequence SEQ ID NO: 70 with no more than 10 (9, 8, 7, 6, 5, 4, 3, 2 or 1) single amino acid modifications (eg substitutions, eg conservative substitutions). In some embodiments, each of the first and second polypeptides contains, consists of, or consists substantially of the sequence of SEQ ID NO: 77 with no more than 10 (e.g., no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9) single amino acid modifications in the region outside the linker (e.g., subsequence GGGGGGGGGGGGGGGGGGGG (SEQ ID NO: 23); the linker in SEQ ID NO: 77 may be replaced by an alternative linker) and each of the third and fourth polypeptides contains, consists of, or consists essentially of the sequence of SEQ ID NO: 70 with no more than 10 single amino acid modifications. In some embodiments, each of the first and second polypeptides contains, consists of, or consists substantially of the sequence of SEQ ID NO: 77 with no more than 10 single amino acid modifications, provided that at position 72 of SEQ ID NO: 77 is a proline, position 134 SEQ ID NO: 77 contains cysteine, position 137 SEQ ID NO: 77 contains lysine, position 146 SEQ ID NO: 77 contains tryptophan, position 280 SEQ ID NO: 77 contains alanine, position 319 SEQ ID NO : 77 is proline, position 426 of SEQ ID NO: 77 is lysine, and position 436 of SEQ ID NO: 77 is aspartic acid, and the third and fourth polypeptides each contain, consist of, or consist essentially of the sequence of SEQ ID NO: : 70 with no more than 10 single amino acid modifications, provided that position 72 of SEQ ID NO: 70 is proline.

In some embodiments, each of the first and second polypeptides contains, consists of, or consists essentially of the sequence

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMASRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPPEEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDKLTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGGGGGGGGGGGGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMASRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPPEEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP SRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 79), and each of the third and fourth polypeptides contains, consists of, or consists essentially of the sequence

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMASRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPPEEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVDGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 73). In some embodiments, each of the first and second polypeptides contains, consists of, or consists substantially of the sequence of SEQ ID NO: 79 with no more than 10 (9, 8, 7, 6, 5, 4, 3, 2, or 1) single amino acid modifications (e.g., substitutions, e.g., conservative substitutions), and each of the third and fourth polypeptides contains, consists of, or consists essentially of the sequence SEQ ID NO: 73 with no more than 10 (9, 8, 7, 6, 5, 4, 3, 2 or 1) single amino acid modifications (eg substitutions, eg conservative substitutions). In some embodiments, each of the first and second polypeptides contains, consists of, or consists substantially of the sequence of SEQ ID NO: 79 with no more than 10 (e.g., no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9) single amino acid modifications in the region outside the linker (e.g., subsequence GGGGGGGGGGGGGGGGGGGG (SEQ ID NO: 23); the linker in SEQ ID NO: 79 may be replaced by an alternative linker) and each of the third and fourth polypeptides contains, consists of, or consists essentially of the sequence of SEQ ID NO: 73 with no more than 10 single amino acid modifications. In some embodiments, each of the first and second polypeptides contains, consists of, or consists substantially of the sequence of SEQ ID NO: 79 with no more than 10 single amino acid modifications, provided that at position 33 of SEQ ID NO: 79 is an alanine, at position 72 of SEQ ID NO: 79 is proline, at position 280 of SEQ ID NO: 79 is alanine, and at position 319 of SEQ ID NO: 79 is proline, and each of the third and fourth polypeptides contains, consists of, or consists essentially of sequences of SEQ ID NO: 73 with no more than 10 single amino acid modifications, provided that position 33 of SEQ ID NO: 73 is an alanine and position 72 of SEQ ID NO: 73 is a proline. In some embodiments, the Fc construct is Design 4, Design 5, Design 6, Design 7, Design 8, Design 9, Design 10, Design 11, Design 12, Design 13, Design 14, Design 15, Design 16, Design 17 , design 18 or design 19.

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.

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

In a second aspect, the Fc construct of the present invention comprises (i) a first Fc domain comprising a first Fc domain monomer and a second Fc domain monomer, and (ii) a second Fc domain comprising a third Fc domain monomer and a fourth Fc monomer -domain, where at least one Fc domain contains an amino acid modification at position I253. In another aspect, the Fc construct of the present invention comprises (i) a first Fc domain comprising a first Fc domain monomer and a second Fc domain monomer, and (ii) a second Fc domain comprising a third Fc domain monomer and a fourth Fc monomer -domain, where at least one monomer of the Fc domain contains an amino acid substitution at position I253. In some embodiments, each of the amino acid modifications (e.g., substitutions) at position I253 is independently selected from the group consisting of I253A, I253C, I253D, I253E, I253F, I253G, I253H, I253I, I253K, I253L, I253M, I253N, I253P, I253Q , I253R, I253S, I253T, I253V, I253W and I253Y. In some embodiments, the amino acid modification (eg, substitution) at position I253 is I253A. In some embodiments, the Fc construct contains at least one amino acid modification (eg, substitution) at position R292. In some embodiments, the amino acid modification (eg, substitution) at position R292 is selected from the group consisting of R292D, R292E, R292L, R292P, R292Q, R292R, R292T, and R292Y. In some embodiments, the amino acid modification (eg, substitution) at position R292 is R292P.

In some embodiments, the first Fc domain monomer and the third Fc domain monomer are connected via a linker (eg, a spacer). In some embodiments, the second Fc domain monomer and the fourth Fc domain monomer are connected via a linker (eg, a spacer). In some embodiments, each linker (e.g., spacer) is independently selected from a polypeptide having the sequence GGGGGGGGGGGGGGGGGGGG (SEQ ID NO: 23), GGGGS (SEQ ID NO: 1), GGSG (SEQ ID NO: 2), SGGG(SEQ ID NO : 3), GSGS (SEQ ID NO: 4), GSGSGSGS (SEQ ID NO: 5), GSGSGSGS (SEQ ID NO: 6), GSGSGSGSGS (SEQ ID NO: 7), GSGSGSGSGSGS (SEQ ID NO: 8), GGSGGS (SEQ ID NO: 9), GGSGGSGGS (SEQ ID NO: 10), GGSGGSGGSGGS (SEQ ID NO: 11), GGSGGGSG (SEQ ID NO: 12), GGSGGGSGGGSG (SEQ ID NO: 13), GGSGGGSGGGSGGGSG (SEQ ID NO: 14), GGSGGGSGGGSGGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 80), GENLYFQSGG (SEQ ID NO: 28), SACYCELS (SEQ ID NO: 29), RSIAT (SEQ ID NO: 30), RPACKIPNDLKQKVMNH (SEQ ID NO: 31), GGSAGGSGSGSSGGSSGASGTGTAGGTGSGSGTGSG ( SEQ ID NO: 32), AAANSSIIDLISVPVDSR (SEQ ID NO: 33), GGSGGGSEGGGSEGGGSEGGGSEGGGSEGGGSGGGS (SEQ ID NO: 34), GGGSGGGSGGGS (SEQ ID NO: 35), SGGGSGGGSGGGSGGGSGGG (SEQ ID NO: 18), GGSGGGSGGGSGGGSGGS (SEQ ID NO: 36 ), GGGG (SEQ ID NO: 19), GGGGGGGG (SEQ ID NO: 20), GGGGGGGGGGGG (SEQ ID NO: 21) or GGGGGGGGGGGGGGGG (SEQ ID NO: 22).

In some embodiments, the first Fc domain monomer and the second Fc domain monomer contain complementary dimerization selectivity modules that facilitate dimerization between the first Fc domain monomer and the second Fc domain monomer. In some embodiments, the third Fc domain monomer and the fourth Fc domain monomer comprise complementary dimerization selectivity modules that facilitate dimerization between the third Fc domain monomer and the fourth Fc domain monomer.

In another aspect, the present invention provides an Fc construct that comprises (i) a first Fc domain comprising a first Fc domain monomer and a second Fc domain monomer, and (ii) a second Fc domain comprising a third Fc domain monomer and a fourth an Fc domain monomer, wherein at least one Fc domain contains an amino acid modification at position R292. In another aspect, the present invention provides an Fc construct that comprises (i) a first Fc domain comprising a first Fc domain monomer and a second Fc domain monomer, and (ii) a second Fc domain comprising a third Fc domain monomer and a fourth an Fc domain monomer, wherein at least one Fc domain monomer contains an amino acid substitution at position R292. In some embodiments, each of the amino acid modifications (eg, substitutions) at position R292 is independently selected from the group consisting of R292D, R292E, R292L, R292P, R292Q, R292R, R292T, and R292Y. In some embodiments, the amino acid modification (eg, substitution) at position R292 is R292P. In some embodiments, the first Fc domain monomer and the third Fc domain monomer are connected via a linker.

In some embodiments, the second Fc domain monomer and the fourth Fc domain monomer are connected via a linker. In some embodiments, each linker is independently selected from a polypeptide having the sequence GGGGGGGGGGGGGGGGGGGG (SEQ ID NO: 23), GGGGS (SEQ ID NO: 1), GGSG (SEQ ID NO: 2), SGGG(SEQ ID NO: 3), GSGS (SEQ ID NO: 4), GSGSGSGS (SEQ ID NO: 5), GSGSGSGSGS (SEQ ID NO: 6), GSGSGSGSGS (SEQ ID NO: 7), GSGSGSGSGSGS (SEQ ID NO: 8), GGSGGS (SEQ ID NO: 9), GGSGGSGGS (SEQ ID NO: 10), GGSGGSGGSGGS (SEQ ID NO: 11), GGSGGGSG (SEQ ID NO: 12), GGSGGGSGGGSG (SEQ ID NO: 13), GGSGGGSGGGSGGGSG (SEQ ID NO: 14), GGSGGGSGGGSGGGGGSGGGGSGGGGSGGGGS ( SEQ ID NO: 80), GENLYFQSGG (SEQ ID NO: 28), SACYCELS (SEQ ID NO: 29), RSIAT (SEQ ID NO: 30), RPACKIPNDLKQKVMNH (SEQ ID NO: 31), GGSAGGSGSGSSGGSSGASGTGTAGGTGSGSGTGSG (SEQ ID NO: 32 ), AAANSSIDLISPVDSR (SEQ ID NO: 33), GGSGGGSEGGGSEGGGSEGGGSEGGGSEGGGSGGGS (SEQ ID NO: 34), GGGSGGGSGGGS (SEQ ID NO: 35), SGGGSGGGSGGGSGGGSGGG (SEQ ID NO: 18), GGSGGGSGGGSGGGSGGS (SEQ ID NO: 36), GGGG (SEQ ID NO: 19), GGGGGGGG (SEQ ID NO: 20), GGGGGGGGGGGG (SEQ ID NO: 21) or GGGGGGGGGGGGGGGG (SEQ ID NO: 22). In some embodiments, the linker is GGGGGGGGGGGGGGGGGGGG (SEQ ID NO: 23).

In some embodiments, the first Fc domain monomer and the second Fc domain monomer contain complementary dimerization selectivity modules that facilitate dimerization between the first Fc domain monomer and the second Fc domain monomer. In some embodiments, the third Fc domain monomer and the fourth Fc domain monomer comprise complementary dimerization selectivity modules that facilitate dimerization between the third Fc domain monomer and the fourth Fc domain monomer.

In another aspect, the present invention provides a method for producing the Fc construct described herein. The method involves: a) providing a host cell containing polynucleotides encoding the polypeptides of the present invention; b) expression of the polypeptides in the host cell under conditions that allow formation of the Fc construct; and c) recovery of the Fc construct.

In another aspect, the present invention provides an Fc construct comprising a) a first polypeptide comprising i) a first Fc domain monomer; ii) a second Fc domain monomer; and iii) a linker connecting the first Fc domain monomer to a second Fc domain monomer; b) a second polypeptide comprising i) a third Fc domain monomer; ii) a fourth Fc domain monomer; and iii) a linker connecting a third Fc domain monomer to a fourth Fc domain monomer; c) the third polypeptide contains a fifth Fc domain monomer; and d) the fourth polypeptide contains a sixth Fc domain monomer; wherein the first Fc domain monomer and the fifth Fc domain monomer are joined to form a first Fc domain, the second Fc domain monomer and the fourth Fc domain monomer are joined to form a second Fc domain, and the third Fc domain monomer and the sixth Fc domain monomer are joined to form a second Fc domain. domains are linked to form a third Fc domain, and wherein the at least one Fc domain contains an amino acid modification at position I253, wherein each of the first and second polypeptides contains the sequence

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMASRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPPEEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDKLTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGGGGGGGGGGGGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPPEEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP SRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 78), and each of the third and fourth polypeptides contains the sequence

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMASRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPPEEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVDGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 73). In another aspect, the present invention provides an Fc construct comprising a) a first polypeptide comprising i) a first Fc domain monomer; ii) a second monomer of the Fc domain; and iii) a linker connecting the first Fc domain monomer to the second Fc domain monomer; b) a second polypeptide comprising i) a third Fc domain monomer; ii) a fourth monomer of the Fc domain; and iii) a linker connecting the third Fc domain monomer to the fourth Fc domain monomer; c) the third polypeptide contains a fifth monomer of the Fc domain; and d) the fourth polypeptide contains a sixth Fc domain monomer; wherein the first Fc domain monomer and the fifth Fc domain monomer are joined to form a first Fc domain, the second Fc domain monomer and the fourth Fc domain monomer are joined to form a second Fc domain, and the third Fc domain monomer and the sixth Fc domain monomer are joined to form a second Fc domain domains are connected to form a third Fc domain, where each of the first and second polypeptides contains the sequence

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMASRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPPEEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDKLTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGGGGGGGGGGGGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPPEEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP SRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 78), and each of the third and fourth polypeptides contains the sequence

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMASRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPPEEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVDGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 73).

In another aspect, the present invention provides an Fc construct comprising a) a first polypeptide comprising i) a first Fc domain monomer; ii) a second monomer of the Fc domain; and iii) a linker connecting the first Fc domain monomer to the second Fc domain monomer; b) a second polypeptide comprising i) a third Fc domain monomer; ii) a fourth monomer of the Fc domain; and iii) a linker connecting the third Fc domain monomer to the fourth Fc domain monomer; c) the third polypeptide contains a fifth monomer of the Fc domain; and d) the fourth polypeptide contains a sixth Fc domain monomer; wherein the first Fc domain monomer and the fifth Fc domain monomer are joined to form a first Fc domain, the second Fc domain monomer and the fourth Fc domain monomer are joined to form a second Fc domain, and the third Fc domain monomer and the sixth Fc domain monomer are joined to form a second Fc domain domains are linked to form a third Fc domain, and wherein the at least one Fc domain contains an amino acid modification at position I253, wherein each of the first and second polypeptides contains the sequence

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMASRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPPEEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDKLTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGGGGGGGGGGGGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMASRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPPEEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP SRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 79), and each of the third and fourth polypeptides contains the sequence

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMASRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPPEEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVDGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 73). In another aspect, the present invention provides an Fc construct comprising a) a first polypeptide comprising i) a first Fc domain monomer; ii) a second monomer of the Fc domain; and iii) a linker connecting the first Fc domain monomer to the second Fc domain monomer; b) a second polypeptide comprising i) a third Fc domain monomer; ii) a fourth monomer of the Fc domain; and iii) a linker connecting the third Fc domain monomer to the fourth Fc domain monomer; c) the third polypeptide contains a fifth monomer of the Fc domain; and d) the fourth polypeptide contains a sixth Fc domain monomer; wherein the first Fc domain monomer and the fifth Fc domain monomer are joined to form a first Fc domain, the second Fc domain monomer and the fourth Fc domain monomer are joined to form a second Fc domain, and the third Fc domain monomer and the sixth Fc domain monomer are joined to form a second Fc domain. domains are connected to form a third Fc domain, where each of the first and second polypeptides contains the sequence

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMASRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPPEEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDKLTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGGGGGGGGGGGGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMASRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPPEEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP SRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 79), and each of the third and fourth polypeptides contains the sequence

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMASRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPPEEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVDGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 73).

In another aspect, the present invention provides an Fc construct comprising: a) a first polypeptide comprising i) a first Fc domain monomer; ii) a second monomer of the Fc domain; and iii) a linker connecting the first Fc domain monomer to the second Fc domain monomer; b) a second polypeptide comprising i) a third Fc domain monomer; ii) a fourth monomer of the Fc domain; and iii) a linker connecting the third Fc domain monomer to the fourth Fc domain monomer; c) the third polypeptide contains a fifth monomer of the Fc domain; and d) the fourth polypeptide contains a sixth Fc domain monomer; wherein the first Fc domain monomer and the fifth Fc domain monomer are joined to form a first Fc domain, the second Fc domain monomer and the fourth Fc domain monomer are joined to form a second Fc domain, and the third Fc domain monomer and the sixth Fc domain monomer are joined to form a second Fc domain domains are linked to form a third Fc domain, and wherein the at least one Fc domain contains an amino acid modification at position R292, wherein each of the first and second polypeptides contains the sequence

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPPEEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDKLTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGGGGGGGGGGGGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPPEEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP SRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 76), and each of the third and fourth polypeptides contains the sequence

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPPEEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVDGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 70). In another aspect, the present invention provides an Fc construct comprising: a) a first polypeptide comprising i) a first Fc domain monomer; ii) a second monomer of the Fc domain; and iii) a linker connecting the first Fc domain monomer to the second Fc domain monomer; b) a second polypeptide comprising i) a third Fc domain monomer; ii) a fourth monomer of the Fc domain; and iii) a linker connecting the third Fc domain monomer to the fourth Fc domain monomer; c) the third polypeptide contains a fifth monomer of the Fc domain; and d) the fourth polypeptide contains a sixth Fc domain monomer; wherein the first Fc domain monomer and the fifth Fc domain monomer are joined to form a first Fc domain, the second Fc domain monomer and the fourth Fc domain monomer are joined to form a second Fc domain, and the third Fc domain monomer and the sixth Fc domain monomer are joined to form a second Fc domain. domains are connected to form a third Fc domain, where each of the first and second polypeptides contains the sequence

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPPEEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDKLTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGGGGGGGGGGGGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPPEEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP SRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 76), and each of the third and fourth polypeptides contains the sequence

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPPEEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVDGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 70).

In some embodiments, in any of the Fc constructs described herein, each Fc domain is independently an IgG1 Fc domain, an IgG2 Fc domain, an IgG3 Fc domain, an IgG4 Fc domain, or a combination thereof. In some embodiments, in any of the Fc constructs described herein, each Fc domain is independently an IgG1 Fc domain, an IgG2 Fc domain, an IgG3 Fc domain, an IgG4 Fc domain, or a combination thereof with no more than 10 (eg, no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) single amino acid modifications. In some embodiments, each Fc domain is an IgG1 Fc domain. In some embodiments, each Fc domain is an IgG2 Fc domain. In some embodiments, each Fc domain is an IgG3 Fc domain. In some embodiments, the first, second, and third Fc domains are each an IgG1 Fc domain. In some embodiments, each Fc domain is a human IgG1 Fc domain. In some embodiments, each Fc domain comprises SEQ ID NO: 42 with no more than 10 (e.g., no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9) single amino acid modifications.

In some embodiments, in any of the Fc constructs described herein, one or more Fc domain monomers is a human IgG Fc domain monomer containing no more than ten (e.g., no more than 1, 2, 3, 4 , 5, 6, 7, 8, 9 or 10) amino acid modifications. In some embodiments, in any of the Fc constructs described herein, each Fc domain monomer has no more than ten (e.g., no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 ) amino acid modifications. In some embodiments, in any of the Fc constructs described herein, each Fc monomer contains the sequence of SEQ ID NO: 42 with no more than ten (e.g., no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acid modifications. In some embodiments, in any of the Fc constructs described herein, each Fc monomer contains the sequence of SEQ ID NO: 42 with 5 amino acid modifications. In some embodiments, in any of the Fc constructs described herein, each Fc monomer contains the sequence of SEQ ID NO: 42 with 10 amino acid modifications. In some embodiments, in any of the Fc constructs described herein, each Fc monomer contains the sequence of SEQ ID NO: 42 with 8 amino acid modifications. In each case, the modifications may include one or both of the amino acid substitutions I253A and R292P.

In another aspect, the present invention provides a host cell that expresses the Fc construct described herein. The host cell contains polynucleotides encoding the polypeptides of the present invention, where the polynucleotides are expressed in the host cell.

In another aspect, the present invention provides a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-79. In another aspect, the present invention provides a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 49 and SEQ ID NO: 61-79.

In another aspect, the present invention provides a composition containing or consisting of the sequence: SEQ ID NO: 78 and a polypeptide containing or consisting of the sequence: SEQ ID NO: 73. In another aspect, the present invention provides a composition containing or consisting of the sequence: SEQ ID NO: 49 and a polypeptide containing or consisting of the sequence: SEQ ID NO: 61. In another aspect, the present invention provides a composition containing or consisting of the sequence: SEQ ID NO: 62 and a polypeptide containing or consisting of the sequence: SEQ ID NO: 61. In another aspect, the present invention provides a composition containing or consisting of the sequence: SEQ ID NO: 64 and a polypeptide containing or consisting of the sequence: SEQ ID NO: 63. In another aspect, the present invention provides a composition containing or consisting of the sequence: SEQ ID NO: 65 and a polypeptide containing or consisting of the sequence: SEQ ID NO: 63. In another aspect, the present invention provides a composition containing or consisting of the sequence: SEQ ID NO: 67 and a polypeptide containing or consisting of the sequence: SEQ ID NO: 61. In another aspect, the present invention provides a composition containing or consisting of the sequence: SEQ ID NO: 68 and a polypeptide containing or consisting of the sequence: SEQ ID NO: 63. In another aspect, the present invention provides a composition containing or consisting of sequence: SEQ ID NO: 69 and a polypeptide containing or consisting of the sequence: SEQ ID NO: 63. In another aspect, the present invention provides a composition containing or consisting of the sequence: SEQ ID NO: 72 and a polypeptide containing or consisting of the sequence: SEQ ID NO: 70. In another aspect, the present invention provides a composition containing or consisting of the sequence: SEQ ID NO: 74 and a polypeptide containing or consisting of the sequence: SEQ ID NO: 73. In another aspect, the present invention provides a composition containing or consisting of the sequence: SEQ ID NO: 75 and a polypeptide containing or consisting of the sequence: SEQ ID NO: 73. In another aspect, the present invention provides a composition containing or consisting of the sequence: SEQ ID NO: 77 and a polypeptide containing or consisting of sequence: SEQ ID NO: 70. In another aspect, the present invention provides a composition containing or consisting of the sequence: SEQ ID NO: 79 and a polypeptide containing or consisting of the sequence: SEQ ID NO: 73. In another aspect, the present invention provides a composition, containing or consisting of the sequence: SEQ ID NO: 76 and a polypeptide containing or consisting of the sequence: SEQ ID NO: 70.

In some embodiments, the compositions comprise the first and second listed polypeptides present in a molar ratio of 1.1:1 to 1:1.1.

In another aspect, the present invention provides a method of treating a patient, comprising administering to the patient any of the compositions described herein.

In another aspect, the present invention provides a pharmaceutical composition comprising a substantially homogeneous (e.g., at least 85%, 90%, 95%, 97%, 98%, 99% homogeneous) population of the Fc constructs described herein (e.g. , Fc constructs containing three Fc domains) or a composition containing a substantially homogeneous collection of Fc constructs described herein (e.g., a composition containing a substantially homogeneous collection of Fc constructs containing three Fc domains) and one or more pharmaceutically acceptable carriers or excipients. Such pharmaceutical compositions can be prepared without significant aggregation or undesirable multimerization of Fc constructs.

In some aspects, the present invention provides a composition containing a polypeptide that contains SEQ ID NO: 76, and a polypeptide that contains SEQ ID NO: 70. In some aspects, the present invention provides a composition containing a polypeptide that contains SEQ ID NO: 78, and a polypeptide that contains SEQ ID NO: 73. In some aspects, the present invention provides a composition comprising a polypeptide that contains SEQ ID NO: 79 and a polypeptide that contains SEQ ID NO: 73.

In some aspects, the present invention provides a method of treating a patient, comprising administering to the patient any of the compositions described herein.

In some aspects, the present invention provides a cell containing a nucleic acid sequence encoding SEQ ID NO: 76 and a nucleic acid sequence encoding SEQ ID NO: 70. In some aspects, the present invention provides a cell containing a nucleic acid sequence encoding SEQ ID NO: 70. 78, and a nucleic acid sequence encoding SEQ ID NO: 73. In some aspects, the present invention provides a cell containing a nucleic acid sequence encoding SEQ ID NO: 79, and a nucleic acid sequence encoding SEQ ID NO: 73. In some aspects, the present The invention provides a cell containing a nucleic acid sequence encoding any of the Fc constructs described herein.

In another aspect, the present invention provides a method of reducing the activation of immune cells that mediate an immune response in a subject, comprising administering to the subject an Fc construct described herein (e.g., an Fc construct containing three Fc domains) or a composition comprising a substantially homogeneous a set of Fc constructs described herein (eg, a composition containing a substantially homogeneous set of Fc constructs containing three Fc domains). In some embodiments, the subject has an autoimmune disease.

In another aspect, the present invention provides a method of treating inflammation or an inflammatory disease in a subject, comprising administering to the subject an Fc construct described herein (e.g., an Fc construct containing three Fc domains) or a composition containing a substantially homogeneous array of Fc- constructs described herein (eg, compositions containing a substantially homogeneous collection of Fc constructs containing three Fc domains).

In another aspect, the present invention provides a method of promoting autoantibody clearance and/or inhibiting antigen presentation in a subject, comprising administering to the subject an Fc construct described herein (e.g., an Fc construct containing three Fc domains) or a composition comprising essentially a homogeneous collection of Fc constructs described herein (eg, a composition comprising a substantially homogeneous collection of Fc constructs comprising three Fc domains).

In some embodiments, exemplary diseases that can be treated by administration of an Fc construct described herein (eg, an Fc construct comprising three Fc domains) include the following: rheumatoid arthritis (RA); systemic lupus erythematosus (SLE); ANCA-associated vasculitis; antiphospholipid antibody syndrome; autoimmune hemolytic anemia; chronic inflammatory demyelinating neuropathy; clearance of allocellular antigens during transplantation; autoreactivity in GVHD; replacement therapy with antibodies, IgG-based therapeutics, IgG-based paraproteins, dermatomyositis; Goodpasture's syndrome; type II organ system hypersensitivity syndromes mediated by antibody-dependent cell-mediated cytotoxicity, eg Guillain-Barré syndrome, CIDP, dermatomyositis, Felty's syndrome, antibody-mediated rejection, autoimmune thyroid disease, ulcerative colitis, autoimmune liver disease; idiopathic thrombocytopenic purpura; myasthenia gravis, neuromyelitis optica; pemphigus and other autoimmune disorders accompanied by the formation of blisters; Sjögren's syndrome; autoimmune cytopenias and other disorders mediated by antibody-dependent phagocytosis; other FcR-dependent inflammatory syndromes, such as synovitis; dermatomyositis, systemic vasculitis, glomerulitis and vasculitis.

In some embodiments, the Fc constructs described herein do not contain an antigen recognition region, such as a variable domain or a complementarity determining region (CDR). In some embodiments, the Fc construct (or the Fc domain within an Fc construct) is formed in whole or in part by the association of Fc domain monomers that are present in different polypeptides. In some embodiments, the Fc constructs described herein contain an antigen recognition region, such as a variable domain or CDR. In certain embodiments, the Fc construct does not contain an additional domain (eg, IgM tail or IgA tail) that facilitates the association of the two polypeptides. In other embodiments, covalent bonds are present in the Fc construct only between the two Fc domain monomers that join to form the Fc domain. In other embodiments, the Fc construct does not contain covalent bonds between the Fc domains. In other embodiments, the Fc construct provides sufficient structural flexibility such that all or substantially all of the Fc domains in the Fc construct are capable of simultaneously interacting with the Fc receptor on the cell surface. In one embodiment, the domain monomers differ in primary sequence from the wild type or from each other in that they contain selectivity modules that enable dimerization.

Fc domain monomers belonging to an Fc domain Fc construct may have the same primary amino acid sequence. For example, both Fc domain monomers belonging to the Fc domain may have the same selectivity modulus, providing dimerization, for example, both Fc domain monomers belonging to the Fc domain may have identical mutations, providing a charge reversal of at least two positions in the ring of charged residues at the interface between the C _H 3 domains. In some embodiments, the first polypeptide and the second polypeptide have the same amino acid sequence. In some embodiments, the third and fourth polypeptides have the same amino acid sequence. In some embodiments, 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 different from the sequence of the sixth Fc domain monomer. In some embodiments, the sequence of the second Fc domain monomer is the same as the sequence of the fourth Fc domain monomer.

In any of the Fc constructs described herein, the Fc domain monomers belonging to the Fc domain construct may have different sequences, such as sequences that differ by no more than 20 amino acids (e.g., no more than 15, 10 amino acids), for example, no more than 20, 15, 10, 8, 7, 6, 5, 4, 3, or 2 amino acids between two Fc domain monomers (ie, between an Fc domain monomer and another Fc construct monomer). For example, the sequences of the Fc domain monomers of a construct described herein may differ because the selectivity modules that provide complementary dimerization of any of the Fc constructs may contain an engineered cavity in the antibody C _H 3 constant domain of one of the domain monomers and an engineered knob in the constant domain C _H 3 domain of the antibody of another of the Fc domain monomers, wherein the engineered cavity and the engineered protrusion are arranged to form a protrusion-to-cavity pair of Fc domain monomers. In some embodiments, the Fc constructs contain amino acid modifications in the C _H 3 domain. In some embodiments, the Fc constructs contain amino acid modifications in the C _H 3 domain of Fc domain monomers (one or more Fc domain monomers) for selective dimerization. Exemplary engineered cavities and protrusions are shown in Table 1. In other embodiments, selectivity modules enabling complementary dimerization comprise an engineered (substituted) negatively charged amino acid in the antibody C _H 3 constant domain of one of the domain monomers and a designed (substituted) positively charged amino acid in the constant C _H 3 antibody domain of another of the Fc domain monomers, where a negatively charged amino acid and a positively charged amino acid are arranged to promote the formation of an Fc domain between complementary domain monomers. Exemplary complementary amino acid substitutions are shown in Tables 2A-2C. In some embodiments, one or more Fc domain monomers have the same sequence. In some embodiments, one or more Fc domain monomers have the same modifications. In some embodiments, only one, two, three, or four Fc domain monomers have the same modifications.

In some cases, the Fc domain contains at least one amino acid modification, wherein the amino acid modifications alter one or more of the following: (i) binding affinity to one or more Fc receptors, (ii) effector functions, (iii) level of Fc sulfation domain, (iv) half-life, (v) protease resistance, (vi) Fc domain stability, and/or (vii) susceptibility to degradation (eg, compared to an unmodified Fc domain). In some cases, the Fc domain contains no more than 16 amino acid modifications (e.g., no more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 amino acid modifications) . In some cases, the Fc domain contains no more than 16 amino acid modifications (for example, no more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 amino acid modifications per CH3 domain).

The present invention also provides a pharmaceutical composition that contains a substantially homogeneous collection of any of the Fc constructs described herein. In one embodiment, a sterile syringe or vial suitable for pharmaceutical use contains a pharmaceutical composition in which the sole or primary active ingredient is a substantially homogeneous combination of any of the Fc constructs described herein. The pharmaceutical composition may contain one or more inactive ingredients, for example, selected from salts, detergents, surfactants, excipients, polymers, preservatives and other pharmaceutical excipients.

In some embodiments, the Fc construct is formed at least in part by association of Fc domain monomers that are present in different polypeptides. In certain embodiments, the Fc construct is formed by association of Fc domain monomers that are present in different polypeptides. In these embodiments, the Fc construct does not contain an additional domain that facilitates the association of two polypeptides (eg, IgM tail or IgA tail). In other embodiments, covalent bonds (eg, disulfide bridges) are present only between the two Fc domain monomers that join to form the Fc domain. In other embodiments, the Fc construct does not contain covalent bonds (eg, disulfide bridges) between the Fc domains. In other embodiments, the Fc construct provides sufficient structural flexibility such that all or substantially all of the Fc domains in the Fc construct are capable of simultaneously interacting with the Fc receptor on the cell surface. In certain examples of any of these embodiments, the Fc construct comprises at least two Fc domains connected by a linker (eg, a flexible amino acid spacer).

In another aspect, the present invention provides compositions and methods for promoting selective dimerization of Fc domain monomers. The present invention provides an Fc domain wherein two Fc domain monomers belonging to the Fc domain contain identical mutations at at least two positions in a ring of charged residues at the interface between antibody C _H 3 constant domains. The present invention also provides a method for creating such an Fc domain, comprising introducing complementary dimerization selectivity modules having identical mutations in two Fc domain monomer sequences at at least two positions in a ring of charged residues at the interface between antibody C _H 3 constant domains . The contact surface between the constant C _H 3 domains of an antibody consists of a hydrophobic segment surrounded by a ring of charged residues. When one C _H 3 constant domain of an antibody is found together with another, these charged residues pair with residues of opposite charge. By reversing the charge on both members of two or more complementary residue pairs, mutated Fc domain monomers remain complementary to Fc domain monomers with the same mutated sequence, but are less complementary to Fc domain monomers without these mutations. In this embodiment, identical dimerization selectivity modules promote homodimerization. Such Fc domains contain Fc domain monomers containing double mutants K409D/D399K, K392D/D399K, E357K/K370E, D356K/K439D, K409E/D399K, K392E/D399K, E357K/K370D, or D356K/K439. In another embodiment, the Fc domain contains Fc domain monomers, including quadruple mutants combining any pair of double mutants, for example K409D/D399K/E357K/K370E.

In another embodiment, in addition to identical dimerization selectivity modules, Fc domain monomers belonging to the Fc domain contain complementary dimerization selectivity modules having non-identical mutations that promote specific association (eg, engineered cavity and overhang). As a result, the two Fc domain monomers contain two selectivity modules that enable dimerization and remain complementary to each other, but have reduced complementarity with respect to other Fc domain monomers. In this embodiment, heterodimerization is achieved between the cavity-containing Fc domain and the knob-containing Fc domain monomer. In one example, complementary dimerization selectivity modules having non-identical mutations in a charged residue pair of both Fc domain monomers are combined with a protrusion on one Fc domain monomer and a cavity on the other Fc domain monomer.

In any of the Fc constructs described herein, it will be seen that the order of the Fc domain monomers is interchangeable. For example, in a polypeptide having a first Fc domain monomer connected to a second Fc domain monomer via a linker, the carboxy terminus of the first Fc domain monomer may be attached to the amino terminus of the linker, which in turn is attached at its carboxy terminus to the amino terminus of the second monomer of the Fc domain. Alternatively, the carboxy terminus of the second Fc domain monomer may be attached to the amino terminus of a linker, which in turn is attached at its carboxy terminus to the amino terminus of the first Fc domain monomer. Both of these configurations are covered by the present invention.

Definitions

As used herein, the term “Fc domain monomer” refers to a polypeptide chain that contains at least a hinge domain (or portion thereof) and second and third antibody constant domains ( _CH 2 and _CH 3) or functional fragments thereof (eg , fragments that are capable of (i) dimerizing with another Fc domain monomer to form an Fc domain and (ii) binding to the Fc receptor). The Fc domain monomer can be of various origins, for example, human, mouse or rat. The Fc domain monomer may be an immunoglobulin, which is an antibody of any isotype, including IgG, IgE, IgM, IgA, or IgD (eg, IgG). In addition, the Fc domain monomer may be an IgG subtype (eg, IgG1, IgG2a, IgG2b, IgG3, or IgG4) (eg, IgG1). The Fc domain monomer does not contain any part of the immunoglobulin that is capable of acting as an antigen recognition region, such as a variable domain or a complementarity determining region (CDR). Fc domain monomers may contain up to ten changes (e.g., single amino acid modifications) compared to the wild-type Fc domain monomer sequence (e.g., 1-10, 1-8, 1-6, 1-4 amino acid substitutions, additions, or deletions ), which alter the interaction between the Fc domain and the Fc receptor. Examples of suitable modifications are known in the art.

As used herein, the term “Fc domain” refers to a dimer of two Fc domain monomers that is capable of binding an Fc receptor. In the wild-type Fc domain, the two Fc domain monomers dimerize through interactions between the two antibody C _H 3 constant domains as well as one or more disulfide bonds that form between the hinge domains of the two dimerizing Fc domain monomers.

As used herein, the term “Fc construct” refers to linked polypeptide chains forming Fc domains as described herein (eg, an Fc construct having three Fc domains). The Fc constructs described herein may contain Fc domain monomers that have the same or different sequences. For example, an Fc construct may have three Fc domains, two of which contain Fc domain monomers derived from IgG1 or IgG1, and the third containing Fc domain monomers derived from IgG2 or IgG2. In another example, an Fc construct may have three Fc domains, two of which contain a projection-to-cavity pair and the third does not contain a projection-to-cavity pair. In the present invention, the Fc domain does not contain an antibody variable region, for example, _VH , _VL , CDR or HVR. The Fc domain forms a minimal structure that binds to the Fc receptor, for example, FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, FcγRIIIb, FcγRIV. In some embodiments, the Fc constructs described herein do not contain an antigen recognition region, such as a variable domain or a complementarity determining region (CDR). In some embodiments, the Fc constructs described herein contain an antigen recognition region, such as a variable domain or CDR.

As used herein, the term “antibody constant domain” refers to a polypeptide that corresponds to an antibody constant region domain (e.g., antibody C _L constant domain, antibody C _H 1 constant domain, antibody C _H 2 constant domain, or antibody C _H 3 constant domain) .

As used herein, the term “facilitate” means to enable and favor, for example, favor the formation of an Fc domain from two Fc domain monomers that have a higher binding affinity for each other than for other, distinct Fc domain monomers. As described herein, two Fc domain monomers that combine to form an Fc domain may have compatible amino acid modifications (eg, engineered knobs and engineered cavities) at the interface of their respective antibody C _H 3 constant domains. Compatible amino acid modifications promote or favor the selective interaction of such Fc domain monomers with each other relative to other Fc domain monomers that lack such amino acid modifications or to incompatible amino acid modifications. This occurs because, due to amino acid modifications at the interface of the two interacting constant domains of an antibody C _H 3 , Fc domain monomers have a higher affinity for each other than for other Fc domain monomers that lack amino acid modifications.

As used herein, the term “dimerization selectivity module” refers to an Fc domain monomer sequence that promotes preferential pairing between two Fc domain monomers. “Complementary” dimerization selectivity modules are dimerization selectivity modules that promote or favor the selective interaction of two Fc domain monomers with each other. The selectivity modules that provide complementary dimerization may have the same or different sequences. Exemplary selectivity modules enabling complementary dimerization are described herein.

As used herein, the term “engineered cavity” refers to the replacement of at least one of the original amino acid residues in the constant C _H 3 domain of an antibody with another amino acid residue having a smaller side chain volume than the original amino acid residue, thereby producing a three-dimensional cavity in the constant domain. domain C _H 3 antibodies. The term "original amino acid residue" refers to the naturally occurring amino acid residue encoded by the genetic code of the constant domain C _H 3 of a wild-type antibody.

As used herein, the term “engineered overhang” refers to the replacement of at least one of the original amino acid residues in the constant C _H 3 domain of an antibody with another amino acid residue having a larger side chain volume than the original amino acid residue, thereby producing a three-dimensional overhang in the constant region. domain C _H 3 antibodies. The term “original amino acid residues” refers to the naturally occurring amino acid residues encoded by the genetic code of the C _H 3 constant domain of a wild-type antibody.

As used herein, the term "protrusion-to-cavity pair" describes an Fc domain containing two Fc domain monomers, where the first Fc domain monomer contains an engineered cavity in its antibody C _H 3 constant domain, while the second Fc domain monomer -domain contains an engineered knob in its constant antibody C _H 3 domain. In a protrusion-to-cavity pair, the engineered protrusion in the antibody C _H 3 constant domain of the first Fc domain monomer is positioned such that it interacts with the antibody C _H 3 constant domain engineered cavity of the second Fc domain monomer without significantly disrupting the normal association of the dimer at the contact surface constant domain C _H 3 antibody.

As used herein, the term “heterodimeric Fc domain” refers to an Fc domain that is formed by heterodimerization of two Fc domain monomers, where the two Fc domain monomers contain different mutations that cause the charge to be reversed (see, for example, mutations in Table 2A), which contribute to the favorable formation of these two Fc domain monomers. As shown in FIG. 1 and 2, in an Fc construct containing three Fc domains—one carboxy-terminal “stem” Fc domain and two amino-terminal “branch” Fc domains—each of the amino-terminal “branch” Fc domains may be a heterodimeric Fc domain ( also called "heterodimeric branch Fc domain") (e.g., heterodimeric Fc domain formed by Fc domain monomers 106 and 114, or Fc domain monomers 112 and 116 in Figure 1; heterodimeric Fc domain formed by Fc domain monomers 206 and 214, or Fc domain monomers 212 and 216 in Fig. 2).

As used herein, the term “homodimeric Fc domain” refers to an Fc domain that is formed by the homodimerization of two Fc domain monomers, where the two Fc domain monomers contain the same mutations to reverse charge (see, for example, mutations in Tables 2B and 2C). As shown in FIG. 1 and 2, in an Fc construct containing three Fc domains—one carboxy-terminal stem Fc domain and two amino-terminal branch Fc domains—the carboxy-terminal stem Fc domain may be a homodimeric Fc domain (also called “stem homodimeric Fc domain”) (e.g., homodimeric Fc domain formed by Fc domain monomers 104 and 110 in Fig. 1; homodimeric Fc domain formed by Fc domain monomers 204 and 210 in Fig. 2).

As used herein, the term “heterodimerization selectivity module” refers to engineered protrusions, engineered cavities, and certain charge-reversing amino acid substitutions that can be made in the antibody C _H 3 constant domains of Fc domain monomers to promote beneficial heterodimerization of two Fc domain monomers that have compatible selectivity modules to enable heterodimerization. Fc domain monomers containing selectivity modules enabling heterodimerization can combine to form a heterodimeric Fc domain. Examples of selectivity modules that enable heterodimerization are shown in Tables 1 and 2A.

As used herein, the term “heterodimerization selectivity module” refers to mutations that cause a charge reversal in an Fc domain monomer at at least two positions in the ring of charged residues at the interface between the _CH 3 domains that promote homodimerization of the monomer Fc domain to form a homodimeric Fc domain. Examples of selectivity modules mediating homodimerization are shown in Tables 2A and 2B.

As used herein, the term “linked” is used to describe the combination or joining of two or more elements, components, or protein domains, such as polypeptides, through methods including chemical conjugation, recombinant means, and chemical bonds, such as disulfide bonds and amide bonds. For example, two separate polypeptides can be joined to form one contiguous protein structure through chemical conjugation, chemical bonding, a peptide linker, or any other means of providing covalent binding. In some embodiments, a first Fc domain monomer is linked to a second Fc domain monomer via a peptide linker, wherein the N-terminus of the peptide linker is linked to the C-terminus of the first Fc domain monomer via a chemical bond, such as a peptide bond, and the C-terminus of the peptide the linker is attached to the N-terminus of the second Fc domain monomer via a chemical bond, such as a peptide bond. In other embodiments, the N-terminus of the albumin-binding peptide is attached to the C-terminus of the antibody C _H 3 constant domain of the Fc domain monomer via a linker in the same manner as above.

As used herein, the term “linked” is used to describe interactions, such as hydrogen bonding, hydrophobic interactions, or ionic interactions, between polypeptides (or sequences within a single polypeptide) such that the polypeptides (or sequences within a single polypeptide) are arranged to form an Fc construct as described herein (eg, an Fc construct containing three Fc domains). For example, in some embodiments, four polypeptides, such as two polypeptides each containing two Fc domain monomers and two polypeptides each containing one Fc domain monomer, are linked to form an Fc construct that has three Fc domains (for example, as shown in Fig. 1 and 2). The four polypeptides can be linked through their respective Fc domain monomers. Bonding between polypeptides does not involve covalent interactions.

As used herein, the term “linker” refers to a connection between two elements, such as protein domains. The linker may be a covalent bond or a spacer. The term "bond" refers to a chemical bond, such as an amide bond or a disulfide bond, or any kind of bond created by a chemical reaction, such as chemical conjugation. The term "spacer" refers to a fragment (eg, polyethylene glycol (PEG) polymer) or amino acid sequence (eg, consisting of 3-200 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, 10-30 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 20-30 amino acids sequences) located between two polypeptides or polypeptide domains, to provide space and/or flexibility between two polypeptides or polypeptide domains. An amino acid spacer is part of the primary sequence of a polypeptide (eg, attached to spaced polypeptides or domains of a polypeptide via a polypeptide backbone). The formation of disulfide bonds, for example between two hinge regions or two Fc domain monomers that form an Fc domain, is not considered a linker.

As used herein, the term “glycine spacer” refers to a linker containing only glycine residues that connects two Fc domain monomers in tandem sequences. The glycine spacer may contain at least 4, 8, 12, 14, 16, 18, or 20 glycine residues (e.g., 4-30, 8-30, 12-30, 12-50, 12-100, or 12-200 glycine residues , 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 residues). In some embodiments, the glycine spacer contains, consists of, or consists essentially of the sequence GGGGGGGGGGGGGGGGGGGG (SEQ ID NO: 27).

As used herein, the term “albumin-binding peptide” refers to an amino acid sequence of 12-16 amino acids that has an affinity for albumin and functions to bind serum albumin. The albumin-binding peptide can have different origins, for example, it can come from humans, mice or rats. In some embodiments of the present invention, the albumin-binding peptide is fused to the C-terminus of the Fc domain monomer to increase the serum half-life of the Fc construct. The albumin-binding peptide can be fused, either directly or via a linker, to the N- or C-terminus of the Fc domain monomer.

As used herein, the term “purification peptide” refers to a peptide of any length that can be used to purify, isolate, or identify a polypeptide. A purification peptide can be attached to a polypeptide to facilitate purification of the polypeptide and/or isolation of the polypeptide, for example, from a mixture of cell lysates. In some embodiments, the purification peptide is coupled to another moiety that has a specific affinity for the purification peptide. In some embodiments, such moieties that specifically bind to the purification peptide are attached to a solid support such as a matrix, resin, or agarose beads. Examples of purification peptides that can be attached to the Fc construct are described in more detail herein.

As used herein, the term “multimer” refers to a molecule containing at least two linked Fc constructs described herein.

As used herein, the term “antigen recognition region” refers to the portions of the light and heavy chains of an antibody that are responsible for recognizing and binding the antibody to an antigen. The antigen recognition region contains light and heavy chain variable domains (Fabs), which contain complementarity determining regions (CDRs, e.g., CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, and CDR H3), and framework regions (FRs). .

As used herein, the expression “activation of immune cells mediating an immune response” refers to an immune response that is induced or activated by binding of an immune complex or Fc construct to an Fcγ receptor (FcγR) (e.g., an activating FcγR, e.g., FcγRI, FcγRIIa, FcγRIIc , FcγRIIIa or FcγRIIIb) on a cell (eg, an immune cell (eg, a monocyte)). An immune complex is an antigen-antibody complex formed by the binding of an antibody to an antigen. The immune complex often has multiple Fc domains that aggregate FcγRs and inhibit or activate cellular processes that play a critical role in inflammation, infection, and other diseases. In some embodiments, the Fc constructs of the present invention are capable of binding to FcγR and inducing activation of FcγR (eg, FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa, or FcγRIIIb) signaling to immune cells (eg, monocyte). Measurement of certain downstream signaling events, such as kinase phosphorylation (eg, Syk phosphorylation) and calcium influx into an FcγR-expressing cell, can be used to detect activation of immune cells mediating the immune response caused by binding of an immune complex or Fc construct. For example, activation of immune cells to mediate an immune response is induced if the level of kinase phosphorylation (e.g., Syk phosphorylation) or the level of calcium influx into the cell is at least 5-fold, e.g., 5-100-fold (e.g., 5-100. 10-95, 15-90, 20-85, 25-80, 30-75, 35-70, 40-65, 45-60 or 50-55 times, for example 5, 6, 7, 8, 9, 10 , 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 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 100 times higher than the level of kinase phosphorylation (eg, Syk phosphorylation) or calcium influx from the cell without any activation by the immune complex or Fc construct.

As used herein, the term “phagocytosis” refers to a form of endocytosis in which a cell, often a phagocyte (eg, a monocyte), engulfs another cell, particle, or pathogen (eg, a microbe or parasite) to form a phagosome. In the immune system, phagocytosis is the primary mechanism used to remove diseased cells (eg, cancer cells, infected cells, or dead cells), pathogens, and cellular debris. A cell that is targeted for phagocytosis by another cell (e.g., a phagocyte (e.g., a monocyte)) is called a target cell. For example, an immune cell (eg, a monocyte) activated by binding an Fc construct of the present invention to FcγRs (eg, FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa, or FcγRIIIb) on the immune cell can phagocytose a target cell, which may be a cancer cell or an infected cell in the subject.

As used herein, the term “increase” or “increase” phagocytosis of a target cell refers to the increase in phagocytosis caused by binding of the Fc construct of the present invention to FcγRs (e.g., FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa, or FcγRIIIb) on an immune cell (e.g., monocyte) relative to the level of phagocytosis that occurs without induction by the Fc construct. For example, phagocytosis of a target cell is increased if the level of phagocytosis is at least 10%, e.g., 10-100% (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 100% above phagocytosis level , which occurs without induction by the Fc construct.

As used herein, the term “cancer treatment” refers to the therapeutic treatment of cancer in a subject. The therapeutic treatment slows the progression of the cancer, improves the subject's outcome, and/or eliminates the cancer.

As used herein, the term “treating an infection” refers to therapeutically treating an infection in a subject. Therapeutic treatment slows the progression of the infection, improves the subject's final condition, and/or eliminates the infection.

As used herein, the term “infection” refers to the entry into cells, tissues and/or organs of a subject by a pathogen such as bacteria, viruses, fungi, helminths, protozoa, arthropods and other microbes, parasites and worms. In some embodiments, the pathogen can grow, multiply, and/or produce toxins in cells, tissues, and/or organs of the subject. In some embodiments, the subject may develop an adverse reaction (ie, an allergic reaction or immune response) to the pathogen. Examples of infections include, but are not limited to, bacterial infection, viral infection, fungal infection, helminth infection, and protozoal infection.

As used herein, the term “bacterial infection” refers to an infection caused by one or more species of bacteria. Examples of bacteria that cause infection are well known in the art and include, but are not limited to, bacteria of the genus Streptococcus (for example, Streptococcus pyogenes ), bacteria of the genus Escherichia (for example, Escherichia coli), bacteria of the genus Vibrio (for example, Vibrio cholerae ), bacteria of the genus Enteritis ( for example, Enteritis salmonella ), as well as bacteria of the genus Salmonella (for example, Salmonella typhi ).

As used herein, the term “viral infection” refers to an infection caused by one or more types of viruses. Examples of infectious viruses are well known in the art and include, but are not limited to, viruses of the family Retroviridae (e.g., human immunodeficiency virus (HIV)), viruses of the family Adenoviridae (e.g., adenovirus), viruses of the family Herpesviridae (e.g., herpes simplex viruses types 1 and 2 ), viruses of the family Papillomaviridae (for example, human papillomavirus (HPV)), viruses of the family Poxviridae (for example, smallpox), viruses of the family Picornaviridae (for example, hepatitis A virus, poliovirus, rhinovirus), viruses of the family Hepadnaviridae (for example, hepatitis B virus) , viruses of the family Flaviviridae (for example, hepatitis C virus, yellow fever virus, West Nile virus), viruses of the family Togaviridae (for example, rubella virus), viruses of the family Orthomyxoviridae (for example, influenza virus), viruses of the family Filoviridae (for example, Ebola virus, Marburg) and viruses of the Paramyxoviridae family (for example, measles virus, mumps virus).

As used herein, the term “fungal infection” refers to an infection caused by one or more species of fungi. Examples of fungi that cause infection are well known in the art and include, but are not limited to, fungi of the genus Aspergillus (e.g., Aspergillus fumigatus , A. flavus , A. terreus , A. niger , A. candidus , A. clavatus , A. ochraceus ), fungi the genus Candida (e.g. Candida albicans , C. parapsilosis , C. glabrata , C. guilliermondii , C. krusei , C. lusitaniae , C. tropicalis ), fungi of the genus Cryptococcus (e.g. Cryptococcus neoformans ), and fungi of the genus Fusarium (e.g. , Fusarium solani , F. verticillioides , F. oxysporum ).

As used herein, the term “helminthic infection” refers to an infection caused by one or more species of helminths. Examples of helminths include, but are not limited to, tapeworms (cestodes), roundworms (nematodes), flukes (flukes), and monogenetic flukes.

As used herein, the term “protozoal infection” refers to an infection caused by one or more species of protozoa. Examples of protozoa include, but are not limited to, protozoans of the genus Entamoeba (e.g., Entamoeba histolytica ), protozoans of the genus Plasmodium (e.g., Plasmodium falciparum, P. malariae ), protozoans of the genus Giardia (e.g., Giardia lamblia ), and protozoans of the genus Trypanosoma (e.g., Trypanosoma brucei ).

As used herein, the term "polynucleotide" refers to an oligonucleotide or nucleotide and fragments or parts thereof, as well as DNA or RNA of genomic or synthetic origin, which may be single-stranded or double-stranded and is either a sense or an antisense strand. One polynucleotide is translated into one polypeptide.

As used herein, the term “polypeptide” describes a single polymer in which the monomers are amino acid residues that are linked together through amide bonds. It is contemplated that any amino acid sequence, whether naturally occurring, recombinant or synthetically produced, may be included in the polypeptide.

As used herein, the term “amino acid positions” refers to the numbers of amino acid positions in a protein or protein domain. Amino acid positions for antibodies or Fc constructs are numbered using the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, MD, ed. 5, 1991).

As used herein, the terms “amino acid mutation,” “amino acid substitution,” and “amino acid modification” are used interchangeably to refer to a change in an Fc domain polypeptide compared to a reference Fc domain polypeptide (e.g., a wild-type, unmutated or unmodified Fc sequence). The reference Fc domain polypeptide may be a wild-type human IgG1 Fc domain polypeptide. Amino acid modification includes amino acid substitutions, deletions and/or insertions. In some embodiments, the amino acid modification is a modification of a single amino acid. In another embodiment, the amino acid modification is a modification of multiple (eg, more than one) amino acids. Amino acid modification may involve a combination of amino acid substitutions, deletions and/or insertions. The description of amino acid modifications includes genetic (i.e., DNA and RNA) changes, such as point mutations (e.g., substitution of one nucleotide for another), insertions and deletions (e.g., addition and/or deletion of one or more nucleotides) of the nucleotide sequence, which encodes an Fc polypeptide. An insertion, unless otherwise noted, is the addition of one or more amino acids immediately after the amino acid position at which the insertion is indicated . Amino acid modification, such as substitution, insertion and/or deletion, unless otherwise indicated, is included in both Fc domain monomers that make up the Fc domain. In other embodiments, the Fc domain comprising the first and second Fc domain monomers may contain, for example, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) amino acid modifications in the first Fc domain monomer that differ from one or more (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) amino acid modifications in the second Fc domain monomer. In some embodiments, the Fc domain may comprise Fc domain monomers, eg, first and second Fc domain monomers, with homogeneous amino acid modifications, eg, at amino acid position I253 and/or R292. In other embodiments, the Fc domain may comprise Fc domain monomers, eg, first and second Fc domain monomers, with heterogeneous amino acid modifications, eg, at amino acid position I253 and/or R292. In certain embodiments, at least one (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) Fc domain in the Fc construct contains an amino acid modification. In some cases, at least one Fc domain contains one or more (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) single amino acid modifications. In some cases, the Fc domain contains no more than sixteen single amino acid modifications (for example, no more than one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen or sixteen amino acids modifications). In some cases, the Fc domain monomer contains no more than ten single amino acid modifications. In some cases, the Fc domain monomer contains no more than 12 single amino acid modifications. In some cases, the Fc domain monomer contains no more than 14 single amino acid modifications.

In certain embodiments, at least one (eg, one, two, or three) Fc domain in the Fc construct contains an amino acid modification. In some cases, at least one Fc domain contains one or more (eg, two, three, four, five, six, seven, eight, nine, ten, or twenty or more) amino acid modifications. In some cases, at least one Fc domain contains no more than ten single amino acid modifications.

As used herein, the term “percentage (%) identity” refers to the percentage of amino acid residues (or nucleic acid residues) of a candidate sequence, e.g., an Fc domain monomer sequence in an Fc construct described herein, that are identical to amino acid residues (or residues) nucleic acids) reference sequence, e.g., wild-type Fc domain monomer sequence, after sequence alignment and introduction of gaps if necessary to achieve maximum percent identity (i.e., gaps may be introduced into one or both of the candidate and reference sequences for optimal alignments and non-homologous sequences may not be taken into account for comparison purposes). Alignment for the purpose of determining percent identity can be achieved in a variety of ways known to one skilled in the art, for example using publicly available computer software such as BLAST, ALIGN or Megalign (DNASTAR). Those skilled in the art can determine appropriate parameters for evaluating the alignment, including any algorithms necessary to achieve maximum alignment over the full length of the sequences being compared. In some embodiments, the percentage of amino acid (or nucleic acid) sequence identity of a given candidate sequence is relative to or against a given reference sequence (which may alternatively be stated as a given candidate sequence that has or contains a certain percentage of amino acid (or nucleic acid) sequence identity to or against a given reference sequence) is calculated as follows:

100 x (fraction A/B)

where A is the number of amino acid residues (or nucleic acid residues) assessed as identical in the alignment of the candidate sequence and the reference sequence, and where B is the total number of amino acid residues (or nucleic acid residues) in the reference sequence. In some embodiments, where the length of the candidate sequence is not equal to the length of the reference sequence, the percentage amino acid (or nucleic acid sequence) identity of the candidate sequence relative to the reference sequence will not be equal to the percentage amino acid (or nucleic acid sequence) identity of the reference sequence of the candidate sequence.

In specific embodiments, a reference sequence aligned for comparison with a candidate sequence may show that the candidate sequence exhibits 50% to 100% identity (e.g., 50% to 100%, 60% to 100%, 70% to 100 %, from 80% to 100%, from 90% to 100%, from 92% to 100%, from 95% to 100%, from 97% to 100%, from 99% to 100% or from 99.5% to 100%) over the entire length of the candidate sequence or a selected portion of contiguous amino acid residues (or nucleic acid residues) of the candidate sequence. The length of the candidate sequence aligned for comparison purposes is at least 30%, eg at least 40%, eg at least 50%, 60%, 70%, 80%, 90% or 100% of the length of the reference sequence. When a position in a candidate sequence is occupied by the same amino acid (or nucleic acid) residue as the corresponding position in the reference sequence, then the molecules at that position are identical.

In some embodiments, an Fc domain monomer in an Fc construct described herein (e.g., an Fc construct containing three Fc domains) may contain, consist of, or consist essentially of a sequence that is at least 95 % identical (eg, at least 97%, 99%, or 99.5 identical) to the wild-type Fc domain monomer sequence (eg, SEQ ID NO: 42). In some embodiments, an Fc domain monomer in an Fc construct described herein (e.g., an Fc construct containing three Fc domains) may contain, consist of, or consist essentially of a sequence that is at least 95 % identical (eg, at least 97%, 99%, or 99.5% identical) to the sequence of any of SEQ ID NOs: 44, 46, 48, and 50-53. In certain embodiments, an Fc domain monomer in an Fc construct may contain, consist of, or consist essentially of a sequence that is at least 95% identical (e.g., at least 97%, 99%, or 99.5 identical) %) sequences under SEQ ID NO: 48, 52 and 53.

In some cases, an Fc domain monomer in an Fc construct described herein may contain, consist of, or consist essentially of a sequence that is at least 95% identical (e.g., at least 97% identical, 99 % or 99.5%) any of the sequences described herein. In some cases, an Fc domain monomer in an Fc construct described herein may contain, consist of, or consist essentially of a sequence that is any of the sequences described herein with no more than 10 (e.g., no more than 9, 8, 7, 6, 5, 4, 3, 2 or 1) single amino acid modifications (eg substitutions, eg conservative substitutions).

In some embodiments, a polypeptide characterized by two Fc domain monomers in an Fc construct described herein (e.g., polypeptides 102 and 108 in FIG. 1, polypeptides 202 and 208 in FIG. 2) may comprise, consist of, or consist of substantially from a sequence that is at least 95% identical (e.g., at least 97%, 99%, or 99.5% identical) to the sequence of any of SEQ ID NOs: 43, 45, 47, and 49. In some In embodiments, a polypeptide containing two Fc domain monomers in an Fc construct described herein may contain, consist of, or consist essentially of a sequence that is at least 95% identical (e.g., at least 97% identical %, 99% or 99.5%) of the sequence under SEQ ID NO: 49.

In some embodiments, a spacer between two Fc domain monomers may contain, consist of, or consist essentially of a sequence that is at least 75% identical (e.g., 75%, 77%, 79%, 81%, 83%, 85% (87%, 89%, 91%, 93%, 95%, 97%, 99%, 99.5% or 100% identical) of the sequence under any of SEQ ID NO: 1-36 (for example, SEQ ID NO : 17, 18, 26 and 27), further described herein.

As used herein, the term “host cell” refers to a carrier that contains the necessary cellular components, such as organelles, necessary for the expression of proteins from their respective nucleic acids. Nucleic acids are typically included in nucleic acid vectors that can be introduced into a host cell through conventional techniques known in the art (transformation, transfection, electroporation, calcium phosphate precipitation, direct microinjection, etc.). The host cell may be a prokaryotic cell, such as a bacterial cell, or a eukaryotic cell, such as a mammalian cell (eg, a CHO cell). As described herein, a host cell is used to express one or more polypeptides encoding the required domains, which can then be combined to form the desired Fc construct.

As used herein, the term “pharmaceutical composition” refers to a medicinal or pharmaceutical composition that contains an active ingredient as well as one or more excipients and diluents to enable the active ingredient to be acceptable for the route of administration. The pharmaceutical composition of the present invention contains pharmaceutically acceptable components that are compatible with the Fc construct. The pharmaceutical composition is usually in aqueous form for intravenous or subcutaneous administration.

As used herein, the term “substantially homogeneous population” of polypeptides or Fc constructs is a group in which at least 50% of the polypeptides or Fc constructs in the composition (eg, cell culture medium or pharmaceutical composition) have the same amount of Fc- domains, as determined by SDS gel electrophoresis under non-reducing conditions or size exclusion chromatography. A substantially homogeneous collection of polypeptides or Fc constructs may be obtained prior to purification, or after purification of Protein A or Protein G, or only after any Fab or Fc specific affinity chromatography. In various embodiments, at least 55%, 60%, 65%, 70%, 75%, 80%, or 85% of the polypeptides or Fc constructs in the composition have the same number of Fc domains. In other embodiments, up to 85%, 90%, 92%, or 95% of the polypeptides or Fc constructs in the composition have the same number of Fc domains. A substantially homogeneous body or composition is at least 85% homogeneous (eg, at least 85%, 90%, or 95% homogeneous).

As used herein, the term “pharmaceutically acceptable carrier” refers to an excipient or diluent in a pharmaceutical composition. The pharmaceutically acceptable carrier must be compatible with the other ingredients of the formulation and not cause harm to the recipient. In the present invention, a pharmaceutically acceptable carrier must provide sufficient pharmaceutical stability of the Fc construct. The nature of the carrier depends on the route of administration. For example, for oral administration, a solid carrier is preferred; for intravenous administration, an aqueous carrier solution (eg, WFI and/or buffered solution) is usually used.

As used herein, the term “therapeutically effective amount” refers to an amount, for example a pharmaceutical dose, effective to induce a desired biological effect in a subject or patient or in treating a patient having a condition or disorder described herein. It should also be understood that a “therapeutically effective amount” can be interpreted as an amount that provides the desired therapeutic effect, either in a single dose or in any dosage or route of administration, taken alone or in combination with other therapeutic agents.

DESCRIPTION OF GRAPHIC MATERIALS

In fig. 1 shows Fc constructs (Fc construct 1, Fc construct 2, or Fc construct 3) containing three Fc domains formed from four polypeptides. The first polypeptide (102) contains one Fc domain monomer (104) containing charged amino acids at the C _H 3 - C _H 3 contact surface, different from those of the wild-type Fc domain monomer sequence, linked in tandem sequence with the Fc domain monomer , containing a protrusion (106). The second polypeptide (108) contains an Fc domain monomer (110) containing charged amino acids at the C _H 3-C _H 3 interface, different from those of the wild-type Fc domain monomer sequence, linked in tandem sequence with another Fc domain monomer , containing a protrusion (112). Each of the third and fourth polypeptides (114 and 116, respectively) contains an Fc domain monomer containing a cavity.

In fig. 2 shows an Fc construct (Fc construct 4), consisting of three Fc domains formed from four polypeptides. The first polypeptide (202) contains one Fc domain monomer (204), containing differently charged amino acids at the C _H 3 - C _H 3 interface, different from those of the wild-type Fc domain monomer sequence, linked in tandem sequence with another Fc monomer -domain (206), containing differently charged amino acids and a protrusion. The second polypeptide (208) contains an Fc domain monomer (210) containing various charged amino acids at the C _H 3 - C _H 3 contact surface, different from those of the wild type Fc domain monomer sequence, linked in tandem sequence with another Fc - monomer domain (212), containing differently charged amino acids and a protrusion. Each of the third and fourth polypeptides (214 and 216, respectively) contains an Fc domain monomer containing differently charged amino acids and a cavity.

In 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.

In fig. 4 shows the level of linker O-xylosylation in an Fc construct containing two Fc domains (the Fc construct shown in Figure 13) as determined by LC-MS/MS.

In fig. 5 shows the formation of monomeric Fc molecules from Fc constructs 2 and 4 when stored at 45°C, as determined by CE-SDS.

In fig. 6 shows the proteolysis products of Fc construct 2 after two weeks of storage at 45°C, as determined by LC-MS.

In fig. 7 shows the proteolysis products of Fc construct 4 after two weeks of storage at 45°C, as determined by LC-MS.

In fig. 8 shows inhibition of IL-8 release by THP-1 cells by Fc construct 2 with different linker lengths.

In fig. Figure 9 shows inhibition of calcium flux in neutrophils by Fc construct 2 with different linker lengths.

In fig. 10 shows the size distribution by SDS-PAGE under non-reducing conditions of Fc construct 2 and Fc construct 4 in crude medium.

In fig. 11 shows the expression and assembly of Fc construct 2 (“electrostatically coordinated”) and another Fc construct containing three Fc domains but without electrostatically coordinated mutations in the stem subunits (“no electrostatically coordinated”).

In fig. 12 shows that removal of the C-terminal lysine to produce Fc construct 2 does not induce complement dependent cytotoxicity (CDC) in vitro.

In fig. 13 shows an Fc construct consisting of two Fc domains formed from six polypeptides.

In fig. 14 shows an Fc construct consisting of five Fc domains formed from six polypeptides (Fc5X).

In fig. 15 shows an Fc construct consisting of five Fc domains formed from six polypeptides (Fc5Y).

In fig. 16 shows an Fc construct consisting of five Fc domains formed from six polypeptides (reverse Fc5Y-).

In fig. 17 shows an Fc construct consisting of three Fc domains formed from two polypeptides.

In fig. 18A depicts an Fc construct (Construct 5) consisting of three Fc domains formed from four polypeptides. Each of the first polypeptide (502) and the second polypeptide (508) contains one Fc domain monomer containing charged amino acids at the C _H 3 -C _H 3 interface, unlike the wild type sequence (506 and 512, respectively), attached via a linker to the Fc domain monomer containing the knob (504 and 510, respectively). Each of the third and fourth polypeptides (514 and 516, respectively) contains an Fc domain monomer containing a cavity. Each of the first polypeptide (502) and the second polypeptide (508) also contains mutations associated with electrostatic coordination, for example, E357K. Likewise, each of the third and fourth polypeptides (514 and 516, respectively) contains mutations associated with electrostatic coordination, for example, K370D. Each of 506 and 512 contains the amino acid modification I253A, which is indicated by an asterisk. Each of 502 and 508 contains the amino acid sequence of SEQ ID NO: 62. Each of 514 and 516 contains the amino acid sequence of SEQ ID NO: 61.

In fig. 18B depicts an Fc construct (Construct 6) consisting of three Fc domains formed from four polypeptides. Each of the first polypeptide (602) and the second polypeptide (608) contains one Fc domain monomer containing charged amino acids at the C _H 3 -C _H 3 interface, unlike the wild type sequence (606 and 612, respectively), attached via a linker to the Fc domain monomer containing the knob (604 and 610, respectively). Each of the third and fourth polypeptides (614 and 616, respectively) contains an Fc domain monomer containing a cavity. Each of the first polypeptide (602) and the second polypeptide (608) also contains mutations associated with electrostatic coordination, for example, E357K. Likewise, each of the third and fourth polypeptides (614 and 616, respectively) contains mutations associated with electrostatic coordination, for example, K370D. Each of 604, 610, 614 and 616 contains the amino acid modification I253A, which is indicated by an asterisk. Each of 602 and 608 contains the amino acid sequence of SEQ ID NO: 64. Each of 614 and 616 contains the amino acid sequence of SEQ ID NO: 63.

In fig. 18C shows an Fc construct (construct 7) consisting of three Fc domains formed from four polypeptides. Each of the first polypeptide (702) and the second polypeptide (708) contains one Fc domain monomer containing charged amino acids at the C _H 3 -C _H 3 interface, unlike the wild type sequence (706 and 712, respectively), attached via a linker to the Fc domain monomer containing the knob (704 and 710, respectively). Each of the third and fourth polypeptides (714 and 716, respectively) contains an Fc domain monomer containing a cavity. Each of the first polypeptide (702) and the second polypeptide (708) also contains mutations associated with electrostatic coordination, for example, E357K. Likewise, each of the third and fourth polypeptides (714 and 716, respectively) contains mutations associated with electrostatic coordination, for example, K370D. Each of 704, 706, 710, 712, 714 and 716 contains the amino acid modification I253A, which is indicated by an asterisk. Each of 702 and 708 contains the amino acid sequence of SEQ ID NO: 65. Each of 714 and 716 contains the amino acid sequence of SEQ ID NO: 63.

In fig. 18D depicts an Fc construct (construct 8) consisting of three Fc domains formed from four polypeptides. Each of the first polypeptide (802) and the second polypeptide (808) contains one Fc domain monomer containing charged amino acids at the C _H 3 -C _H 3 interface, unlike the wild type sequence (806 and 812, respectively), attached via a linker to the Fc domain monomer containing the knob (804 and 810, respectively). Each of the third and fourth polypeptides (814 and 816, respectively) contains an Fc domain monomer containing a cavity. Each of the first polypeptide (802) and the second polypeptide (808) also contains mutations associated with electrostatic coordination, for example, E357K. Likewise, each of the third and fourth polypeptides (814 and 816, respectively) contains mutations associated with electrostatic coordination, for example, K370D. Each of 806 and 812 contains the amino acid modification R292P, which is indicated by a diamond. Each of 802 and 808 contains the amino acid sequence of SEQ ID NO: 66. Each of 814 and 816 contains the amino acid sequence of SEQ ID NO: 61.

In fig. 18E shows an Fc construct (construct 9) consisting of three Fc domains formed from four polypeptides. Each of the first polypeptide (902) and the second polypeptide (908) contains one Fc domain monomer containing charged amino acids at the C _H 3-C _H 3 interface, unlike the wild type sequence (906 and 912, respectively), attached via a linker to the Fc domain monomer containing the knob (904 and 910, respectively). Each of the third and fourth polypeptides (914 and 916, respectively) contains an Fc domain monomer containing a cavity. Each of the first polypeptide (902) and the second polypeptide (908) also contains mutations associated with electrostatic coordination, for example, E357K. Likewise, each of the third and fourth polypeptides (914 and 916, respectively) contains mutations associated with electrostatic coordination, for example, K370D. Each of 906 and 912 contains the amino acid modifications I253A, which is indicated by an asterisk, and R292P, indicated by a diamond. Each of 902 and 908 contains the amino acid sequence of SEQ ID NO: 67. Each of 914 and 916 contains the amino acid sequence of SEQ ID NO: 61.

In fig. 18F shows an Fc construct (construct 10) consisting of three Fc domains formed from four polypeptides. Each of the first polypeptide (1002) and the second polypeptide (1008) contains one Fc domain monomer containing charged amino acids at the C _H 3-C _H 3 interface, unlike the wild type sequence (1006 and 1012, respectively), attached via a linker to the Fc domain monomer containing the knob (1004 and 1010, respectively). Each of the third and fourth polypeptides (1014 and 1016, respectively) contains an Fc domain monomer containing a cavity. Each of the first polypeptide (1002) and the second polypeptide (1008) also contains mutations associated with electrostatic coordination, for example, E357K. Likewise, each of the third and fourth polypeptides (1014 and 1016, respectively) contains mutations associated with electrostatic coordination, for example, K370D. Each of 1006 and 1012 contains the amino acid modification R292P, which is represented by a diamond, and each of 1004, 1010, 1014 and 1016 contains the amino acid modification I253A, which is represented by an asterisk. Each of 1002 and 1008 contains the amino acid sequence of SEQ ID NO: 68. Each of 1014 and 1016 contains the amino acid sequence of SEQ ID NO: 63.

In fig. 18G depicts an Fc construct (construct 11) consisting of three Fc domains formed from four polypeptides. Each of the first polypeptide (1102) and the second polypeptide (1108) contains one Fc domain monomer containing charged amino acids at the C _H 3 -C _H 3 interface, unlike the wild type sequence (1106 and 1112, respectively), attached via a linker to the Fc domain monomer containing the knob (1104 and 1110, respectively). Each of the third and fourth polypeptides (1114 and 1116, respectively) contains an Fc domain monomer containing a cavity. Each of the first polypeptide (1102) and the second polypeptide (1108) also contains mutations associated with electrostatic coordination, for example, E357K. Likewise, each of the third and fourth polypeptides (1114 and 1116, respectively) contains mutations associated with electrostatic coordination, for example, K370D. Each of 1106 and 1112 contains the amino acid modification R292P, which is represented by a diamond, and each of 1104, 1110, 1114 and 1116 contains the amino acid modification I253A, which is represented by an asterisk. Each of 1102 and 1108 contains the amino acid sequence of SEQ ID NO: 69. Each of 1114 and 1116 contains the amino acid sequence of SEQ ID NO: 63.

In fig. 18H shows an Fc construct (construct 12) consisting of three Fc domains formed from four polypeptides. Each of the first polypeptide (1202) and the second polypeptide (1208) contains one Fc domain monomer containing charged amino acids at the C _H 3-C _H 3 interface, unlike the wild type sequence (1206 and 1212, respectively), attached via a linker to the Fc domain monomer containing the knob (1204 and 1210, respectively). Each of the third and fourth polypeptides (1214 and 1216, respectively) contains an Fc domain monomer containing a cavity. Each of the first polypeptide (1202) and the second polypeptide (1208) also contains mutations associated with electrostatic coordination, for example, E357K. Likewise, each of the third and fourth polypeptides (1214 and 1216, respectively) contains mutations associated with electrostatic coordination, for example, K370D. Each of 1204, 1210, 1214 and 1216 contains the amino acid modification R292P, which is indicated by a diamond. Each of 1202 and 1208 contains the amino acid sequence of SEQ ID NO: 71. Each of 1214 and 1216 contains the amino acid sequence of SEQ ID NO: 70.

In fig. 18I shows an Fc construct (construct 13) consisting of three Fc domains formed from four polypeptides. Each of the first polypeptide (1302) and the second polypeptide (1308) contains one Fc domain monomer containing charged amino acids at the C _H 3 -C _H 3 interface, unlike the wild type sequence (1306 and 1312, respectively), attached via a linker to the Fc domain monomer containing the knob (1304 and 1310, respectively). Each of the third and fourth polypeptides (1314 and 1316, respectively) contains an Fc domain monomer containing a cavity. Each of the first polypeptide (1302) and the second polypeptide (1308) also contains mutations associated with electrostatic coordination, for example, E357K. Similarly, each of the third and fourth polypeptides (1314 and 1316, respectively) contains mutations associated with electrostatic coordination, for example, K370D. Each of 1304, 1310, 1314 and 1316 contains the amino acid modification R292P, which is represented by a diamond, and each of 1306 and 1132 contains the amino acid modification I253A, which is represented by an asterisk. Each of 1302 and 1308 contains the amino acid sequence of SEQ ID NO: 72. Each of 1314 and 1316 contains the amino acid sequence of SEQ ID NO: 70.

In fig. 18J depicts an Fc construct (construct 14) consisting of three Fc domains formed from four polypeptides. Each of the first polypeptide (1402) and the second polypeptide (1408) contains one Fc domain monomer containing charged amino acids at the C _H 3 -C _H 3 interface, unlike the wild type sequence (1406 and 1412, respectively), attached via a linker to the Fc domain monomer containing the knob (1404 and 1410, respectively). Each of the third and fourth polypeptides (1414 and 1416, respectively) contains an Fc domain monomer containing a cavity. Each of the first polypeptide (1402) and the second polypeptide (1408) also contains mutations associated with electrostatic coordination, for example, E357K. Likewise, each of the third and fourth polypeptides (1414 and 1416, respectively) contains mutations associated with electrostatic coordination, for example, K370D. Each of 1404, 1410, 1414 and 1416 contains the amino acid modifications R292P, which is indicated by a diamond, and I253A, indicated by an asterisk. Each of 1402 and 1408 contains the amino acid sequence of SEQ ID NO: 74. Each of 1414 and 1416 contains the amino acid sequence of SEQ ID NO: 73.

In fig. 18K shows an Fc construct (construct 15) consisting of three Fc domains formed from four polypeptides. Each of the first polypeptide (1502) and the second polypeptide (1508) contains one Fc domain monomer containing charged amino acids at the C _H 3-C _H 3 interface, unlike the wild type sequence (1506 and 1512, respectively), attached via a linker to the Fc domain monomer containing the knob (1504 and 1510, respectively). Each of the third and fourth polypeptides (1514 and 1516, respectively) contains an Fc domain monomer containing a cavity. Each of the first polypeptide (1502) and the second polypeptide (1508) also contains mutations associated with electrostatic coordination, for example, E357K. Likewise, each of the third and fourth polypeptides (1514 and 1516, respectively) contains mutations associated with electrostatic coordination, for example, K370D. Each of 1504, 1510, 1514 and 1516 contains the amino acid modification R292P, which is represented by a diamond, and each of 1506 and 1512 contains the amino acid modification I253A, which is represented by an asterisk. Each of 1502 and 1508 contains the amino acid sequence of SEQ ID NO: 75. Each of 1514 and 1516 contains the amino acid sequence of SEQ ID NO: 73.

In fig. 18L depicts an Fc construct (construct 16) consisting of three Fc domains formed from four polypeptides. Each of the first polypeptide (1602) and the second polypeptide (1608) contains one Fc domain monomer containing charged amino acids at the C _H 3 -C _H 3 interface, unlike the wild type sequence (1606 and 1612, respectively), attached via a linker to the Fc domain monomer containing the knob (1604 and 1610, respectively). Each of the third and fourth polypeptides (1614 and 1616, respectively) contains an Fc domain monomer containing a cavity. Each of the first polypeptide (1602) and the second polypeptide (1608) also contains mutations associated with electrostatic coordination, for example, E357K. Likewise, each of the third and fourth polypeptides (1614 and 1616, respectively) contains mutations associated with electrostatic coordination, for example, K370D. Each of 1604, 1606, 1610, 1612, 1614 and 1616 contains the amino acid modification R292P, which is indicated by a diamond. Each of 1602 and 1608 contains the amino acid sequence of SEQ ID NO: 76. Each of 1614 and 1616 contains the amino acid sequence of SEQ ID NO: 70.

In fig. 18M depicts an Fc construct (construct 17) consisting of three Fc domains formed from four polypeptides. Each of the first polypeptide (1702) and the second polypeptide (1708) contains one Fc domain monomer containing charged amino acids at the C _H 3 -C _H 3 interface, unlike the wild type sequence (1706 and 1712, respectively), attached via a linker to the Fc domain monomer containing the knob (1704 and 1710, respectively). Each of the third and fourth polypeptides (1714 and 1716, respectively) contains an Fc domain monomer containing a cavity. Each of the first polypeptide (1702) and the second polypeptide (1708) also contains mutations associated with electrostatic coordination, for example, E357K. Likewise, each of the third and fourth polypeptides (1714 and 1716, respectively) contains mutations associated with electrostatic coordination, for example, K370D. Each of 1704, 1706, 1710, 1712, 1714 and 1716 contains the amino acid modification R292P, which is represented by a diamond, and each of 1706 and 1712 contains the amino acid modification I253A, which is represented by an asterisk. Each of 1702 and 1708 contains the amino acid sequence of SEQ ID NO: 77. Each of 1714 and 1716 contains the amino acid sequence of SEQ ID NO: 70.

In fig. 18N depicts an Fc construct (construct 18) consisting of three Fc domains formed from four polypeptides. Each of the first polypeptide (1802) and the second polypeptide (1808) contains one Fc domain monomer containing charged amino acids at the C _H 3-C _H 3 interface, unlike the wild type sequence (1806 and 1812, respectively), attached via a linker to the Fc domain monomer containing the knob (1804 and 1810, respectively). Each of the third and fourth polypeptides (1814 and 1816, respectively) contains an Fc domain monomer containing a cavity. Each of the first polypeptide (1802) and the second polypeptide (1808) also contains mutations associated with electrostatic coordination, for example, E357K. Likewise, each of the third and fourth polypeptides (1814 and 1816, respectively) contains mutations associated with electrostatic coordination, for example, K370D. Each of 1804, 1806, 1810, 1812, 1814 and 1816 contains the amino acid modification R292P, which is represented by a diamond, and each of 1804, 1810, 1814 and 1816 contains the amino acid modification I253A, which is represented by an asterisk. Each of 1802 and 1808 contains the amino acid sequence of SEQ ID NO: 78. Each of 1814 and 1816 contains the amino acid sequence of SEQ ID NO: 73.

In fig. 18O depicts an Fc construct (construct 19) consisting of three Fc domains formed from four polypeptides. Each of the first polypeptide (1902) and the second polypeptide (1908) contains one Fc domain monomer containing charged amino acids at the C _H 3-C _H 3 interface, unlike the wild type sequence (1906 and 1912, respectively), attached via a linker to the Fc domain monomer containing the knob (1904 and 1910, respectively). Each of the third and fourth polypeptides (1914 and 1916, respectively) contains an Fc domain monomer containing a cavity. Each of the first polypeptide (1902) and the second polypeptide (1908) also contains mutations associated with electrostatic coordination, for example, E357K. Likewise, each of the third and fourth polypeptides (1914 and 1916, respectively) contains mutations associated with electrostatic coordination, for example, K370D. Each of 1904, 1906, 1910, 1912, 1914 and 1916 contains the amino acid modifications R292P, which is indicated by a diamond, and I253A, indicated by an asterisk. Each of 1902 and 1908 contains the amino acid sequence of SEQ ID NO: 79. Each of 1914 and 1916 contains the amino acid sequence of SEQ ID NO: 73.

In fig. 18P is a Western blot showing expression of construct 4, construct 5, and construct 7.

In fig. 19A is a graph showing cellular binding of IgG1 and construct 4, construct 6, construct 16, and construct 18 by TR-FRET to FcγRIIb (mean ± standard deviation shown for 3-8 replicates).

In fig. 19B is a graph showing cellular binding of IgG1 and construct 4, construct 6, construct 16, and construct 18 by TR-FRET to FcγRI (mean ± standard deviation shown for 3-8 replicates).

In fig. 19C is a graph showing cellular binding of IgG1 and construct 4, construct 6, construct 16, and construct 18 by TR-FRET to FcγRIIa (mean ± standard deviation shown for 3-8 replicates).

In fig. 19D is a graph showing cellular binding of IgG1 and construct 4, construct 6, construct 16, and construct 18 by TR-FRET to FcγRIIIa (mean ± standard deviation shown for 3-8 replicates).

In fig. 20 is a graph showing the binding of construct 4, construct 7, construct 6, construct 16, construct 18, and construct 19 to FcRn at pH 6.0 via surface plasmon resonance (SPR). The normalized maximum level of binding is proportional to the number of domains that functionally bind FcRn.

In fig. 21 is a graph comparing the pharmacokinetics of construct 4, construct 6, construct 16, and IVIg in mice.

In 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 exhibiting reduced FcRn binding, such as the I253A mutation, on pharmacokinetics in mice.

In fig. 23 is a graph comparing the pharmacokinetics of construct 20 (construct 4), construct 16, construct 18, and construct 19 in mice.

In 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.

In fig. 25 is a graph comparing the inhibition of IL-8 release in monocytes induced by pacific-blue labeled IgG1, construct 4, construct 6, construct 16, and construct 18.

In 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.

In fig. 27 is a graph comparing the effectiveness 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 scores on Day 12.

In fig. 28 is a graph comparing the effectiveness of Design 4 and Design 18 in the CAIA model administered prophylactically on Day 1, as measured by clinical endpoints.

In fig. 29 is a graph comparing the effectiveness of Design 4 and Design 18 in the CAIA model, administered prophylactically on Day 3, as measured by clinical endpoints.

In fig. 30 is a graph comparing the effectiveness of Design 4 and Design 18 in the CAIA model, administered prophylactically on Day 7, as measured by clinical indicators.

In fig. 31 is a graph comparing the effectiveness of design 4 and design 18 in the CAIA model administered prophylactically on day 10, as measured by clinical indicators.

In fig. Figure 32 shows a graph comparing the effectiveness of Design 4 (AA: black squares, solid line), Design 18 (Q1: black triangles, dashed line), Design 19 (Q2: black diamonds, dotted line) or saline (gray circles, dashed line). at a dose of 100 mg/kg on day 1 in the collagen antibody-induced arthritis (CAIA) model. An equivalent volume of saline (gray circles, dashed line) was administered on day 1. The mean and standard error of the mean are shown for each time point.

In fig. 33 is a graph comparing the effectiveness of Design 4 (AA: black squares, solid line), Design 18 (Q1: black triangles, dashed line), or Design 19 (Q2: black diamonds, dotted line) at a dose of 100 mg/kg on day 3 on collagen antibody-induced arthritis (CAIA) model. An equivalent volume of saline (gray circles, dashed line) was administered on day 1. The mean and standard error of the mean are shown for each time point.

In fig. 34 is a graph comparing the effectiveness of Design 4 (AA: black squares, solid line), Design 18 (Q1: black triangles, dashed line), or Design 19 (Q2: black diamonds, dotted line) at a dose of 100 mg/kg on day 7 on collagen antibody-induced arthritis (CAIA) model. An equivalent volume of saline (gray circles, dashed line) was administered on day 1. The mean and standard error of the mean are shown for each time point.

In fig. 35 is a graph comparing the effectiveness of Design 4 (AA: black squares, solid line), Design 18 (Q1: black triangles, dashed line), or Design 19 (Q2: black diamonds, dotted line) at a dose of 100 mg/kg on day 10 on collagen antibody-induced arthritis (CAIA) model. An equivalent volume of saline (gray circles, dashed line) was administered on day 1. The mean and standard error of the mean are shown for each time point.

In fig. 36 is a graph comparing the size exclusion chromatography profile of purified constructs X1 (gray) and X2 (black) normalized to peak maxima.

In fig. 37 shows the result of sodium dodecyl sulfate-polyacrylamide gel electrophoresis under non-reducing conditions for purified construct X1 (right), purified construct X2 (center), and molecular weight standards (right). Equal masses of the two structures were loaded.

In fig. 38 shows 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 evaluate the effect of reduced binding to FcRn and FcγRIIb on pharmacokinetics. The mean and standard deviation are shown for each time point.

In fig. 39 is a graph depicting 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), at a dose of 10 mg. /kg in cynomolgus macaques. Construct 4 (inverted triangles; SEQ ID NOs: 49 and 48) was administered at a dose of 20 mg/kg to cynomolgus monkeys.

In fig. 40 is a graph depicting 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) at a dose of 30 mg/kg in cynomolgus macaques. Construct 4 (SEQ ID NOs: 49 and 48) was administered at a dose of 20 mg/kg (squares), 30 mg/kg (circles) and 50 mg/kg (triangles) to cynomolgus monkeys.

DETAILED DESCRIPTION OF THE INVENTION

Therapeutic proteins that contain IgG Fc domains can be used to treat inflammation and immunological and inflammatory diseases, cancer and infections. The present invention describes compositions and methods for preparing Fc constructs containing Fc domains (e.g., Fc constructs having 2-10 Fc domains, e.g. Fc constructs having 2, 3, 4, 5, 6, 7, 8 , 9 or 10 Fc domains). The Fc constructs described herein facilitate the preparation of homogeneous pharmaceutical compositions by incorporating structural elements (eg, glycine spacers) that significantly improve manufacturing results.

Accordingly, the present invention provides pharmaceutical compositions that contain a substantially homogeneous collection of Fc constructs described herein (eg, an Fc construct containing three Fc domains). Homogeneity is an important aspect of a pharmaceutical composition as it affects the pharmacokinetics and in vivo efficacy of the composition. Traditionally, in the production of pharmaceutical products, there is the problem of product heterogeneity, which can be caused by several factors depending on how the product is manufactured. For example, a pharmaceutical product may be subject to random degradation, proteolysis, product degradation and/or aggregation, off-target subunit association, and/or inefficient protein folding. Different organisms with different biosynthetic modes or cellular mechanisms that are used to produce a pharmaceutical product may also cause heterogeneity in the product. Often, the original culture containing the desired pharmaceutical product must be subjected to a stringent purification process to obtain a less heterogeneous composition containing the pharmaceutical product.

The present invention provides, in one aspect, Fc constructs having structural elements that significantly improve the folding efficiency of the Fc constructs and minimize off-target association of subunits, thereby producing pharmaceutical compositions containing such Fc constructs with high homogeneity. A high degree of homogeneity ensures the safety, effectiveness, uniformity and reliability of the pharmaceutical composition. The presence of a high degree of homogeneity also minimizes potential aggregation or degradation of the pharmaceutical product caused by undesirable materials (eg, degradation products and/or aggregates or multimers), and also ensures that undesirable and adverse side effects caused by undesirable materials are limited.

As described in detail herein, the invention provides substantially homogeneous compositions containing Fc constructs that contain the same number of Fc domains, as well as methods for preparing such substantially homogeneous compositions.

The Fc constructs described herein contain glycine spacers between the Fc domains. As is well known in the art, linkers containing both serine and glycine residues provide structural flexibility in a protein and are commonly used to join two polypeptides. During experiments (see Example 4), the present inventors observed that linkers containing both serine and glycine residues undergo O-glycosylation (e.g., O-xylosylation) at several serine residues in the linker and proteolysis at the N-terminal parts of serine. The present inventors sought to optimize the sequence and linker length to further improve the homogeneity of the Fc constructs. The present inventors have created Fc constructs in which all linkers in the constructs are glycine spacers containing only glycine residues (eg, at least 12 glycine residues, eg, 12-30 glycine residues; SEQ ID NO: 27). The presence of all-glycine spacers in the Fc constructs further improved the homogeneity of the Fc constructs by preventing O-glycosylation at serine residues, as well as reducing the rate of proteolysis of the constructs (see Example 4). Consequently, the authors were able to achieve a more homogeneous population of Fc constructs by using all-glycine spacers in the Fc constructs.

Homogeneity results from the use of Fc construct components. For example, the first approach (“approach (a)”) can use the inclusion of linkers containing only glycine residues to attach Fc domain monomers. As observed experimentally, all-glycine spacers (e.g., at least 12 glycine residues, e.g., 12-30 glycine residues; SEQ ID NO: 27) in the Fc construct do not undergo O-glycosylation and are less susceptible to proteolysis compared to traditional linkers that contain serine and glycine residues (see example 4).

Additionally, in another approach (“approach (b)”), the homogeneity of a composition containing an Fc construct described herein (eg, an Fc construct having three Fc domains) is improved by removing C-terminal lysine residues. Such C-terminal lysine residues are highly conserved in immunoglobulins across many species and can be completely or partially removed by cellular machinery during protein production. Removal of the C-terminal lysine residues in the Fc constructs of the present invention improves the homogeneity of the resulting composition and allows for a more homogeneous preparation of the Fc construct (see Example 8). For example, in some embodiments of the Fc constructs described herein (e.g., an Fc construct containing three Fc domains), the C-terminal lysine codon is removed, thereby producing an Fc construct containing polypeptides without C-terminal residues lysine, and resulting in a homogeneous aggregate.

In a complementary approach (“approach (c)”), two sets of heterodimerization selectivity modules were used to improve the homogeneity of a composition containing an Fc construct described herein (e.g., an Fc construct having three Fc domains): (i ) heterodimerization selectivity modules having various charge reversal mutations, and (ii) heterodimerization selectivity modules having engineered cavities and protrusions. In experiments, for example, see Example 6, the present inventors observed that when attempting to form a heterodimeric Fc domain in an Fc construct, in the presence of both (i) and (ii), further improvement in the homogeneity of the pharmaceutical composition was achieved by reducing uncontrolled association of Fc domain monomers and hence unwanted oligomers and multimers. In specific examples, an Fc domain monomer may be produced comprising (i) at least one charge reversal mutation and (ii) at least one engineered cavity or at least one engineered knob and will selectively combine with another Fc domain monomer comprising (i) at least one charge reversal mutation and (ii) at least one engineered knob or at least one engineered cavity to form an Fc domain. In another example, an Fc domain monomer containing a charge reversal mutation, K370D, and engineered cavities Y349C, T366S, L368A, and Y407V, and another Fc domain monomer containing a charge reversal mutation, E357K, and engineered protrusions S354C and T366W can be produced and selectively combined to form an Fc domain.

As described in detail herein, a substantially homogeneous composition comprising an Fc construct of the present invention (eg, an Fc construct having three Fc domains) can be prepared by using all-glycine spacers between two Fc domain monomers in an Fc domain. constructs (approach (a)), by using polypeptides that lack the C-terminal lysine residues in the Fc construct (approach (b)), and/or by using two sets of heterodimerization selectivity modules ((i) selectivity modules , providing heterodimerization, having various mutations, providing a change in charge to the opposite, and (ii) selectivity modules, providing heterodimerization, having designed cavities and protrusions), in order to promote the formation of a heterodimeric Fc domain by some monomers of the Fc domain in the Fc construct (approach (With)).

In some embodiments, a substantially homogeneous composition comprising an Fc construct described herein (eg, an Fc construct having three Fc domains) can be prepared using approach (a).

In some embodiments, a substantially homogeneous composition comprising an Fc construct described herein (eg, an Fc construct having three Fc domains) can be prepared using approach (b).

In some embodiments, a substantially homogeneous composition comprising an Fc construct described herein (eg, an Fc construct having three Fc domains) can be prepared using approach (c).

In some embodiments, a substantially homogeneous composition comprising an Fc construct described herein (eg, an Fc construct having three Fc domains) can be prepared by a combination of approaches (a) and (b).

In some embodiments, a substantially homogeneous composition comprising an Fc construct described herein (eg, an Fc construct having three Fc domains) can be prepared by a combination of approaches (a) and (c).

In some embodiments, a substantially homogeneous composition comprising an Fc construct described herein (eg, an Fc construct having three Fc domains) can be prepared by a combination of approaches (b) and (c).

In some embodiments, a substantially homogeneous composition comprising an Fc construct described herein (eg, an Fc construct having three Fc domains) can be prepared by a combination of approaches (a), (b), and (c).

In some embodiments, to further improve the homogeneity of a pharmaceutical composition containing an Fc construct described herein, the N-terminal Asp in one or more polypeptides in the Fc construct in the composition is replaced by a Gln by mutation. In some embodiments of a composition comprising a substantially homogeneous collection of Fc constructs described herein, the N-terminal Asp in each of the polypeptides in the Fc construct in the composition is replaced by a Gln as a result of mutation.

In addition, in the Fc constructs of the present invention (eg, an Fc construct containing three Fc domains), the length of the linkers that connect the Fc domain monomers affects the folding efficiency of the Fc constructs. In some embodiments, a linker containing at least 4, 8, or 12 glycine residues (e.g., 4-30, 8-30, 12-30 glycine residues; SEQ ID NOs: 26 and 27) can be used to connect Fc- monomers domain in the Fc construct of the present invention.

I. Fc domain monomers

The Fc domain monomer contains a hinge domain, an antibody C _H 2 constant domain, and an antibody C _H 3 constant domain. The Fc domain monomer can be of different origins, for example, human, mouse or rat. The Fc domain monomer may be an immunoglobulin, which is an antibody of the IgG, IgE, IgM, IgA or IgD isotype. An Fc domain monomer can also refer to an immunoglobulin, which is an antibody of any isotype (eg, IgG1, IgG2a, IgG2b, IgG3, or IgG4). Fc domain monomers can also be hybrids, for example with the hinge and _CH 2 of IgG1 and the _CH 3 of IgA, or with the hinge and _CH 2 of IgG1 but with the _CH 3 of IgG3. A dimer of Fc domain monomers is an Fc domain (further defined herein) that can bind to an Fc receptor, for example, FcγRIIIa, which is a receptor located on the surface of leukocytes. In the present invention, the antibody C _H 3 constant domain of an Fc domain monomer may contain amino acid substitutions at the interface between the antibody C _H 3 -C _H 3 constant domains to promote their association with each other. In other embodiments, the Fc domain monomer contains an additional moiety, such as an albumin-binding peptide or a purification peptide, attached to the N- or C-terminus. In the present invention, the Fc domain monomer does not contain any type of antibody variable region, for example _VH , _VL , complementarity determining region (CDR) or hypervariable region (HVR).

In some embodiments, an Fc domain monomer in an Fc construct described herein (e.g., an Fc construct containing three Fc domains) may contain, consist of, or consist essentially of a sequence that is at least 95 % identical (e.g., at least 97%, 99%, or 99.5 identical) to the wild-type Fc domain monomer sequence (SEQ ID NO: 42). In some embodiments, an Fc domain monomer in an Fc construct described herein (e.g., an Fc construct containing three Fc domains) may contain, consist of, or consist essentially of a sequence that is an Fc domain monomer wild type (SEQ ID NO: 42) with no more than 10 (9, 8, 7, 6, 5, 4, 3, 2 or 1) single amino acid modifications (eg, substitutions, eg, conservative substitutions). In some embodiments, an Fc domain monomer in an Fc construct described herein (e.g., an Fc construct containing three Fc domains) may contain, consist of, or consist essentially of a sequence that is at least 95 % identical (e.g., at least 97%, 99%, or 99.5% identical) to the sequence of any of SEQ ID NOs: 44, 46, 48, and 50-53 (see Example 1, Tables 4 and 5). In some embodiments, an Fc domain monomer in an Fc construct described herein (e.g., an Fc construct containing three Fc domains) may contain, consist of, or consist essentially of a sequence that is a sequence under any of SEQ ID NO: 44, 46, 48 and 50-53 (see example 1, tables 4 and 5) with no more than 10 (9, 8, 7, 6, 5, 4, 3, 2 or 1) single amino acid modifications (eg substitutions, eg conservative substitutions). In some cases, these amino acid modifications are in addition to changes in the length of the glycine spacer, i.e., no more than 10 (9, 8, 7, 6, 5, 4, 3, 2, or 1) single amino acid modifications are in addition to changes in the length of the entire glycine spacer. spacer (SEQ ID NO: 23). In certain embodiments, an Fc domain monomer in an Fc construct may contain, consist of, or consist essentially of a sequence that is at least 95% identical (e.g., at least 97%, 99%, or 99.5 identical) %) sequence of any of SEQ ID NOs: 48, 52, and 53. In certain embodiments, an Fc domain monomer in an Fc construct may contain, consist of, or consist essentially of a sequence that is a sequence of any of SEQ ID NOs : 48, 52 and 53 with no more than 10 (9, 8, 7, 6, 5, 4, 3, 2 or 1) single amino acid modifications (eg substitutions, eg conservative substitutions).

SEQ ID NO: 42: Amino acid sequence of wild-type human IgG1 Fc domain monomer

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO: 44

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO: 46

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

SEQ ID NO: 48

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVDGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

SEQ ID NO: 50

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO: 51

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSDL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO: 52

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSDL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

SEQ ID NO: 53

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDKLTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

II. Fc domains

As defined herein, the Fc domain contains two Fc domain monomers that dimerize as a result of interactions between the antibody C _H 3 constant domains. In the present invention, the Fc domain does not contain an antibody variable region, for example, _VH , _VL , CDR or HVR. The Fc domain forms a minimal structure that binds to an Fc receptor, e.g., Fc gamma receptors (i.e., Fcγ receptors (FcγR)), Fc alpha receptors (i.e., Fcα receptors (FcαR)), Fc receptors -epsilon (i.e., Fcε receptors (FcεR)) and/or neonatal Fc receptor (FcRn). In some embodiments, the Fc domain of the present invention binds to an Fcγ receptor (e.g., FcγRI (CD64), FcγRIIa (CD32), FcγRIIb (CD32), FcγRIIIa (CD16a), FcγRIIIb (CD16b)), and/or FcγRIV, and/ or neonatal Fc receptor (FcRn).

III. Fc domain modifications

The unmodified Fc domain monomer may be a native human Fc domain monomer or a WT human Fc domain monomer. The Fc domain monomer may be a natural human Fc domain monomer comprising a hinge, a CH2 domain, and a CH3 domain; or a variant thereof having no more than 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., single amino acid modifications) to provide or promote directional dimerization. The Fc domain monomer may be an IgG1 Fc domain, an IgG2 Fc domain, an IgG3 Fc domain, an IgG4 Fc domain, or a combination thereof. The Fc domain monomer may be an IgG1 Fc domain, an IgG2 Fc domain, an IgG3 Fc domain, an IgG4 Fc domain, or a combination thereof with no more than 10 (9, 8, 7, 6, 5, 4, 3, 2 or 1) single amino acid modifications (eg, substitutions, eg, conservative substitutions). In some cases, the Fc domain monomer is a human IgG Fc domain monomer having up to ten amino acid modifications (e.g., no more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 amino acid modifications). In some cases, the Fc domain monomer contains, consists of, or consists substantially of the sequence of SEQ ID NO: 42 with no more than ten amino acid modifications (e.g., no more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 amino acid modifications). In some cases, the Fc domain contains at least one amino acid modification, wherein the amino acid modifications alter one or more of the following: (i) binding affinity to one or more Fc receptors, (ii) effector functions, (iii) level of Fc sulfation domain, (iv) half-life, (v) protease resistance, (vi) Fc domain stability, and/or (vii) susceptibility to degradation (eg, compared to an unmodified Fc domain). In some cases, the Fc domain contains no more than 16 amino acid modifications (for example, no more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 amino acid modifications per CH3 domain).

At least one Fc domain of the Fc construct of the present invention contains an amino acid modification at position I253 (e.g., I253A, I253C, I253D, I253E, I253F, I253G, I253H, I253I, I253K, I253L, I253M, I253N, I253P, I253Q, I253R, I253S, I253T, I253V, I253W or I253Y) and/or in position R292 (for example, R292P, R292D, R292E, R292L, R292Q, R292R, R292T and R292Y). In some cases, at least one Fc domain contains an amino acid modification at position I253, for example I253A. In some cases, at least one Fc domain contains an amino acid modification at position R292, for example R292P. The Fc domain may contain an amino acid modification at both position I253 (eg, I253A) and position R292 (eg, R292P). For example, an Fc construct having three Fc domains may contain an amino acid modification at position I253 (e.g., I253A) in one, two, or all three Fc domains and may additionally or alternatively contain an amino acid modification at position R292 (e.g., R292P) in one, two or all three Fc domains. Representative Fc constructs having the amino acid modifications I253A and/or R292P are depicted in FIG. 2 and fig. 18B-18O.

In some embodiments, modifications to the Fc domain that alter the half-life may reduce the extent of binding of the modified Fc domain to FcRn, for example, by modifying the Fc domain at position I253. Modifications at position I253 may include an amino acid substitution, where the amino acid at position I253 is replaced by a natural or unnatural amino acid; deletion of the amino acid at position I253; or the insertion of one or more amino acid residues at position I253 of the Fc domain. The I253 amino acid modification may be part of a combination involving multiple modifications (eg, at other residue positions, eg, R292), eg, a combination of one or more amino acid substitutions, deletions and/or insertions. In specific embodiments, the Fc construct may comprise, for example, three Fc domains, wherein at least one Fc domain contains a modification at position I253. For example, a wild-type amino acid residue, such as isoleucine (I), at position I253 may be replaced by a natural or unnatural amino acid, such as alanine (A). In some cases, each amino acid modification at position I253 is independently selected from, for example, I253A, I253C, I253D, I253E, I253F, I253G, I253H, I253I, I253K, I253L, I253M, I253N, I253P, I253Q, I253R, I253S, I253T, I 253V , I253W and I253Y.

In other embodiments, modifications to the Fc domain that alter the half-life may alter the extent of binding of the modified Fc domain to FcγRIIb, for example, by modifying the Fc domain at position R292. Modifications at position R292 may include an amino acid substitution, where the amino acid at position R292 is replaced by a natural or unnatural amino acid; deletion of the amino acid at position R292; or the insertion of one or more amino acid residues at position R292 of the Fc domain. The modification to amino acid 292 may be part of a combination involving multiple modifications (eg, at other residue positions, eg, I253), eg, a combination of one or more amino acid substitutions, deletions and/or insertions. In specific embodiments, the Fc construct may comprise, for example, three Fc domains, wherein at least one Fc domain contains a modification at position R292. For example, a wild-type amino acid residue, such as arginine (R), at position 292 may be replaced by a natural or unnatural amino acid, such as proline (P). In some cases, each amino acid modification at position R292 is independently selected from, for example, R292P, R292D, R292E, R292L, R292Q, R292R, R292T, and R292Y.

Typical Fc domains with altered binding affinity for Fc receptors include Fc monomers containing the S267E/L328F double mutants. The S267E/L328F mutations have previously been shown to significantly and specifically enhance IgG1 binding to the FcγRIIb receptor (Chu et al. Molecular Immunology 45 2008).

Amino acid modification, for example to change the half-life, at position I253, for example I253A, can occur in at least one (for example, 1, 2, 3, 4 or 5) Fc domain of the Fc construct, for example construct 4 (Fig. 2 ). In other embodiments, an amino acid modification at position R292, such as R292P, may occur in at least one (eg, 1, 2, 3, 4, or 5) Fc domain of an Fc construct, such as construct 4 (FIG. 2). In some embodiments, for example, amino acid modification may occur at both position I253, such as I253A, and position R292, such as R292P. For example, an Fc construct, such as construct 4 (FIG. 2), may contain one Fc domain with an amino acid modification at I253, such as I253A, and contain at least one (e.g., 1, 2, or 3) Fc domain with an amino acid modification modification to R292, for example R292P. In another embodiment, an Fc construct, such as construct 4 (FIG. 2), may contain two Fc domains with an amino acid modification at I253, such as I253A, and contain at least one (eg, 1, 2, or 3) Fc domain with an amino acid modification at R292, for example R292P. In another embodiment, an Fc construct, such as construct 4, may contain three Fc domains with an amino acid modification at I253, such as I293A, and contain at least one (e.g., 1, 2, or 3) Fc domain with an amino acid modification at R292, for example R292P. Representative Fc constructs having modifications at amino acid position I253 and/or R292, eg I293A and/or R292P, respectively, are depicted in FIG. 18A-18O. Not shown in Fig. 18A-18O, but disclosed herein are Fc constructs containing heterogeneous combinations of amino acid modifications, for example at amino acid positions I253 and/or R292, in the Fc domain monomers constituting the Fc domain.

IV. Selectivity modules providing dimerization

In the present invention, the dimerization selectivity module is a portion of an Fc domain monomer that promotes preferential pairing of two Fc domain monomers to form an Fc domain. Specifically, the dimerization selectivity module is that portion of the antibody C _H 3 constant domain of an Fc domain monomer that contains amino acid substitutions located at the interface between the interacting antibody C _H 3 constant domains of two Fc domain monomers. In the dimerization selectivity module, amino acid substitutions favor dimerization of the two C _H 3 constant domains of an antibody as a result of the compatibility of the amino acids selected for the substitutions. The final formation of a preferred Fc domain is selective relative to other Fc domains that are formed from Fc domain monomers that lack dimerization selectivity modules or have incompatible amino acid substitutions in dimerization selectivity modules. This type of amino acid substitution can be accomplished using conventional molecular cloning techniques well known in the art, such as QuikChange ^® mutagenesis.

In some embodiments, the dimerization selectivity module comprises an engineered cavity (described later herein) in the C _H 3 constant domain of the antibody. In other embodiments, the dimerization selectivity module comprises an engineered knob (described later herein) in the C _H 3 constant domain of the antibody. For selective formation of an Fc domain, two Fc domain monomers with compatible selectivity modules enabling dimerization, e.g., one antibody C _H 3 constant domain containing an engineered cavity and another antibody C _H 3 constant domain containing an engineered knob, are combined to form a pair protrusion-to-cavity Fc domain monomers. Engineered protrusions and engineered cavities are examples of heterodimerization selectivity modules that can be created in the antibody C _H 3 constant domains of Fc domain monomers to promote favorable heterodimerization of two Fc domain monomers that have compatible heterodimerization selectivity modules.

In other embodiments, an Fc domain monomer with a dimerization selectivity module containing amino acid substitutions that provide a positive charge and an Fc domain monomer with a dimerization selectivity module containing amino acid substitutions that provide a negative charge can selectively combine to form an Fc- domain by facilitating electrostatic coordination (described later herein) of charged amino acids. In some embodiments, the Fc domain monomer may contain one of the following amino acid substitutions that provide a positive charge and amino acid substitutions that provide a negative charge: K392D, K392E, D399K, K409D, K409E, K439D, and K439E. In one example, an Fc domain monomer containing an amino acid substitution conferring a positive charge, such as D356K or E357K, and an Fc domain monomer containing an amino acid substitution conferring a negative charge, such as K370D or K370E, can selectively combine to form an Fc domain by promoting this is the electrostatic coordination of charged amino acids. In another example, an E357K-containing Fc domain monomer and a K370D-containing Fc domain monomer can selectively combine to form an Fc domain by facilitating electrostatic coordination of charged amino acids. In some embodiments, charge-reversing amino acid substitutions can be used as heterodimerization selectivity modules, wherein two Fc domain monomers containing different but compatible charge-reversing amino acid substitutions combine to form a heterodimeric Fc -domain. Specific selectivity modules providing dimerization are further listed, without limitation, in Tables 1 and 2A, further described below.

In other embodiments, the two Fc domain monomers include homodimerization selectivity modules containing identical charge reversal mutations at at least two positions in the ring of charged residues at the interface between the C _H 3 domains. Homodimerization selectivity modules , are amino acid substitutions that provide a charge reversal that promote homodimerization of Fc domain monomers to form a homodimeric Fc domain. When the charge is reversed in both representatives of two or more complementary pairs of residues in two Fc domain monomers, the mutated Fc domain monomers remain complementary to Fc domain monomers with the same mutated sequence, but are characterized by less complementarity with respect to Fc domain monomers without these mutations. In one embodiment, the Fc domain comprises Fc domain monomers containing double mutants K409D/D399K, K392D/D399K, E357K/K370E, D356K/K439D, K409E/D399K, K392E/D399K, E357K/K370D, or D356K/K439E. In another embodiment, the Fc domain contains Fc domain monomers, including quadruple mutants combining any pair of double mutants, for example K409D/D399K/E357K/K370E. Examples of selectivity modules enabling homodimerization are further shown in Tables 2B and 2C.

In further embodiments, an Fc domain monomer comprising (i) at least one charge reversal mutation and (ii) at least one engineered cavity or at least one engineered overhang can be selectively combined with another Fc monomer -domain containing (i) at least one mutation causing a charge reversal, and (ii) at least one engineered protrusion or at least one engineered cavity, to form an Fc domain. For example, an Fc domain monomer containing a charge reversal mutation, K370D, and engineered cavities Y349C, T366S, L368A, and Y407V, and another Fc domain monomer containing a charge reversal mutation, E357K, and engineered overhangs S354C and T366W can selectively combine to form an Fc domain.

The formation of such Fc domains is facilitated by compatible amino acid substitutions in the constant C _H 3 domains of the antibody. Two dimerization selectivity modules containing incompatible amino acid substitutions, for example, both containing engineered cavities, both containing engineered overhangs, or both containing the same charged amino acids at the C _H 3 -C _H 3 interface, will not contribute to the formation of a heterodimeric Fc domain.

Additionally, other methods used to promote the formation of Fc domains from certain Fc domain monomers include, but are not limited to, the LUZ-Y approach (U.S. Patent Application Publication No. WO 2011034605), which involves the C-terminal fusion of monomeric leucine α-helices. lightning" with each of the monomers of the Fc domain, ensuring heterodimer formation, as well as the strand exchange engineered domain (SEED) approach (Davis et al., Protein Eng Des Sel. 23: 195-202, 2010), with by which the Fc domain is produced from heterodimeric Fc domain monomers, each of which contains alternating segments of the C _H 3 IgA and IgG sequences.

V. Constructed Cavities and Constructed Protrusions

The use of engineered cavities and engineered protrusions (or the ridge-to-groove strategy) has been described by Carter et al. (Ridgway et al., Protein Eng. 9:617-612, 1996; Atwell et al., J Mol Biol. 270:26 -35, 1997; Merchant et al., Nat Biotechnol. 16:677-681, 1998). Keg-to-valley interactions promote heterodimer formation, whereas peak-to-keg and valley-tooth interactions prevent homodimer formation due to steric clash and removal of favorable interactions. The ridge-to-groove technique is also disclosed in US Pat. No. 5,731,168.

In the present invention, engineered cavities and engineered protrusions are used in the preparation of the Fc constructs described herein. An engineered cavity is a void that is created when the original amino acid in a protein is replaced by another amino acid that has a smaller side chain volume. An engineered knob is a knob that is created when the original amino acid in a protein is replaced by another amino acid that has a larger side chain. In particular, the amino acid being replaced is located in the antibody constant domain C _H 3 of the Fc domain monomer and is involved in the dimerization of two Fc domain monomers. In some embodiments, an engineered cavity in one antibody C _H 3 constant domain is created to accommodate an engineered overhang in another antibody C _H 3 constant domain such that both antibody C _H 3 constant domains act as selectivity modules to enable dimerization (e.g., selectivity modules heterodimerization agents) (described above), which promote or favor the dimerization of two Fc domain monomers. In other embodiments, an engineered cavity in one C _H 3 constant domain of an antibody is created to better accommodate the parent amino acid in another C _H 3 constant domain of the antibody. In other embodiments, an engineered knob in one antibody C _H 3 constant domain is designed to form additional interactions with parent amino acids in another antibody C _H 3 constant domain.

The engineered cavity can be constructed by replacing amino acids containing large side chains, such as tyrosine or tryptophan, with amino acids containing smaller side chains, such as alanine, valine or threonine. In particular, some dimerization selectivity modules (eg, heterodimerization selectivity modules) (described in more detail above) contain engineered cavities, such as the Y407V mutation in the antibody C _H 3 constant domain. Likewise, a designed protrusion can be constructed by replacing amino acids containing smaller side chains with amino acids containing larger side chains. In particular, some dimerization selectivity modules (eg, heterodimerization selectivity modules) (described in more detail above) contain engineered overhangs, such as the T366W mutation in the antibody C _H 3 constant domain. In the present invention, engineered cavities and engineered protrusions are also combined with the engineering of disulfide bonds between _CH 3 domains to improve heterodimer formation. In one example, an Fc domain monomer containing the engineered cavities Y349C, T366S, L368A, and Y407V can selectively combine with another Fc domain monomer containing the engineered knobs S354C and T366W to form an Fc domain. In another example, an Fc domain monomer containing an engineered Y349C cavity and an Fc domain monomer containing an engineered S354C knob can selectively combine to form an Fc domain. Other engineered cavities and engineered protrusions in combination with both disulfide bond engineering and structure calculations (mixed HA-TF) are contained, without limitation, in Table 1.

Table 1

Strategy CH constant domain ₃ Fc domain monomer 1 antibodies CH constant domain ₃ Fc domain monomer 2 antibodies Link Engineered cavities and protrusions (“peak-to-valve”) Y407T T366Y US 8216805 Y407A T366W US 8216805 F405A T394W US 8216805 Y407T T366Y US 8216805 T394S F405W US 8216805 T394W:Y407T T366Y:F405A US 8216805 T394S:Y407A T366W:F405W US 8216805 T366W:T394S F405W:Y407A US 8216805 Engineered cavities and protrusions (“peak-to-valve”), S-S design T366S:L368A: Y407V:Y349C T366W:S354C Zeidler et al., J Immunol. 163:1246-52, 1999 Mixed HA-TF S346H:F405A Y349T:T394F WO 2006106905

Replacing the original amino acid residue in the C _H 3 constant domain of an antibody with another amino acid residue can be achieved by altering the nucleic acid encoding the original amino acid residue. The upper limit on the number of original amino acid residues that can be replaced is the total number of residues at the contact surface between the C _H 3 constant domains of an antibody such that there is still sufficient interaction at the contact surface. In some cases, the CH3 constant domain of an antibody has no more than 16 (eg, no more than 2, 4, 6, 8, 10, 12, 14, or 16) single amino acid modifications.

VI. Electrostatic coordination

Electrostatic coordination is the use of favorable electrostatic interactions between oppositely charged amino acids in peptides, protein domains, and proteins to control the formation of higher order protein molecules. A method for using electrostatic coordination effects to alter the interaction of antibody domains to reduce the formation of homodimers in favor of the formation of heterodimers in the production of bispecific antibodies is disclosed in US Patent Application Publication No. 2014-0024111.

In the present invention, electrostatic coordination is used to control the dimerization of Fc domain monomers and the formation of Fc constructs. In particular, to control the dimerization of Fc domain monomers using electrostatic coordination, one or more amino acid residues that constitute the C _H 3 -C _H 3 interface are replaced with positively or negatively charged amino acid residues such that the interaction becomes electrostatically favorable or unfavorable depending from the specific charged amino acids introduced. In some embodiments, a positively charged amino acid at the contact surface, such as lysine, arginine, or histidine, is replaced with a negatively charged amino acid, such as aspartic acid or glutamic acid. In other embodiments, the negatively charged amino acid on the contact surface is replaced with a positively charged amino acid. Charged amino acids can be introduced into one or both of the interacting constant C _H 3 domains of the antibody. By introducing charged amino acids into the interacting C _H 3 constant domains of antibodies, dimerization selectivity modules (described in more detail above) are created that can selectively form dimers of Fc domain monomers, controlled by electrostatic coordination effects resulting from interactions between charged amino acids.

In some embodiments, to create a dimerization selectivity module containing reversed charges that can selectively form dimers of Fc domain monomers that are controlled by electrostatic coordination effects, two Fc domain monomers can be selectively formed through heterodimerization or homodimerization.

Heterodimerization of Fc domain monomers

Heterodimerization of Fc domain monomers can be promoted by introducing different but compatible mutations in the two Fc domain monomers, such as the charge-compatible residue pairing mutations contained, without limitation, in Table 2A. In some embodiments, the Fc domain monomer may contain one of the following amino acid substitutions that provide a negative charge and amino acid substitutions that provide a positive charge: D356K, D356R, E357K, E357R, K370D, K370E, K392D, K392E, D399K, K409D, K409E, K439D and K439E. In one example, an Fc domain monomer containing an amino acid substitution conferring a positive charge, such as D356K or E357K, and an Fc domain monomer containing an amino acid substitution conferring a negative charge, such as K370D or K370E, can selectively combine to form an Fc domain by promoting this is the electrostatic coordination of charged amino acids. In another example, an E357K-containing Fc domain monomer and a K370D-containing Fc domain monomer can selectively combine to form an Fc domain by facilitating electrostatic coordination of charged amino acids.

For example, in an Fc construct containing three Fc domains, two of the three Fc domains can be formed by heterodimerization of two Fc domain monomers, facilitated by electrostatic coordination effects. The term “heterodimeric Fc domain” refers to an Fc domain that is formed through the heterodimerization of two Fc domain monomers, where the two Fc domain monomers contain different charge-reversing mutations (heterodimerization selectivity modules) (see, e.g. , mutations in Table 2A) that favor the formation of these two Fc domain monomers. As shown in FIG. 1 and 2, in an Fc construct containing three Fc domains—one carboxy-terminal “stem” Fc domain and two amino-terminal “branch” Fc domains—each of the amino-terminal “branch” Fc domains may be a heterodimeric Fc domain ( also called "heterodimeric branch Fc domain") (e.g., heterodimeric Fc domain formed by Fc domain monomers 106 and 114, or Fc domain monomers 112 and 116 in Figure 1; heterodimeric Fc domain formed by Fc domain monomers 206 and 214, or Fc domain monomers 212 and 216 in Fig. 2). A heterodimeric Fc domain branch can be formed by an Fc domain monomer containing E357K and another Fc domain monomer containing K370D.

Table 2A

Mutation(s) causing charge reversal in constant domain C _H 3 antibodies monomer 1 Fc domain Mutation(s) causing charge reversal in constant domain C _H 3 antibody monomers 2 Fc domains K409D D399K K409D D399R K409E D399K K409E D399R K392D D399K K392D D399R K392E D399K K392E D399R K370D E357K K370D E357R K370E E357K K370E E357R K370D D356K K370D D356R K370E D356K K370E D356R K409D, K392D D399K, E356K K370E, K409D, K439E E356K, E357K, D399K

Homodimerization of Fc domain monomers

Homodimerization of Fc domain monomers can be facilitated by introducing identical electrostatic coordination mutations (homodimerization selectivity modules) into both Fc domain monomers in a symmetrical manner. In some embodiments, the two Fc domain monomers contain homodimerization selectivity modules containing identical charge reversal mutations at at least two positions in the ring of charged residues at the interface between the C _H 3 domains. in both representatives of two or more complementary residue pairs in two Fc domain monomers, the mutated Fc domain monomers remain complementary to Fc domain monomers with the same mutated sequence, but are less complementary to Fc domain monomers without these mutations. Electrostatic coordination mutations that can be introduced into the Fc domain monomer to promote its homodimerization are listed, without limitation, in Tables 2B and 2C. In one embodiment, the Fc domain contains two Fc domain monomers, each of which includes charge reversal double mutants (Table 2B), for example K409D/D399K. In another embodiment, the Fc domain contains two Fc domain monomers, each of which includes quadruple charge reversal mutants (Table 2C), for example K409D/D399K/K370D/E357K.

For example, in an Fc construct containing three Fc domains, one of the three Fc domains can be formed by homodimerization of two Fc domain monomers, facilitated by electrostatic coordination effects. “Homodimeric Fc domain” refers to an Fc domain that is formed by homodimerization of two Fc domain monomers, where the two Fc domain monomers contain the same charge-reversing mutations (see, for example, mutations in Tables 2B and 2C) . As shown in FIG. 1 and 2, in an Fc construct containing three Fc domains—one carboxy-terminal stem Fc domain and two amino-terminal branch Fc domains—the carboxy-terminal stem Fc domain may be a homodimeric Fc domain (also called “stem homodimeric Fc domain”) (e.g., homodimeric Fc domain formed by Fc domain monomers 104 and 110 in Fig. 1; homodimeric Fc domain formed by Fc domain monomers 204 and 210 in Fig. 2). The homodimeric stem Fc domain can be formed by two Fc domain monomers, each containing the K409D/D399K double mutants.

Table 2B

Mutation(s) causing charge reversal in constant domain C _H 3 antibodies from each of the two Fc domain monomers in a homodimeric Fc domain K409D/D399K K409D/D399R K409E/D399K K409E/D399R K392D/D399K K392D/D399R K392E/D399K K392E/D399R K370D/E357K K370D/E357R K370E/E357K K370E/E357R K370D/D356K K370D/D356R K370E/D356K K370E/D356R

Table 2C

Mutation(s) causing charge reversal in constant domain C _H 3 antibodies from each of the two Fc domain monomers in a homodimeric Fc domain Mutation(s) causing charge reversal in constant domain C _H 3 antibodies from each of the two Fc domain monomers in a homodimeric Fc domain K409D/D399K/K370D/E357K K392D/D399K/K370D/E357K K409D/D399K/K370D/E357R K392D/D399K/K370D/E357R K409D/D399K/K370E/E357K K392D/D399K/K370E/E357K K409D/D399K/K370E/E357R K392D/D399K/K370E/E357R K409D/D399K/K370D/D356K K392D/D399K/K370D/D356K K409D/D399K/K370D/D356R K392D/D399K/K370D/D356R K409D/D399K/K370E/D356K K392D/D399K/K370E/D356K K409D/D399K/K370E/D356R K392D/D399K/K370E/D356R K409D/D399R/K370D/E357K K392D/D399R/K370D/E357K K409D/D399R/K370D/E357R K392D/D399R/K370D/E357R K409D/D399R/K370E/E357K K392D/D399R/K370E/E357K K409D/D399R/K370E/E357R K392D/D399R/K370E/E357R K409D/D399R/K370D/D356K K392D/D399R/K370D/D356K K409D/D399R/K370D/D356R K392D/D399R/K370D/D356R K409D/D399R/K370E/D356K K392D/D399R/K370E/D356K K409D/D399R/K370E/D356R K392D/D399R/K370E/D356R K409E/D399K/K370D/E357K K392E/D399K/K370D/E357K K409E/D399K/K370D/E357R K392E/D399K/K370D/E357R K409E/D399K/K370E/E357K K392E/D399K/K370E/E357K K409E/D399K/K370E/E357R K392E/D399K/K370E/E357R K409E/D399K/K370D/D356K K392E/D399K/K370D/D356K K409E/D399K/K370D/D356R K392E/D399K/K370D/D356R K409E/D399K/K370E/D356K K392E/D399K/K370E/D356K K409E/D399K/K370E/D356R K392E/D399K/K370E/D356R K409E/D399R/K370D/E357K K392E/D399R/K370D/E357K K409E/D399R/K370D/E357R K392E/D399R/K370D/E357R K409E/D399R/K370E/E357K K392E/D399R/K370E/E357K K409E/D399R/K370E/E357R K392E/D399R/K370E/E357R K409E/D399R/K370D/D356K K392E/D399R/K370D/D356K K409E/D399R/K370D/D356R K392E/D399R/K370D/D356R K409E/D399R/K370E/D356K K392E/D399R/K370E/D356K K409E/D399R/K370E/D356R K392E/D399R/K370E/D356R

Replacing the original amino acid residue in the C _H 3 constant domain of an antibody with another amino acid residue can be achieved by altering the nucleic acid encoding the original amino acid residue. The upper limit on the number of original amino acid residues that can be replaced is the total number of residues at the contact surface between the C _H 3 constant domains of an antibody such that there is still sufficient interaction at the contact surface. In some cases, the CH3 constant domain of an antibody has no more than 16 (eg, no more than 2, 4, 6, 8, 10, 12, 14, or 16) single amino acid modifications.

VII. Linkers

In the present invention, the term linker is used to describe a connection or connection between polypeptides or protein domains and/or associated non-protein fragments. In some embodiments, the linker is a linkage or connection between at least two Fc domain monomers, wherein the linker connects the C-terminus of the antibody C _H 3 constant domain of the first Fc domain monomer to the N-terminus of the hinge domain of a second Fc domain monomer such such that the two Fc domain monomers are linked to each other in a tandem sequence. In other embodiments, the linker is a link between the Fc domain monomer and any other protein domains that are attached to it. For example, a linker may attach the C-terminus of an antibody C _H 3 constant domain of an Fc domain monomer to the N-terminus of an albumin-binding peptide.

The linker may be a simple covalent bond, such as a peptide bond, a synthetic polymer, such as a polyethylene glycol (PEG) polymer, or any type of bond resulting from a chemical reaction, such as chemical conjugation. In the case where the linker is a peptide bond, a carboxylic acid group at the C-terminus of one protein domain can react with an amino group at the N-terminus of another protein domain in a condensation reaction to form a peptide bond. In particular, the peptide bond can be formed from synthetic means using a conventional organic chemistry reaction well known in the art, or by natural production from a host cell, wherein the polynucleotide sequence encoding the DNA sequences of both proteins, for example, two monomers The Fc domain, as a tandem sequence, can be directly transcribed and translated in the host cell into a continuous polypeptide encoding both proteins by the necessary molecular machinery, such as DNA polymerase and ribosomes.

In the case where the linker is a synthetic polymer, such as a PEG polymer, the polymer may be functionalized with reactive functional groups at each end to react with the terminal amino acids at the joining ends of the two proteins.

In case the linker (other than the above-mentioned peptide bond) is obtained through a chemical reaction, chemical functional groups, for example, amine, carboxylic acid, ester, azide or other functional groups commonly used in the art, can be synthetically attached to C -end of one protein and N-end of another protein, respectively. The two functional groups can then react using synthetic chemistry to form a chemical bond, thereby linking the two proteins to each other. Such chemical conjugation techniques are common to those skilled in the art.

Spacer

In the present invention, the linker between two Fc domain monomers may be an amino acid spacer containing 3-200 amino acids (for example, 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, 9-200, 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, 160-200 or 180-200 amino acids) (e.g. 3-150, 3-100, 3-60, 3-50, 3-40, 3-30, 3- 20, 3-10, 3-8, 3-5, 4-30, 5-30, 6-30, 8-30, 10-20, 10-30, 12-30, 14-30 20-30, 15 -25, 15-30, 18-22 and 20-30 amino acids). In some embodiments, the linker between two Fc domain monomers is an amino acid spacer containing at least 12 amino acids, such as 12-200 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, 12-15, 12-14 or 12-13 amino acids) (e.g. 14-200, 16-200, 18-200, 20-200, 30-200, 40-200, 50-200, 60-200 , 70-200, 80-200, 90-200, 100-200, 120-200, 140-200, 160-200, 180-200 or 190-200 amino acids). In some embodiments, the linker between two Fc domain monomers is an amino acid spacer containing 12-30 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). Suitable peptide spacers are known in the art and include, for example, peptide linkers containing flexible amino acid residues such as glycine and serine. In certain embodiments, the spacer may contain motifs, such as multiple or repeat motifs, from GS, GGS, GGGGS (SEQ ID NO: 1), GGSG (SEQ ID NO: 2) or SGGG (SEQ ID NO: 3). In certain embodiments, the spacer may contain from 2 to 12 amino acids, including motifs from GS, for example GS, GSGS (SEQ ID NO: 4), GSGSGS (SEQ ID NO: 5), GSGSGSGS (SEQ ID NO: 6), GSGSGSGSGS (SEQ ID NO: 7) or GSGSGSGSGSGS (SEQ ID NO: 8). In certain other embodiments, the spacer may contain from 3 to 12 amino acids, including motifs from GGS, for example, GGS, GGSGGS (SEQ ID NO: 9), GGSGGSGGS (SEQ ID NO: 10) and GGSGGSGGSGGS (SEQ ID NO: 11 ). In still other embodiments, the spacer may contain from 4 to 20 amino acids, including motifs from GGSG (SEQ ID NO: 2), for example, GGSGGGSG (SEQ ID NO: 12), GGSGGGSGGGSG (SEQ ID NO: 13), GGSGGGSGGGSGGGSG ( SEQ ID NO: 14) or GGSGGGSGGGSGGGSGGGSG (SEQ ID NO: 15). In other embodiments, the spacer may contain motifs from GGGGS (SEQ ID NO: 1), such as GGGGSGGGGS (SEQ ID NO: 16) or GGGGSGGGGSGGGGS (SEQ ID NO: 17). In certain embodiments, the spacer is SGGGSGGGSGGGSGGGSGGG (SEQ ID NO: 18).

In some embodiments, the spacer between two Fc domain monomers contains only glycine residues, e.g., at least 4 glycine residues (e.g., 4-200, 4-180, 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, 4-7, 4-6 or 4-5 glycine residues) (e.g. 4- 200, 6-200, 8-200, 10-200, 12-200, 14-200, 16-200, 18-200, 20-200, 30-200, 40-200, 50-200, 60-200, 70-200, 80-200, 90-200, 100-200, 120-200, 140-200, 160-200, 180-200 or 190-200 glycine residues). In certain embodiments, the spacer contains 4-30 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 some embodiments, a spacer containing only glycine residues may not be glycosylated (e.g., does not involve O-linked glycosylation, also referred to as O-glycosylation) or may have a reduced level of glycosylation (e.g., reduced O-glycosylation) (e.g. , reduced level of O-glycosylation by glycans such as xylose, mannose, sialic acids, fucose (Fuc) and/or galactose (Gal) (eg xylose)) compared to, for example, a spacer containing one or more serine residues (eg, SGGGSGGGSGGGSGGGSGGG (SEQ ID NO: 18)) (See Example 4).

In some embodiments, a spacer containing only glycine residues may not be O-glycosylated (e.g., not intended to be O-xylosylated) or may have a reduced level of O-glycosylation (e.g., reduced level of O-xylosylation) compared to, for example, with a spacer containing one or more serine residues (eg, SGGGSGGGSGGGSGGGSGGG (SEQ ID NO: 18)).

In some embodiments, a spacer containing only glycine residues may not undergo proteolysis or may have a reduced level of proteolysis compared to, for example, a spacer containing one or more serine residues (e.g., SGGGSGGGSGGGSGGGSGGG (SEQ ID NO: 18)) (see example 4).

In certain embodiments, the spacer may contain motifs from GGGG (SEQ ID NO: 19), for example, GGGGGGGG (SEQ ID NO: 20), GGGGGGGGGGGG (SEQ ID NO: 21), GGGGGGGGGGGGGGGG (SEQ ID NO: 22) or GGGGGGGGGGGGGGGGGGGG (SEQ ID NO: 23). In certain embodiments, the spacer may contain motifs from GGGGG (SEQ ID NO: 24), for example, GGGGGGGGGG (SEQ ID NO: 25) or GGGGGGGGGGGGGGG (SEQ ID NO: 26). In certain embodiments, the spacer is GGGGGGGGGGGGGGGGGGGG (SEQ ID NO: 27).

In other embodiments, the spacer may also contain amino acids other than glycine and serine, for example, GENLYFQSGG (SEQ ID NO: 28), SACYCELS (SEQ ID NO: 29), RSIAT (SEQ ID NO: 30), RPACKIPNDLKQKVMNH (SEQ ID NO: : 31), GGSAGGSGSGSSGGSSGASGTGTAGGTGSGSGTGSG (SEQ ID NO: 32), AAAANSSIDLISVPVDSR (SEQ ID NO: 33) or GGSGGGSEGGGSEGGGSEGGGSEGGGSEGGGSGGGS (SEQ ID NO: 34).

In certain embodiments of the present invention, a peptide spacer of 12 or 20 amino acids is used to join two Fc domain monomers in tandem sequence (e.g., polypeptides 102 and 108 in FIG. 1; polypeptides 202 and 208 in FIG. 2), wherein the peptide the 12 and 20 amino acid spacers consist of the sequences GGGSGGGSGGGS (SEQ ID NO: 35) and SGGGSGGGSGGGSGGGSGGGG (SEQ ID NO: 18), respectively. In other embodiments, an 18 amino acid peptide spacer consisting of the sequence GGSGGGSGGGSGGGSGGS (SEQ ID NO: 36) may be used.

In some embodiments, a spacer between two Fc domain monomers may contain, consist of, or consist essentially of a sequence that is at least 75% identical (e.g., at least 77%, 79%, 81%, 83% identical). 85%, 87%, 89%, 91%, 93%, 95%, 97%, 99%, or 99.5% identical) to the sequence of any of SEQ ID NO: 1-36 described above. In some embodiments, a spacer between two monomers, the Fc domain may contain, consist of, or consist essentially of a sequence that is the sequence of any of SEQ ID NO: 1-36 described herein, with no more than 10 (9, 8, 7, 6, 5, 4, 3, 2, or 1) single amino acid modifications (e.g., substitutions, such as conservative substitutions). In certain embodiments, a spacer between two Fc domain monomers may contain, consist of, or consist essentially of a sequence that is at least 80% identical (e.g., at least 82%, 85%, 87%, 90%, 92%, 95%, 97%, 99%, or 99.5% identical) to the sequence of any of SEQ ID NO : 17, 18, 26, and 27. In certain embodiments, the spacer between two Fc domain monomers may contain, consist of, or consist essentially of a sequence that is the sequence of any of SEQ ID NOs: 17, 18, 26, and 27 , with no more than 10 (9, 8, 7, 6, 5, 4, 3, 2 or 1) single amino acid modifications (eg, substitutions, such as conservative substitutions). In certain embodiments, a spacer between two Fc domain monomers may contain, consist of, or consist essentially of a sequence that is at least 80% (e.g., at least 82%, 85%, 87%, 90%, 92 %, 95%, 97%, 99%, or 99.5%) identical to the sequence of SEQ ID NO: 18 or 27. In certain embodiments, the spacer between two Fc domain monomers may contain, consist of, or consist essentially of the sequence which is the sequence of SEQ ID NO: 18 or 27, with no more than 10 (9, 8, 7, 6, 5, 4, 3, 2 or 1) single amino acid modifications (eg, substitutions, eg conservative substitutions).

VIII. Whey Protein Binding Peptides

By binding to whey proteins, peptides can provide improvements in the pharmacokinetics of protein pharmaceuticals, and in particular, the Fc constructs described herein can be fused to whey protein binding peptides.

As one example, albumin-binding peptides that can be used in the methods and compositions described herein are well known in the art. In one embodiment, the albumin binding peptide contains the sequence DICLPRWGCLW (SEQ ID NO: 37). In some embodiments, the albumin binding peptide contains, consists of, or consists essentially of a sequence that is at least 80% identical (e.g., 80%, 90%, or 100% identical) to the sequence of SEQ ID NO: 37. In some embodiments, the albumin binding peptide contains, consists of, or consists essentially of a sequence that is the sequence of SEQ ID NO: 37, with no more than 10 (9, 8, 7, 6, 5, 4, 3 , 2 or 1) single amino acid modifications (eg, substitutions, eg conservative substitutions).

In the present invention, albumin binding peptides can be attached to the N- or C-terminus of certain polypeptides in an Fc construct. In one embodiment, an albumin binding peptide may be attached to the C-terminus of one or more polypeptides in Fc constructs 1-4 (FIGS. 1 and 2). In another embodiment, the albumin binding peptide may be fused to the C-terminus of a polypeptide encoding two Fc domain monomers linked in Fc constructs 1-4 as a tandem sequence (e.g., polypeptides 102 and 108 in FIG. 1 and polypeptides 202 and 208 in Fig. 2). In yet another embodiment, an albumin binding peptide may be attached to the C-terminus of an Fc domain monomer (e.g., Fc domain monomers 114 and 116 in FIG. 1; Fc domain monomers 214 and 216 in FIG. 2) that attached to a second Fc domain monomer in a polypeptide encoding two Fc domain monomers linked as a tandem sequence. Albumin-binding peptides can be fused to Fc constructs at the genetic sequence level or attached to Fc constructs by chemical means, such as chemical conjugation. If necessary, a spacer can be inserted between the Fc construct and the albumin binding peptide. Without being limited by any theory, it is expected that the inclusion of an albumin binding peptide in the Fc construct of the present invention may result in an increase in the retention time of the therapeutic protein in the body due to its binding to serum albumin.

IX. Fc constructions

In general, the present invention provides Fc constructs containing Fc domains (eg, an Fc construct containing three Fc domains). They may have greater binding affinity and/or avidity to an Fc receptor, such as FcγRIIIa, than a single wild-type Fc domain. The present invention discloses methods for designing amino acids at the interface between two interacting C _H 3 constant domains of an antibody such that two Fc domain monomers belonging to the Fc domain selectively form a dimer with each other, thereby preventing the formation of undesirable multimers or aggregates. The Fc construct contains an even number of Fc domain monomers, with each pair of Fc domain monomers forming an Fc domain. The Fc construct contains at least one functional Fc domain formed from a dimer of two Fc domain monomers. In some embodiments, the Fc constructs described herein do not contain an antigen recognition region, such as a variable domain (eg, _VH , _VL , hypervariable region (HVR)) or a complementarity determining region (CDR). In some embodiments, the Fc constructs described herein comprise an antigen recognition region, such as a variable domain (eg, _VH , _VL , HVR) or CDR.

An Fc construct containing three Fc domains can be formed from four polypeptides (FIGS. 1 and 2). The first and second polypeptides (e.g., polypeptides 102 and 108 in FIG. 1; polypeptides 202 and 208 in FIG. 2) may be the same or different, as can the third and fourth polypeptides (e.g., polypeptides 114 and 116 in FIG. 1; polypeptides 214 and 216 in Fig. 2). In fig. 1, both the first and second polypeptides encode two Fc domain monomers (for example, Fc domain monomers 104, 106, 110 and 112), connected by a linker in the form of a tandem sequence, with one Fc domain monomer containing substitutions , which are charged amino acids, in the antibody C _H 3 constant domain (e.g., Fc domain monomers 104 and 110), while another Fc domain monomer contains a knob in the antibody C _H 3 constant domain (e.g., Fc domain monomers 106 and 112). Both the third and fourth polypeptides encode an Fc domain monomer with a cavity (eg, Fc domain monomers 114 and 116). A homodimeric stem Fc domain can be formed by combining Fc domain monomers 104 and 110, each containing the same charge-reversing mutations in its antibody constant C _H 3 domain (e.g., Fc monomers 104 and 110 each domain contains D399K and K409D. A first branch heterodimeric Fc domain can be formed by combining Fc domain monomers 106 and 114 (e.g., Fc domain monomer 106 contains engineered knobs S354C and T366W, and Fc domain monomer 114 contains engineered cavities Y349C, T366S, L368A and Y409V) A second heterodimeric Fc domain can be formed by combining Fc domain monomers 112 and 116 (e.g., Fc domain monomer 112 contains engineered knobs S354C and T366W, and Fc domain monomer 116 contains engineered cavities Y349C, T366S, L368A and Y409V).

In fig. 2, both the first and second polypeptides encode two Fc domain monomers (for example, Fc domain monomers 204, 206, 210 and 212), connected by a linker in the form of a tandem sequence, where one Fc domain monomer contains substitutions, representing charged amino acids in the antibody C _H 3 constant domain (eg, Fc domain monomers 204 and 210), while another Fc domain monomer contains an overhang and substitutions representing charged amino acids in the antibody C _H 3 constant domain (eg , Fc domain monomers 206 and 212). Both the third and fourth polypeptides encode an Fc domain monomer with a cavity and substitutions that represent charged amino acids (eg, Fc domain monomers 214 and 216). A homodimeric stem Fc domain can be formed by combining Fc domain monomers 204 and 210, each containing the same charge-reversing mutations in its antibody constant C _H 3 domain (e.g., Fc monomers 204 and 210 each domain contains D399K and K409D. A first branch heterodimeric Fc domain can be formed by combining Fc domain monomers 206 and 214 (e.g., Fc domain monomer 206 contains the engineered overhangs S354C and T366W and a charge reversal mutation, E357K, and Fc domain monomer 214 contains the engineered cavities Y349C, T366S, L368A and Y409V, as well as the charge-reversing mutation K370D.) A heterodimeric second branch Fc domain can be formed by combining Fc domain monomers 212 and 216 (e.g. Fc domain monomer 212 contains engineered overhangs S354C and T366W and a charge reversal mutation, E357K, and Fc domain monomer 216 contains engineered cavities Y349C, T366S, L368A and Y409V, and a charge reversal mutation, K370D ).

In further embodiments, an Fc domain monomer comprising (i) at least one charge reversal mutation and (ii) at least one engineered cavity or at least one engineered overhang can be selectively combined with another Fc monomer -domain containing (i) at least one mutation causing a charge reversal, and (ii) at least one engineered protrusion or at least one engineered cavity, to form an Fc domain. For example, an Fc domain monomer containing a charge reversal mutation, K370D, and engineered cavities Y349C, T366S, L368A, and Y407V, and another Fc domain monomer containing a charge reversal mutation, E357K, and engineered overhangs S354C and T366W can selectively combine to form an Fc domain.

In some embodiments, an Fc construct comprising a) a first polypeptide having the formula A-L-B; where i) A contains the first monomer of the Fc domain; ii) L is a linker; and iii) B contains a second monomer of the Fc domain; b) a second polypeptide having the formula A'-L'-B'; where i) A' contains the third monomer of the Fc domain; ii) L' is a linker; and iii) B' contains a fourth monomer of the Fc domain; c) a third polypeptide that contains a fifth monomer of the Fc domain; and d) a fourth polypeptide that contains a sixth Fc domain monomer; where B and B' combine to form the first Fc domain, A and the fifth Fc domain monomer combine to form the second Fc domain, and A' and the sixth Fc domain monomer combine to form the third Fc domain, with examples of some amino acids mutations that can be included in the Fc domain monomers in the Fc construct are shown in Tables 3A-3D. In some embodiments, each of the first, second, third and fourth polypeptides in the Fc construct lacks a C-terminal lysine. In some embodiments, the N-terminal Asp in each of the first, second, third, and fourth polypeptides in the Fc construct is replaced by Gln as a result of mutation. In some embodiments, L and L' each contain, consist of, or consist essentially of the sequence GGGGGGGGGGGGGGGGGGGG (SEQ ID NO: 27).

In some embodiments, the Fc construct contains two Fc domains formed from three polypeptides. The first polypeptide contains two Fc domain monomers connected in tandem sequence to a linker (eg, a glycine spacer; SEQ ID NOs: 26 and 27), and the second and third polypeptides each contain one Fc domain monomer. The second and third polypeptides may be the same polypeptide or may be different polypeptides. In fig. 13 shows an example of such an Fc design. The first polypeptide (1302) contains two Fc domain monomers (1304 and 1306) joined in tandem sequence by a linker (eg, a glycine spacer; SEQ ID NOs: 26 and 27). Both Fc domain monomers 1304 and 1306 contain engineered overhangs in the antibody C _H 3 constant domains. Each of the second and third polypeptides (1308 and 1310) contains one Fc domain monomer containing engineered cavities in the antibody C _H 3 constant domain. One of the Fc domain monomers (1304) in the first polypeptide forms a first heterodimeric Fc domain with a second polypeptide (1308), while the other Fc domain monomer (1306) in the first polypeptide forms a second heterodimeric Fc domain with a third polypeptide (1310) . The second and third polypeptides are not attached to each other or linked to each other. The designed "protrusion-to-cavity" C _H 3 -C _H 3 contact surface favors the formation of heterodimers from Fc domain monomers and prevents the uncontrolled formation of unwanted multimers. In some embodiments, each of the Fc domain monomers may further contain charge reversal mutations to promote heterodimerization. For example, Fc domain monomer 1304 containing engineered knobs and charge reversal mutations (e.g., E357K) may preferably form a heterodimeric Fc domain with Fc domain monomer 1308 containing engineered cavities and charge reversal mutations (eg K370D).

In still other embodiments, the Fc constructs may contain five Fc domains formed from six polypeptides. Two examples are shown in Fig. 14 and 15. Although these illustrated Fc constructs consist of six polypeptides, four of these polypeptides may be encoded by the same nucleic acid, and the remaining two polypeptides may also be encoded by the same nucleic acid. As a result, these Fc constructs can be produced by expressing the two nucleic acids in a suitable host cell.

In fig. 14 shows an Fc construct containing five Fc domains formed from six polypeptides. Each of the first and second polypeptides (1402 and 1410) contains three Fc domain monomers (1404, 1406, 1408 and 1412, 1414, 1416, respectively) connected in tandem sequence via a linker (e.g., glycine spacer; SEQ ID NO: 26 and 27). Specifically, in polypeptide 1402 or 1410, a first Fc domain monomer containing a knob (1404 or 1412) is connected to a second Fc domain monomer containing differently charged amino acids at a _CH3 - _CH3 contact surface (1406 or 1414), in contrast to the wild-type sequence, which is associated with the third monomer of the Fc domain containing the overhang (1408 or 1416). Fc domain monomers 1406 and 1414 may each contain the same charge-reversing mutations (eg, D399K/K409D) that promote the formation of a homodimeric Fc domain. Each of the third to sixth polypeptides (1418, 1420, 1422 and 1424) contains an Fc domain monomer containing a cavity and forms a heterodimeric Fc domain with each of the Fc domain monomers 1404, 1408, 1412 and 1416, respectively. In some embodiments, each of the Fc domain monomers 1404, 1408, 1412, 1416, 1418, 1420, 1422, and 1424 may further contain charge-reversing mutations to promote heterodimerization. For example, an Fc domain monomer 1408 containing engineered knobs and charge reversal mutations (e.g., E357K) may preferentially form a heterodimeric Fc domain with an Fc domain monomer 1420 containing engineered cavities and charge reversal mutations (eg K370D).

In fig. 15 depicts an Fc construct containing five Fc domains formed from six polypeptides. Each of the first and second polypeptides (1502 and 1510) contains three Fc domain monomers (1504, 1506, 1508 and 1512, 1514, 1516, respectively) connected in tandem sequence via a linker (e.g., glycine spacer; SEQ ID NO: 26 and 27). Specifically, in polypeptide 1502 or 1510, a first Fc domain monomer containing a knob (1504 or 1512) is linked to a second Fc domain monomer containing a knob (1506 or 1514), which is linked to a third Fc domain monomer containing differently charged amino acids at the C _H 3-C _H 3 contact surface (1508 or 1516), unlike the wild type sequence. Fc domain monomers 1508 and 1516 may each contain the same charge-reversing mutations (eg, D399K/K409D) that promote the formation of a homodimeric Fc domain. Each of the third to sixth polypeptides (1518, 1520, 1522 and 1524) contains an Fc domain monomer containing a cavity and forms a heterodimeric Fc domain with each of the Fc domain monomers 1504, 1506, 1512 and 1514, respectively. In some embodiments, each of Fc domain monomers 1504, 1506, 1512, 1514, 1518, 1520, 1522, and 1524 may further contain charge-reversing mutations to promote heterodimerization. For example, an Fc domain monomer 1504 containing engineered knobs and charge reversal mutations (e.g., E357K) may preferentially form a heterodimeric Fc domain with an Fc domain monomer 1518 containing engineered cavities and charge reversal mutations (eg K370D).

In fig. 16 shows another Fc construct consisting of five Fc domains formed from six polypeptides. Each of the first and second polypeptides (1602 and 1610) contains three Fc domain monomers (1604, 1606, 1608 and 1612, 1614, 1616, respectively) connected in tandem sequence via a linker (e.g., glycine spacer; SEQ ID NO: 26 and 27). Specifically, in polypeptide 1602 or 1610, the first Fc domain monomer containing the differentially charged amino acids at the _CH3 - _CH3 interface (1604 or 1612) is linked to the second Fc domain monomer containing the knob (1606 or 1614), which is associated with the third monomer of the Fc domain containing an overhang (1608 or 1616), unlike the wild type sequence. Fc domain monomers 1604 and 1612 may each contain the same charge-reversing mutations (eg, D399K/K409D) that promote the formation of a homodimeric Fc domain. Each of the third to sixth polypeptides (1618, 1620, 1622 and 1624) contains an Fc domain monomer containing a cavity and forms a heterodimeric Fc domain with each of the Fc domain monomers 1606, 1608, 1614 and 1616, respectively. In some embodiments, each of the Fc domain monomers 1606, 1608, 1614, 1616, 1618, 1620, 1622, and 1624 may further contain charge-reversing mutations to promote heterodimerization. For example, an Fc domain monomer 1608 containing engineered knobs and charge reversal mutations (e.g., E357K) may preferentially form a heterodimeric Fc domain with an Fc domain monomer 1620 containing engineered cavities and charge reversal mutations (eg K370D).

In another embodiment, an Fc construct comprising two or more Fc domains may be formed from two polypeptides having the same primary sequence. Such a construct can be formed by expressing a single polypeptide sequence in a host cell. An example is shown in FIG. 17. In this example, one nucleic acid is sufficient to encode an Fc construct containing three Fc domains. Two Fc domain monomers that are part of the same polypeptide can form a heterodimeric Fc domain by including a flexible linker of sufficient length and flexibility. The same polypeptide also contains a third Fc domain monomer attached via a flexible linker (eg, a glycine spacer; SEQ ID NOs: 26 and 27). This third Fc domain monomer (1708) can combine with another Fc domain monomer (1716) to form a homodimeric Fc domain and produce the Y-shaped Fc construct shown in FIG. 17. The formation of Fc domains can be controlled by dimerization selectivity modules, as also shown in FIG. 17. In some embodiments, each of the Fc domain monomers 1704, 1706, 1712 and 1714 may further contain charge reversal mutations to promote heterodimerization. For example, an Fc domain monomer 1704 containing engineered knobs and charge reversal mutations (e.g., E357K) may preferentially form a heterodimeric Fc domain with an Fc domain monomer 1706 containing engineered cavities and charge reversal mutations (eg K370D).

In some embodiments, one or more Fc polypeptides in the Fc construct (eg, Fc constructs 1-3 in FIG. 1; Fc construct 4 in FIG. 2) lack a C-terminal lysine residue. In some embodiments, all Fc polypeptides in the Fc construct lack a C-terminal lysine residue. In some embodiments, the absence of a C-terminal lysine in one or more Fc polypeptides in an Fc construct may improve the homogeneity of a plurality of Fc constructs (e.g., Fc constructs containing three Fc domains), e.g., a plurality of Fc constructs containing three Fc domain, which is essentially homogeneous (see example 8). In one example, the C-terminal lysine residue in an Fc polypeptide having the sequence of any of SEQ ID NOs: 43 and 44 (see Example 1, Table 6) can be removed to produce a corresponding Fc polypeptide that does not contain a C- terminal lysine residue.

In some embodiments, an Fc domain monomer in an Fc construct described herein (e.g., an Fc construct containing three Fc domains) may contain, consist of, or consist essentially of a sequence that is at least 95 % identical (eg, at least 97%, 99%, or 99.5 identical) to the wild-type Fc domain monomer sequence (eg, SEQ ID NO: 42). In some embodiments, an Fc domain monomer in an Fc construct described herein (e.g., an Fc construct comprising three Fc domains) may contain, consist of, or consist essentially of a sequence that is an Fc monomer sequence wild-type domain (SEQ ID NO: 42) with no more than 10 (9, 8, 7, 6, 5, 4, 3, 2 or 1) single amino acid modifications (eg, substitutions, eg, conservative substitutions). In some embodiments, an Fc domain monomer in an Fc construct described herein (e.g., an Fc construct containing three Fc domains) may contain, consist of, or consist essentially of a sequence that is at least 95 % identical (eg, at least 97%, 99%, or 99.5% identical) to the sequence of any of SEQ ID NOs: 44, 46, 48, and 50-53. In some embodiments, an Fc domain monomer in an Fc construct described herein (e.g., an Fc construct containing three Fc domains) may contain, consist of, or consist essentially of a sequence that is a sequence under any of SEQ ID NO: 44, 46, 48 and 50-53 with no more than 10 (9, 8, 7, 6, 5, 4, 3, 2 or 1) single amino acid modifications (eg, substitutions, such as conservative substitutions). In certain embodiments, an Fc domain monomer in an Fc construct may contain, consist of, or consist essentially of a sequence that is at least 95% identical (e.g., at least 97%, 99%, or 99.5 identical) %) sequences of SEQ ID NOs: 48, 52, and 53. In certain embodiments, an Fc domain monomer in an Fc construct may contain, consist of, or consist essentially of a sequence that is the sequence of SEQ ID NOs: 48, 52 and 53 with no more than 10 (no more than 9, 8, 7, 6, 5, 4, 3, 2 or 1) single amino acid modifications (eg, substitutions, eg conservative substitutions).

In some embodiments, a polypeptide comprising two Fc domain monomers in an Fc construct described herein (e.g., polypeptides 102 and 108 in FIG. 1, polypeptides 202 and 208 in FIG. 2) may comprise, consist of, or consist of substantially from a sequence that is at least 95% identical (e.g., at least 97%, 99%, or 99.5% identical) to the sequence of any of SEQ ID NOs: 43, 45, 47, and 49 (see example 1, table 6). In some embodiments, a polypeptide comprising two Fc domain monomers in an Fc construct described herein (e.g., polypeptides 102 and 108 in FIG. 1, polypeptides 202 and 208 in FIG. 2) may comprise, consist of, or consist of essentially from a sequence that is a sequence under any of SEQ ID NO: 43, 45, 47 and 49 (see example 1, table 6) with no more than 10 (no more than 9, 8, 7, 6, 5 , 4, 3, 2 or 1) single amino acid modifications (eg, substitutions, eg conservative substitutions). In certain embodiments, a polypeptide comprising two Fc domain monomers in an Fc construct described herein may contain, consist of, or consist substantially of a sequence that is at least 95% identical (e.g., identical in at least at least 97%, 99%, or 99.5%) of the sequence of SEQ ID NO: 49. In certain embodiments, a polypeptide comprising two Fc domain monomers in an Fc construct described herein may contain, consist of, or consist of essentially from a sequence that is the sequence of SEQ ID NO: 49 with no more than 10 (e.g., no more than 9, 8, 7, 6, 5, 4, 3, 2, or 1) single amino acid modifications (e.g., substitutions , for example conservative substitutions). In some embodiments, amino acid mutations in a polypeptide containing two Fc domain monomers in an Fc construct described herein (e.g., polypeptides 102 and 108 in Figure 1; polypeptides 202 and 208 in Figure 2) occur only in the monomers Fc domain (e.g., Fc domain monomers 104, 106, 110, and 112 in Figure 1; Fc domain monomers 204, 206, 210, and 212 in Figure 2) and do not occur in the spacer. For example, in the polypeptides shown in Table 8, additional amino acid mutations can be introduced into the Fc domain monomers having the sequences SEQ ID NO: 50-53, while the spacers having the sequence SEQ ID NO: 18, 26 and 27 remain unchanged.

In some embodiments, an N-terminal Asp in one or more of the first, second, third, and fourth polypeptides in an Fc construct described herein (e.g., polypeptides 102, 108, 114, and 116 in FIG. 1; 202, 208, 214 and 216 in Fig. 2) can be replaced by Gln as a result of mutation. In some embodiments, the N-terminal Asp in each of the first, second, third and fourth polypeptides in the Fc construct described herein is replaced by Gln as a result of mutation. In other embodiments, an Fc construct described herein (eg, an Fc construct comprising three Fc domains) may comprise one or more Fc domain monomers in which the N-terminal Asp is replaced by Gln by mutation. In some embodiments, replacement of an N-terminal Asp with a Gln resulting from mutation in one or more of the first, second, third, and fourth polypeptides in an Fc construct described herein may improve the homogeneity of a population of Fc constructs (e.g., Fc constructs , containing three Fc domains), for example, a collection of Fc constructs containing three Fc domains, which is essentially homogeneous. For example, Table 4 shows the amino acid sequences of the first, second, third and fourth polypeptides in which the N-terminal Asp in the Fc construct containing three Fc domains is replaced by Gln as a result of mutation.

Table 4

Fc-construct, where in all four polypeptides the N-terminal Asp is replaced by Gln as a result of mutation First and second polypeptides QKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSGGGSGGGSGGGSGGGSGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR DELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 54) Third and fourth polypeptides QKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 55) Fc-construct, where in all four polypeptides the N-terminal Asp is replaced by Gln as a result of mutation First and second polypeptides QKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSGGGSGGGSGGGSGGGSGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR DELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 56) Third and fourth polypeptides QKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 57) Fc-construct, where in all four polypeptides the N-terminal Asp is replaced by Gln as a result of mutation First and second polypeptides QKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDKLTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGGGGGGGGGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDE LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 58) Third and fourth polypeptides QKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVDGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 59) Fc-construct, where in all four polypeptides the N-terminal Asp is replaced by Gln as a result of mutation First and second polypeptides QKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDKLTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGGGGGGGGGGGGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP SRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 60) Third and fourth polypeptides QKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVDGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 59)

X. Host Cells and Protein Production

In the present invention, a host cell refers to a carrier that contains the necessary cellular components, for example, organelles, necessary for the expression of the polypeptides and constructs based on their corresponding nucleic acids described herein. Nucleic acids can be incorporated into nucleic acid vectors that can be introduced into a host cell through conventional techniques known in the art (transformation, transfection, electroporation, calcium phosphate precipitation, direct microinjection, etc.). The host cells may be from a mammal, bacteria, fungus, or insect. Mammalian host cells include, but are not limited to, CHO (or cell strains derived from CHO, e.g., CHO-K1, CHO-DXB11, CHO-DG44), mouse host cells (e.g., NS0, Sp2/0), VERY, HEK ( e.g. HEK293), BHK, HeLa, COS, MDCK, 293, 3T3, W138, BT483, Hs578T, HTB2, BT20 and T47D, CRL7O3O and HsS78Bst cells. Host cells may also be selected that modulate the expression of protein constructs or modify and process the protein product in the desired specific manner. Different host cells have characteristic and specific mechanisms for post-translational processing and modification of protein products. Suitable cell lines or host expression systems can be selected to ensure proper modification and processing of the expressed protein.

To express and secrete protein products from appropriate plasmid DNA constructs, host cells can be transfected or transformed with DNA under the control of appropriate expression control elements known in the art, including promoter, enhancer sequences, transcription terminators, polyadenylation sites, and selectable markers. Methods for expressing therapeutic proteins are known in the art. See, for example, Paulina Balbas, Argelia Lorence (eds.) Recombinant Gene Expression: Reviews and Protocols (Methods in Molecular Biology) , Humana Press; 2nd edition 2004 edition (July 20, 2004); Vladimir Voynov and Justin A. Caravella (eds.) Therapeutic Proteins: Methods and Protocols (Methods in Molecular Biology) Humana Press; 2nd ed. 2012 edition (June 28, 2012).

XI. Cleaning

The Fc construct can be purified by any method known in the art of protein purification, e.g., chromatography (e.g., ion exchange, affinity (e.g., protein A affinity), and size exclusion column chromatography), centrifugation, differential solubility, or any other standard technique protein purification. For example, the Fc construct can be isolated and purified by proper selection and combination of affinity columns, such as a protein A column, chromatography columns, filtration, ultrafiltration, salting out and dialysis (see, for example, Process Scale Purification of Antibodies , Uwe Gottschalk ( eds.) John Wiley & Sons, Inc., 2009; and Subramanian (eds.) Antibodies-Volume I-Production and Purification , Kluwer Academic/Plenum Publishers, New York (2004)).

In some cases, the Fc construct can be conjugated to one or more purification peptides to facilitate purification and isolation of the Fc construct, for example, from a mixture of whole cell lysates. In some embodiments, the purification peptide is coupled to another moiety that has a specific affinity for the purification peptide. In some embodiments, such moieties that specifically bind to the purification peptide are attached to a solid support such as a matrix, resin, or agarose beads. Examples of purification peptides that can be attached to the Fc construct include, but are not limited to, hexahistidine peptide, FLAG peptide, myc peptide, and hemagglutinin (HA) peptide. The hexahistidine peptide (HHHHHH (SEQ ID NO: 38)) binds to a nickel-functionalized agarose affinity chromatography column with micromolar affinity. In some embodiments, the FLAG peptide includes the sequence DYKDDDDK (SEQ ID NO: 39). In some embodiments, the FLAG peptide contains integer multiples of the sequence DYKDDDDK arranged in a tandem sequence, such as 3xDYKDDDDK. In some embodiments, the myc peptide contains the sequence EQKLISEEDL (SEQ ID NO: 40). In some embodiments, the myc peptide comprises integer multiples of the sequence EQKLISEEDL arranged in a tandem sequence, such as 3xEQKLISEEDL. In some embodiments, the HA peptide contains the sequence YPYDVPDYA (SEQ ID NO: 41). In some embodiments, the HA peptide contains integer multiples of the sequence YPYDVPDYA arranged in tandem sequence, for example 3xYPYDVPDYA. Antibodies that specifically recognize and bind to a purification peptide that is FLAG, myc or HA are well known in the art and are often commercially available. A solid support (eg, matrix, resin, or agarose beads) functionalized with such antibodies can be used to purify an Fc construct that contains a FLAG, myc, or HA peptide.

For Fc constructs, Protein A column chromatography can be used as a purification method. Protein A ligands interact with Fc constructs through the Fc region, making Protein A binding chromatography a highly selective capture method that can remove most cellular proteins. owner. In the present invention, the Fc constructs can be purified using Protein A column chromatography as described in Example 2.

XII. Pharmaceutical compositions/preparations

The present invention provides pharmaceutical compositions that contain one or more Fc constructs described herein. In one embodiment, the pharmaceutical composition contains a substantially homogeneous collection of Fc constructs. In various examples, the pharmaceutical composition contains a substantially homogeneous collection of any of the Fc constructs 1-4.

A therapeutic protein construct, eg, an Fc construct described herein (eg, an Fc construct containing three Fc domains) of the present invention may be included in a pharmaceutical composition. Pharmaceutical compositions containing therapeutic proteins can be formulated using methods known to those skilled in the art. The pharmaceutical composition can be administered parenterally in the form of an injectable formulation, including a sterile solution or suspension in water or other pharmaceutically acceptable liquid. For example, the pharmaceutical composition can be formulated by suitably combining the Fc construct with pharmaceutically acceptable carriers or vehicles such as sterile water for injection (WFI), saline, emulsifier, suspending agent, surfactant, stabilizer, diluent, binder , an excipient, followed by mixing to obtain the dosage form required for standard pharmaceutical practice. The amount of active ingredient included in the pharmaceutical preparations is such that a suitable dose is provided within the specified range.

The sterile injectable composition may be formulated in accordance with standard pharmaceutical practice using distilled water for injection as the carrier. For example, saline or isotonic solution containing glucose and other additives such as D-sorbitol, D-mannose, D-mannitol and sodium chloride can be used as an aqueous injection solution, optionally in combination with a suitable solubilizing agent, e.g. an alcohol such as ethanol and a polyhydric alcohol such as propylene glycol or polyethylene glycol, and a nonionic surfactant such as polysorbate 80™, HCO-50, and the like well known in the art. Methods for formulating therapeutic protein products are known in the art, see, for example, Banga (ed.) Therapeutic Peptides and Proteins: Formulation, Processing and Delivery Systems (2nd ed.) Taylor & Francis Group, CRC Press (2006 G.).

XIII. Dosage

The pharmaceutical compositions are administered in a manner consistent with the dosage formulation and in an amount that is therapeutically effective to improve or eliminate symptoms. Pharmaceutical compositions are administered in various dosage forms, such as intravenous dosage forms, subcutaneous dosage forms, oral dosage forms such as oral solutions, drug release capsules and the like. The appropriate dosage for an individual subject depends on the therapeutic goals, route of administration, and the condition of the patient. Typically, recombinant proteins are administered at a dose of 1-200 mg/kg, for example 1-100 mg/kg, for example 20-100 mg/kg. Accordingly, it will be necessary for the healthcare provider to adjust and titrate the dosage and modify the route of administration to obtain optimal therapeutic effect.

XIV. Indications

The pharmaceutical compositions of the present invention (e.g., containing Fc constructs containing 2, 3, or 4 Fc domains) are useful for reducing inflammation in a subject, for stimulating the clearance of autoantibodies in a subject, for inhibiting antigen presentation in a subject, for reducing an immune response, e.g. , blocking immune complex activation of an immune response in a subject, and treating immunological and inflammatory conditions or diseases in a subject. Exemplary conditions and diseases include the following: rheumatoid arthritis (RA); systemic lupus erythematosus (SLE); ANCA-associated vasculitis; antiphospholipid antibody syndrome; autoimmune hemolytic anemia; chronic inflammatory demyelinating neuropathy; clearance of antibodies to foreign cells during transplantation; autoreactivity in GVHD; replacement therapy with antibodies, IgG-based therapeutics, IgG-based paraproteins, dermatomyositis; Goodpasture's syndrome; type II organ system hypersensitivity syndromes mediated by antibody-dependent cell-mediated cytotoxicity, eg Guillain-Barré syndrome, CIDP, dermatomyositis, Felty's syndrome, antibody-mediated rejection, autoimmune thyroid disease, ulcerative colitis, autoimmune liver disease; idiopathic thrombocytopenic purpura; myasthenia gravis, neuromyelitis optica; pemphigus and other autoimmune disorders accompanied by the formation of blisters; Sjögren's syndrome; autoimmune cytopenias and other disorders mediated by antibody-dependent phagocytosis; other FcR-dependent inflammatory syndromes; synovitis; dermatomyositis, systemic vasculitis, glomerulitis and vasculitis.

In some embodiments, the pharmaceutical compositions of the present invention containing Fc constructs containing 5-10 Fc domains are also useful, for example, for inducing immune cell activation of an immune response in a subject, to enhance phagocytosis of a target cell (i.e., cancer cell or infected cell) in a subject and for treating diseases such as cancer and infections in the subject. The Fc constructs and homogeneous pharmaceutical compositions of the present invention can bind to activating Fcγ receptors (eg, FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa and FcγRIIIb) to induce an immune response. The Fc constructs and homogeneous pharmaceutical compositions of the present invention can activate Syk phosphorylation and calcium flux from THP-1 primary monocytes. Activated monocytes and differentiated macrophages derived from them have the ability to phagocytose or destroy target cells. Thus, the present invention provides methods of treatment that can be used to treat subjects suffering from diseases and disorders such as cancer and infections. In some embodiments, the Fc constructs and homogeneous pharmaceutical compositions described herein can be administered to a subject in a therapeutically effective amount to promote phagocytosis or destruction of cancer cells or infected cells in the subject.

Types of cancer that are treatable in accordance with the methods of the present invention include, but are not limited to, bladder cancer, pancreatic cancer, lung cancer, liver cancer, ovarian cancer, colon cancer, stomach cancer, breast cancer, prostate cancer, kidney cancer , testicular cancer, thyroid cancer, uterine cancer, rectal cancer, respiratory system cancer, urinary system cancer, oral cancer, skin cancer, leukemia, sarcoma, carcinoma, basal cell cancer, non-Hodgkin's lymphoma, acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), B-cell chronic lymphocytic leukemia (B-CLL), multiple myeloma (MM), erythroleukemia, renal cell carcinoma, astrocytoma, oligoastrocytoma, biliary tract cancer, choriocarcinoma, central nervous system cancer, laryngeal cancer, small cell lung cancer, adenocarcinoma, giant cell (or oat cell) carcinoma, squamous cell carcinoma, anaplastic large cell lymphoma, non-small cell lung cancer, neuroblastoma, rhabdomyosarcoma, neuroectodermal carcinoma, glioblastoma, breast carcinoma, melanoma, inflammatory myofibroblastic tumor and soft tissue tumor.

Infections that are treatable in accordance with the methods of the present invention include, but are not limited to, bacterial infection, viral infection, fungal infection, helminthic infection and protozoal infection.

Examples of bacteria that cause infection are well known in the art and include, but are not limited to, bacteria of the genus Streptococcus (for example, Streptococcus pyogenes ), bacteria of the genus Escherichia (for example, Escherichia coli ), bacteria of the genus Vibrio (for example, Vibrio cholerae ), bacteria of the genus Enteritis ( for example, Enteritis salmonella ), as well as bacteria of the genus Salmonella (for example, Salmonella typhi ).

Examples of infectious viruses are well known in the art and include, but are not limited to, viruses of the family Retroviridae (e.g., human immunodeficiency virus (HIV)), viruses of the family Adenoviridae (e.g., adenovirus), viruses of the family Herpesviridae (e.g., herpes simplex viruses types 1 and 2 ), viruses of the family Papillomaviridae (for example, human papillomavirus (HPV)), viruses of the family Poxviridae (for example, smallpox), viruses of the family Picornaviridae (for example, hepatitis A virus, poliovirus, rhinovirus), viruses of the family Hepadnaviridae (for example, hepatitis B virus) , viruses of the family Flaviviridae (for example, hepatitis C virus, yellow fever virus, West Nile virus), viruses of the family Togaviridae (for example, rubella virus), viruses of the family Orthomyxoviridae (for example, influenza virus), viruses of the family Filoviridae (for example, Ebola virus, Marburg) and viruses of the Paramyxoviridae family (for example, measles virus, mumps virus).

Examples of fungi that cause infection are well known in the art and include, but are not limited to, fungi of the genus Aspergillus (e.g., Aspergillus fumigatus , A. flavus , A. terreus , A. niger , A. candidus , A. clavatus , A. ochraceus ), fungi the genus Candida (e.g. Candida albicans , C. parapsilosis , C. glabrata , C. guilliermondii , C. krusei , C. lusitaniae , C. tropicalis ), fungi of the genus Cryptococcus (e.g. Cryptococcus neoformans ), and fungi of the genus Fusarium (e.g. , Fusarium solani , F. verticillioides , F. oxysporum ).

Examples of helminths include, but are not limited to, tapeworms (cestodes), roundworms (nematodes), flukes (flukes), and monogenetic flukes.

Examples of protozoa include, but are not limited to, protozoans of the genus Entamoeba (e.g., Entamoeba histolytica ), protozoans of the genus Plasmodium (e.g., Plasmodium falciparum, P. malariae ), protozoans of the genus Giardia (e.g., Giardia lamblia ), and protozoans of the genus Trypanosoma (e.g., Trypanosoma brucei ).

EXAMPLES

Example 1. Development of Fc structures

The goal of developing Fc constructs is to increase folding efficiency, minimize uncontrolled association of subunits that can create undesirable high molecular weight oligomers and multimers, and produce compositions that are essentially homogeneous. With these goals in mind, we designed four Fc constructs (FIGS. 1 and 2), each containing a long polypeptide containing two Fc domain monomers separated by a spacer (polypeptides 102 and 108 in FIG. 1 and polypeptides 202 and 208 in FIG. 2), and a short polypeptide containing one monomer of the Fc domain (polypeptides 114 and 116 in Fig. 1 and polypeptides 214 and 216 in Fig. 2). Each is based on an IgG1 Fc sequence that has been modified to provide engineered cavity, engineered protrusion, and/or electrostatic coordination to control polypeptide assembly. DNA sequences encoding long and short polypeptides were optimized for expression in mammalian cells and cloned into the mammalian expression vector pcDNA3.4. The plasmid DNA constructs were transfected via liposomes into human embryonic kidney (HEK) 293 cells. A total of eight plasmid DNA constructs were used to assemble four Fc constructs, each containing three Fc domains.

In the case of each Fc construct, co-expression of long and short polypeptides produces a branched molecule containing three Fc domains, with the C-terminal Fc monomers of the long polypeptides specifically associating with each other to form one C-terminal Fc domain, and N -terminal Fc monomers of long polypeptides specifically associate with short polypeptides to form two N-terminal Fc domains. Fc constructs 1-4 and their development are described in Table 5 and FIG. 1 and 2. The sequences used in each Fc construct are shown in Table 6. Table 7 below further briefly describes the characteristics of the long and short polypeptides in each of constructs 1-4.

Table 5

Fc design Long polypeptide nos. (SEQ ID NO) No. of short polypeptides (SEQ ID NO) Figure Fc-construction 1 102 and 108 (SEQ ID NO: 43) 114 and 116 (SEQ ID NO: 44) Fig. 1 Fc-construction 2 102 and 108 (SEQ ID NO: 45) 114 and 116 (SEQ ID NO: 46) Fig. 1 Fc-construction 3 102 and 108 (SEQ ID NO: 47) 114 and 116 (SEQ ID NO: 48) Fig. 1 Fc-construction 4 202 and 208 (SEQ ID NO: 49) 214 and 216 (SEQ ID NO: 48) Fig. 2

Table 6

SEQ ID NO Amino acid sequence SEQ ID NO: 43 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSGGGSGGGSGGGSGGGSGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR DELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 44 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 45 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSGGGSGGGSGGGSGGGSGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR DELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 46 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 47 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDKLTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGGGGGGGGGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDE LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 48 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVDGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 49 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDKLTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGGGGGGGGGGGGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP SRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

Long polypeptides 102 and 108 in Fc constructs 1-3 (FIG. 1) and long polypeptides 202 and 208 in Fc construct 4 (FIG. 2) each contain two Fc domain monomers connected as a tandem sequence by a spacer. Table 8 below shows the sequences of the Fc domain monomers in long polypeptides and the spacers in Fc constructs 1-4.

Table 8

Polypeptides 102/108 Fc constructs 1 N-terminal monomer of Fc domain DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 50) Spacer SGGGSGGGSGGGSGGGSGGG (SEQ ID NO: 18) C-terminal monomer of the Fc domain DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSDL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 51) Polypeptides 102/108 Fc constructs 2 N-terminal monomer of Fc domain DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 50) Spacer SGGGSGGGSGGGSGGGSGGG (SEQ ID NO: 18) C-terminal monomer of the Fc domain DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSDL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 52) Polypeptides 102/108 Fc constructs 3 N-terminal monomer of Fc domain DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDKLTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 53) Spacer GGGGGGGGGGGGGGG (SEQ ID NO: 26) C-terminal monomer of the Fc domain DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSDL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 52) Polypeptides 202/208 Fc constructs 4 N-terminal monomer of Fc domain DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDKLTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 53) Spacer GGGGGGGGGGGGGGGGGGGG (SEQ ID NO: 27) C-terminal monomer of the Fc domain DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSDL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 52)

Example 2. Expression of Fc constructs

Expressed proteins were purified from cell culture supernatant by protein A binding affinity column chromatography using a Poros MabCapture A column (LifeTechnologies). Captured Fc constructs were washed with phosphate buffered saline (low salt wash) and eluted with 100 mM glycine, pH 3. The eluate was quickly neutralized by adding 1 M TRIS pH 7.4 and filtered under sterile conditions through a 0.2 μm filter .

Proteins were further fractionated by ion exchange chromatography using Poros XS resin (Applied Biosciences). The column was pre-equilibrated with 50 mM MES, pH 6 (buffer A), and the sample was eluted using a step gradient using 50 mM MES, 400 mM sodium chloride, pH 6 (buffer B) as the elution buffer.

After ion exchange, the target fraction buffer was replaced with PBS buffer using a polyethersulfone (PES) membrane cartridge with a 10 kDa cutoff on a tangential flow filtration system. Samples were concentrated to approximately 30 mg/mL and filtered under sterile conditions through a 0.2 μm filter.

Example 3 Experimental Assays Used to Characterize Fc Constructs

Liquid Chromatography-MS/MS for Peptide and Glycopeptide Analysis

Proteins were diluted to 1 μg/μl in 6 M guanidine (Sigma). Dithiothreitol (DTT) was added to reach a concentration of 10 mM to reduce the number of disulfide bonds under denaturing conditions at 65 °C for 30 min. After cooling on ice, samples were incubated with 30 mM iodoacetamide (IAM) for 1 h in the dark to alkylate (carbamidomethylate) the free thiols. The protein was then dialyzed through a 10 kDa membrane into 25 mM ammonium bicarbonate buffer (pH 7.8) to remove IAM, DTT, and guanidine. Protein was digested with trypsin in a Barocycler (NEP 2320; Pressure Biosciences, Inc.). The pressure was cycled from 20,000 psi. inch to ambient pressure at 37°C for 30 cycles over 1 hour. Peptide analysis by LC-MS/MS was performed on an Ultimate 3000 chromatography system (Dionex) and a Q-Exactive mass spectrometer (Thermo Fisher Scientific). Peptides were separated on a BEH PepMap column (Waters) using 0.1% FA in water and 0.1% FA in acetonitrile as mobile phases. The linker peptide, xylosylated at a single residue, was recognized by a doubly charged ion (z=2) m/z 842.5 with a quadrupole selection window width of ±1.5 Da.

Intact mass spectrometry

The protein was diluted to a concentration of 2 μg/μl in a running buffer consisting of 78.98% water, 20% acetonitrile, 1% formic acid (FA), and 0.02% trifluoroacetic acid. Separation by size exclusion chromatography was performed on two Zenix-C SEC-300 columns (Sepax Technologies, Newark, DE) 2.1 × 350 mm, arranged in series so that the total length of the columns was 700 mm. Proteins were eluted from the SEC column using the running buffer described above at a flow rate of 80 μL/min. Mass spectra were obtained on a Q-ToF QSTAR Elite mass spectrometer (Applied Biosystems) operating in positive mode. The mass values of neutral particles in individual fractions when separated by particle size were deconvoluted using Bayesian peak deconvolution by summing the spectra over the entire width of the chromatographic peak.

Analysis by capillary electrophoresis with sodium dodecyl sulfate (CE-SDS)

Samples were diluted to 1 mg/mL and mixed with HT Protein Express denaturing buffer (PerkinElmer). The mixture was incubated at 40°C for 20 min. Samples were diluted with 70 μL of water and transferred to a 96-well plate. Samples were analyzed using a Caliper GXII instrument (PerkinElmer) equipped with an HT Protein Express LabChip (PerkinElmer). Fluorescence intensity was used to calculate the relative abundance of each size variant.

SDS-PAGE under non-reducing conditions

Samples were denatured in Laemmli sample buffer (4% SDS, Bio-Rad) at 95°C for 10 min. Samples were run on a Criterion TGX gel without staining (4-15% polyacrylamide, Bio-Rad). Protein spots were visualized using UV light or Coomassie blue staining. Gels were visualized using a ChemiDoc MP Imaging System (Bio-Rad). Spot quantification was performed using Imagelab 4.0.1 software (Bio-Rad).

Complement dependent cytotoxicity (CDC)

CDC was assessed using a colorimetric assay in which Raji cells (ATCC) were coated with serial dilutions of rituximab, Fc construct 4, or IVIg. Human serum complement (Quidel) was added to all wells at 25% v/v. and incubated for 2 hours at 37°C. Cells were incubated for 12 h at 37°C after addition of cell proliferation reagent WST-1 (Roche Applied Science). The plates were placed on a shaker for 2 min. and absorbance was measured at 450 nm.

Example 4 O-glycosylation and proteolysis of linker serine residues

O-glycosylation of linker serine residues

As described in Example 1, the inventors of the present invention pursued development of the Fc construct to increase folding efficiency, minimize uncontrolled subunit association, and provide compositions for pharmaceutical use that are substantially homogeneous. In an attempt to achieve these goals, we examined various linkers between two Fc domain monomers in a long polypeptide (102 and 108 in Fig. 1; 202 and 208 in Fig. 2). Fc construct 1 and Fc construct 2 each contain a serine-glycine linker (SGGGSGGGSGGGSGGGSGGG (SEQ ID NO: 18)) between two Fc domain monomers in a long polypeptide.

When analyzing Fc construct 2, which contains the linker SGGGSGGGSGGGSGGGSGGG (SEQ ID NO: 18) between two Fc domain monomers in a long polypeptide, the presence of O-xylosylation was detected using LC-MS/MS for peptide analysis (Fig. 3). However, since fragments y2 to y9 do not contain xylose, the fifth serine in the linker is not O-xylosylated. There may be multiple sites that are O-xylosylated, but each peptide is O-xylosylated at only one residue. The prevalence and location of this post-translational modification may depend on both the sequence and the expression system.

Similarly, O-xylosylation was found in an Fc construct containing two Fc domains (Fc construct shown in FIG. 13) containing the same linker (SG ₃ ) ₅ (FIG. 4). The modification was found in different positions, with no more than two xylose modifications present in each linker. In addition, the extent of modification varied between series.

After discovering O-xylosylation at serine residues in the serine-glycine linker, we explored alternative linkers that contained only glycine residues to further optimize the linker sequence and improve the homogeneity of the Fc construct. As a result, an all-glycine spacer was selected for use in Fc construct 3 and Fc construct 4. Fc construct 3 contains a 15-mer all-glycine spacer (GGGGGGGGGGGGGGGG (SEQ ID NO: 26)) between two Fc domain monomers in a long polypeptide . Fc construct 4 contains a 20-mer all-glycine spacer (GGGGGGGGGGGGGGGGGGGG (SEQ ID NO: 27)) between two Fc domain monomers in a long polypeptide.

Proteolysis at serine residues of the linker

In some embodiments, Fc constructs have been found to undergo proteolysis at linkers when incubated at 45°C in phosphate-buffered saline to form monomeric Fc products. The rate of monomer formation for Fc construct 2 (which contains the SGGGSGGGSGGGSGGGSGGG linker in each of polypeptides 102 and 108) was higher than for Fc construct 4 (which contains the all-glycine spacer GGGGGGGGGGGGGGGGGGGG in each of polypeptides 202 and 208) (Fig. 5), which indicates that the all-glycine spacer is less susceptible to proteolysis. This effect was found to be general for branched Fc constructs containing three Fc domains at different linker lengths; Proteolysis of all-glycine spacers occurs more slowly than of serine-glycine linkers (Table 9).

Table 9

Linker sequence Monomer formation rate (% monomers/day) G ₈ 0.17 G ₁₅ 0.21 G ₂₀ 0.24 (SG ₄ ) ₄ 0.33 (SG ₃ ) ₅ 0.34

In addition, mass spectrometry analyzes of monomeric Fc products in the case of Fc construct 2 with linker (SG ₃ ) ₅ in each of polypeptides 102 and 108 showed that the dominant products were cleaved at the N-terminal serine and were susceptible to proteolysis all serine residues except the first (Fig. 6). In contrast, in the case of the cleavage products of Fc construct 4 with spacer G ₂₀ in each of polypeptides 202 and 208, no strong specificity for any particular spacer residue was observed (Fig. 7). Together, these results indicated that the all-glycine spacer had reduced susceptibility to proteolysis. To limit proteolysis, a serine-free spacer, such as the G ₂₀ spacer used in Fc construct 4, can be used. The use of such a glycine spacer provides a significant improvement in the homogeneity of the final Fc construct composition.

Example 5: Linker Length Optimization

To further optimize for homogeneity, linker length was examined by generating sequence variations in Fc construct 2 in which the (SG ₃ ) ₅ linker was replaced with a G ₈ , G ₁₅ or G ₂₀ spacer. Analyzes using in vitro assays indicated that linker length affected biological activity, presumably by altering the ability of the Fc construct to interact with Fcγ receptors.

It was found that the inhibition of IL-8 release by THP-1 cells stimulated by plate-bound IgG was dependent on linker length (Fig. 8). At low concentrations of the Fc construct, the degree of inhibition followed the order _G8 < _G15 < _G20 , with IL-8 release by THP-1 cells being most strongly inhibited by Fc construct 2 containing the _G20 spacer.

In addition, inhibition of calcium flux in neutrophils was found to be dependent on linker length (Fig. 9). The degree of inhibition followed the order _G8 < _G15 < _G20 , with Fc construct 2 containing the _G20 spacer demonstrating the greatest degree of inhibition of calcium flux in neutrophils.

Example 6. Optimization of heterodimerization using protrusion-to-recess technology

Plasmids expressing long and short polypeptides of Fc construct 2 (polypeptides 102, 108, 114 and 116 in Fig. 1) or long and short polypeptides of Fc construct 4 (polypeptides 202, 208, 214 and 216 in Fig. 2) were transfected into HEK293 cell line. After seven days of culture, the cells were purified by centrifugation and the supernatant from the crude medium was separated by SDS-PAGE under non-reducing conditions (Fig. 10). Densitometric analysis of the visualized protein spots showed that Fc construct 2, containing three Fc domains, and Fc construct 4, containing three Fc domains (Fc3), were expressed at similar levels. However, in the case of Fc construct 2, the constructs showed significantly higher levels of dimer contaminant molecules (Fc2) (Figure 10). Both sets of constructs expressed similar levels of monomeric molecules (Fc1). Additional spots present in the image represent media components that are present in mock-transfected controls.

These results indicate that the presence of both electrostatic coordination mutations that promote heterodimerization and knob-to-groove mutations that promote heterodimerization in branch subunits (e.g., Fc domain monomers 106 , 114, 112 and 116 in Fig. 1; Fc domain monomers 206, 214, 212 and 216 in Fig. 2), provides an improvement regarding the formation of a heterodimeric Fc domain in an Fc construct, optimizing the assembly of an Fc construct containing three Fc domain, and improvement in homogeneity of the composition containing the Fc construct.

Example 7 Electrostatic coordination to control homodimerization

To minimize the association of subunits in unintended ways that result in the formation of undesirable high molecular weight oligomers and multimers, into “branch” subunits (e.g., Fc domain monomers 106, 112, 114, and 116 in Figure 1; Fc domain monomers 206, 212, 214 and 216 in Fig. 2) introduced mutations that promote heterodimerization (eg, peaks and valleys). These amino acid substitutions maintain the affinity of the knob subunits (e.g., Fc domain monomers 106 and 112 in Figure 1; Fc domain monomers 206 and 212 in Figure 2) with their trench counterparts (e.g., Fc domain monomers 114 and 116 in Fig. 1; Fc domain monomers 214 and 216 in Fig. 2) and at the same time prevent the association of subunits with protrusions. Because overhang mutations also inhibit assembly with wild-type Fc sequences, this calls into question the need to include additional mutations to further reduce the affinity of Fc stem subunits (e.g., Fc domain monomers 104 and 110 in Figure 1; monomers Fc domains 204 and 210 in Fig. 2) in relation to the “branch” subunits containing peaks and troughs. To address this issue, a long Fc polypeptide construct was created that contained a wild-type Fc domain monomer sequence in the carboxy-terminal “stem” subunit and an Fc domain monomer carrying overhang mutations in the amino-terminal “branch” subunit. The corresponding short polypeptide was a monomer of the Fc domain carrying mutations that ensured the formation of a cavity. This Fc construct is based on the polypeptide sequences in Fc construct 2, but contains the wild-type Fc domain monomer sequence in the carboxy-terminal stem subunit of each of the long polypeptides.

HEK293 cells were co-transfected with plasmids expressing Fc construct 2 (which contains mutations allowing homodimerization by electrostatic coordination in the Fc domain monomer in the carboxy-terminal stem subunit of each of the long polypeptides; see Tables 5 and 6 in Example 1) , or an Fc construct based on Fc construct 2 in which the Fc domain monomer in the carboxy-terminal stem subunit in each of the long polypeptides was replaced with a wild-type Fc domain monomer sequence (SEQ ID NO: 42) (as described above) . After seven days of culture, the cells were purified by centrifugation and the supernatant from the crude medium was separated by SDS-PAGE under non-reducing conditions. Visualization of the stained proteins showed that the Fc construct without the electrostatic coordination mutations in the stem subunits (labeled “no electrostatic coordination” (lanes 1-3) in Fig. 11) contained much higher levels of monomer (Fc1) and dimer (Fc2) than the analogue of Fc construct 2 (denoted “electrostatically coordinated” (lanes 4 and 5) in Fig. 11). In addition, many more spots with higher molecular weight than the trimer can be detected (lanes 1-3 in Fig. 11).

These results indicate that the presence of electrostatic coordination mutations that promote homodimerization in the stem subunits (e.g., Fc domain monomers 104 and 110 in Fig. 1; Fc domain monomers 204 and 210 in Fig. 2) is additional provides improvement in the formation of a homodimeric Fc domain in the Fc construct, optimization of the assembly of the Fc construct containing three Fc domains, and improvement in the homogeneity of the composition containing the Fc construct.

Example 8: Optimization of composition homogeneity by removing C-terminal lysine residues

The C-terminal lysine residue of immunoglobulins is highly conserved in many species. In some cases, C-terminal lysine residues in polypeptides are removed by cellular mechanisms during protein production. The present inventors sought to further improve the homogeneity of the Fc constructs in the composition and to provide a composition containing the Fc construct described herein with a greater degree of homogeneity by removing the C-terminal lysine from each of the polypeptides in the Fc construct. Fc construct 2 does not contain C-terminal lysine residues either in its long polypeptides (102 and 108; see example 1, tables 5-7; Fig. 1) or in its short polypeptides (114 and 116). Figure 12 shows that removal of the C-terminal lysine to produce Fc construct 2 does not induce complement dependent cytotoxicity (CDC) in vitro . Thus, by removing the C-terminal lysine residue, the present inventors were able to improve the homogeneity of the pharmaceutical composition of the Fc construct without causing adverse immunological side effects.

Example 9. Development of Fc constructs containing amino acid modifications I253 and/or R292

Fc constructs characterized by an altered (eg, extended) half-life have been developed based on construct 4 (M230) and contain amino acid modifications (eg, single mutations or combinations of mutations) that alter the binding affinity to FcRn (eg, reduce the degree of binding to FcRn, e.g., by including an amino acid modification at position I253, eg I253A) and/or which alters the binding affinity to FcRIIb (e.g., reducing the extent of binding to FcRIIb, e.g., by including an amino acid modification at position R292, eg, R292P) (Fig. 18A -18O).

Six Fc constructs were generated (FIG. 2, 18B, 18C, 18L, 18N, and 18O), each comprising a long polypeptide containing two Fc domain monomers separated by a spacer, e.g., an all-glycine linker, e.g., GGGGGGGGGGGGGGGGGGGG (SEQ ID NO: 23) and a short polypeptide containing one monomer of the Fc domain and characterized by different valence of amino acid modifications at positions I253 (for example, I253A) and/or R292 (for example, R292P). Each Fc construct was generated from an IgG1 Fc sequence, which included modifications to provide engineered cavity, engineered protrusion, and/or electrostatic coordination to control polypeptide assembly. DNA sequences encoding long and short polypeptides were optimized for expression in mammalian cells and cloned into the mammalian expression vector pcDNA3.4. Plasmid DNA constructs were transfected via liposomes into human embryonic kidney (HEK) 293 cells.

In the case of each Fc construct, co-expression of long and short polypeptides produces a branched molecule containing three Fc domains, with the C-terminal Fc monomers of the long polypeptides specifically associating with each other to form one C-terminal Fc domain, and N -terminal Fc monomers of long polypeptides specifically associate with short polypeptides to form two N-terminal Fc domains. Fc constructs 12-15, 24, 26 and 27 and their development are described in Table 10 and FIG. 2 and 18B-18O. The sequences used in each Fc construct are shown in Table 11, where each amino acid modification I253A and/or R292P is shown in bold and underlined.

In design 4, each of the long polypeptides contains one Fc domain monomer containing charged amino acids (D399K and K409D) at the CH3-CH3 interface, connected via a linker to a knob-containing (formed by modifications S354C and T366W) Fc domain monomer. The knob-containing Fc domain monomer contains an amino acid modification (E357K) that provides improved Fc domain assembly. Each of the short polypeptides contains an Fc domain monomer containing a cavity (formed by the Y349C/T366S/L368A/Y407V modifications). Short polypeptides also contain an amino acid modification (K370D), which improves the assembly of Fc domains. Construct 4 is formed by expressing the first and second polypeptides having the amino acid sequence of SEQ ID NO: 49, and the third and fourth polypeptides having the amino acid sequence of SEQ ID NO: 61.

Designs 13-27 (FIGS. 18A-18O) are identical to design 4, except for certain modifications to positions I253 and/or R292 described herein. Construct 5 is formed by expressing the first and second polypeptides having the amino acid sequence of SEQ ID NO: 62, and the third and fourth polypeptides having the amino acid sequence of SEQ ID NO: 61. Construct 6 is formed by expressing the first and second polypeptides having the amino acid sequence under SEQ ID NO: 64, as well as the third and fourth polypeptides having the amino acid sequence under SEQ ID NO: 57. Construct 7 is formed by expressing the first and second polypeptides having the amino acid sequence under SEQ ID NO: 65, as well as the third and fourth polypeptides having the amino acid sequence of SEQ ID NO: 57. Construct 8 is formed by expressing the first and second polypeptides having the amino acid sequence of SEQ ID NO: 66, as well as the third and fourth polypeptides having the amino acid sequence of SEQ ID NO: 61. Construct 9 is formed by expressing the first and second polypeptides having the amino acid sequence of SEQ ID NO: 67, and the third and fourth polypeptides having the amino acid sequence of SEQ ID NO: 61. Construct 10 is formed by expressing the first and second polypeptides having the amino acid sequence of SEQ ID NO: 68, as well as the third and fourth polypeptides having the amino acid sequence of SEQ ID NO: 57. Construct 11 is formed by expressing the first and second polypeptides having the amino acid sequence of SEQ ID NO: 69, as well as the third and fourth polypeptides having amino acid sequence of SEQ ID NO: 57. Construct 12 is formed by expressing the first and second polypeptides having the amino acid sequence of SEQ ID NO: 71, and the third and fourth polypeptides having the amino acid sequence of SEQ ID NO: 70. Construct 13 is formed by expression of the first and second polypeptides having the amino acid sequence of SEQ ID NO: 72, as well as the third and fourth polypeptides having the amino acid sequence of SEQ ID NO: 70. Construct 14 is formed by expressing the first and second polypeptides having the amino acid sequence of SEQ ID NO: : 74, as well as third and fourth polypeptides having the amino acid sequence of SEQ ID NO: 73. Construct 15 is formed by expressing the first and second polypeptides having the amino acid sequence of SEQ ID NO: 75, as well as the third and fourth polypeptides having the amino acid sequence under SEQ ID NO: 73. Construct 16 is formed by expressing the first and second polypeptides having the amino acid sequence of SEQ ID NO: 76, and the third and fourth polypeptides having the amino acid sequence of SEQ ID NO: 70. Construct 17 is formed by expressing the first and a second polypeptide having the amino acid sequence of SEQ ID NO: 77, and a third and fourth polypeptide having the amino acid sequence of SEQ ID NO: 70. Construct 18 is formed by expressing the first and second polypeptides having the amino acid sequence of SEQ ID NO: 78 , as well as third and fourth polypeptides having the amino acid sequence of SEQ ID NO: 73. Construct 19 is formed by expressing the first and second polypeptides having the amino acid sequence of SEQ ID NO: 79, as well as the third and fourth polypeptides having the amino acid sequence of SEQ ID NO: 73.

Table 10. Fc constructs containing amino acid modifications I253 and/or R292

Fc design Mutations affecting FcγRIIb binding ¹ Mutations affecting FcRn binding ² Long polypeptide nos. (SEQ ID NO) No. of short polypeptides (SEQ ID NO) Figure Fc-construction 4 Absent Absent 1202 and 1208 (SEQ ID NO: 49) 1214 and 1216 (SEQ ID NO: 61) Fig. 2 Fc-construction 5 Absent C-terminal Fc domain only 502 and 508 (SEQ ID NO: 62) 514 and 516 (SEQ ID NO: 61) Fig. 18A Fc design 6 Absent Two N-terminal Fc domains 602 and 608 (SEQ ID NO: 64) 614 and 616 (SEQ ID NO: 57) Fig. 18B Fc-construction 7 Absent All three Fc domains 702 and 708 (SEQ ID NO: 65) 714 and 716 (SEQ ID NO: 57) Fig. 18C Fc-construction 8 C-terminal Fc domain only Absent 802 and 808 (SEQ ID NO: 66) 814 and 816 (SEQ ID NO: 61) Fig. 18D Fc-construction 9 C-terminal Fc domain only C-terminal Fc domain only 902 and 908 (SEQ ID NO: 67) 914 and 916 (SEQ ID NO: 61) Fig. 18E Fc-construction 10 C-terminal Fc domain only Two N-terminal Fc domains 1002 and 1008 (SEQ ID NO: 68) 1014 and 1016 (SEQ ID NO: 57) Fig. 18F

Fc-construction 11 C-terminal Fc domain only All three Fc domains 1102 and 1108 (SEQ ID NO: 69) 1114 and 1116 (SEQ ID NO: 57) Fig. 18G Fc-construction 12 Two N-terminal Fc domains Absent 1202 and 1208 (SEQ ID NO: 71) 1214 and 1216 (SEQ ID NO: 70) Fig. 18H Fc-construction 13 Two N-terminal Fc domains C-terminal Fc domain only 1302 and 1308 (SEQ ID NO: 72) 1314 and 1316 (SEQ ID NO: 70) Fig. 18I Fc-construction 14 Two N-terminal Fc domains Two N-terminal Fc domains 1402 and 1408 (SEQ ID NO: 74) 1414 and 1416 (SEQ ID NO: 73) Fig. 18J Fc-construction 15 Two N-terminal Fc domains All three Fc domains 1502 and 1508 (SEQ ID NO: 75) 1514 and 1516 (SEQ ID NO: 73) Fig. 18K Fc-construction 16 All three Fc domains Absent 1602 and 1608 (SEQ ID NO: 76) 1614 and 1616 (SEQ ID NO: 70) Fig. 18L Fc-construction 17 All three Fc domains C-terminal Fc domain only 1702 and 1708 (SEQ ID NO: 77) 1714 and 1716 (SEQ ID NO: 70) Fig. 18M Fc-construction 18 All three Fc domains Two N-terminal Fc domains 1804 and 1808 (SEQ ID NO: 78) 1814 and 1816 (SEQ ID NO: 73) Fig. 18N Fc-construction 19 All three Fc domains All three Fc domains 1904 and 1908 (SEQ ID NO: 79) 1914 and 1916 (SEQ ID NO: 73) Fig. 18O ¹ R292P mutation
²mutation I253A

Table 11. Amino acid sequences

Amino acid sequence SEQ ID NO: 61 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVDGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 49 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDKLTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGGGGGGGGGGGGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP SRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 62 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDKLTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGGGGGGGGGGGGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLM A SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 57 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLM A SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVDGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 64 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLM A SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDKLTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGGGGGGGGGGGGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 65 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLM A SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDKLTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGGGGGGGGGGGGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLM A SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY TLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 66 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDKLTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGGGGGGGGGGGGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP P EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY TLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 67 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDKLTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK A YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 68 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLM A SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDKLTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGGGGGGGGGGGGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP P EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 69 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLM A SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDKLTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGGGGGGGGGGGGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLM A SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP P EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 70 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP P EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVDGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 71 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP P EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVDGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 72 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP P EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDKLTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGGGGGGGGGGGGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLM A SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 73 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLM A SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP P EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVDGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 74 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLM A SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP P EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDKLTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGGGGGGGGGGGGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 75 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLM A SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP P EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDKLTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGGGGGGGGGGGGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLM A SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 76 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP P EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDKLTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGGGGGGGGGGGGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP P EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 77 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP P EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDKLTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY A QVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 78 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLM A SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP P EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDKLTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGGGGGGGGGGGGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP P EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP QVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 79 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLM A SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP P EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDKLTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGGGGGGGGGGGGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLM A SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP P EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG I253A and R292P mutations (modifications) are in bold and underlined (if present)

Example 10. Evaluation of the effect of amino acid modifications I253A and R292P on the specificity of binding to the Fc receptor

Cell-based binding assays were used to confirm that amino acid modifications at positions I253, eg I253A, and R292, eg R292P, are specific for the putative receptors, eg reduced binding affinity for the FcRn receptor and the FcγRIIb receptor, respectively. The relative binding of Fc constructs and controls to Fc gamma receptors (FcγRs) was measured using time-resolved fluorescence resonance energy transfer (TR-FRET) homogeneous cell competition assays (CISBIO®) for the FcγRI, FcγRIIa H131, FcγRIIb and FcγRIIIa V158. Assay reagents were prepared according to the manufacturer's instructions. Using an automatic liquid handler (Freedom EVOware 150, TECAN®), a series of 3-fold serial dilutions with 10 points was prepared, plus one blank per sample. Assay plates were read on a PHERAstar fluorescence reader (BMG Labtech GmbH) at 665 and 620 nm. The IgG1 sample was used as a control. 19 A-D shows that the R292P mutations in construct 16 and construct 18 dramatically reduce binding to FcγRIIb-expressing cells compared to IgG1 controls, while having minimal effect on binding of FcγRI, FcγRIIa, and FcγRIIIa. However, the I253A mutations had minimal effect on binding to any Fc-gamma receptor, as evidenced by the similarity of the binding profiles of construct 6 and construct 4 and the similarity of the binding profiles of construct 18 and construct 16.

To evaluate the effect of amino acid modifications at position I253, such as I253A, and R292, such as R292P, on FcRn binding, a surface plasmon resonance (SPR) binding experiment was designed to measure the affinity and normalized binding level of liquid-phase human FcRn to sensor-bound Fc - designs at pH 6.0. The goat anti-human IgG antibody was immobilized on the surfaces of the control and test sensors using the chemical principles of the amine immobilization reaction. Fc constructs were captured on the surface of the sensor under study. Recombinant human FcRn was passed over the sensor surface in a series of dilutions with a maximum concentration of 1.0 μM. The sensor surface was restored at the end of each cycle using 10 mM glycine at pH 1.7. Sensograms with the subtraction of two control values were subjected to equilibrium binding analysis; The maximum level of binding (RMax) and the equilibrium dissociation constant ( _KD ) were assessed. The normalized maximum binding level was calculated by dividing RMax by the uptake level of the FC construct. Construct 16 was captured at the same level as construct 4, indicating no loss of FcRn binding as expected (Figure 20). Construct 6 and Construct 18 showed significantly reduced uptake levels, consistent with the two Fc domains losing affinity for FcRn as predicted (Figure 20). Similarly, construct 7 and construct 19, which had mutations in all three domains to limit FcRn binding, did not bind to FcRn (Fig. 20).

Together, these data demonstrate that mutation to reduce binding to FcγRIIb (eg, an amino acid modification at position R292, eg, R292P) had the intended effect, with little effect on binding to other Fc-gamma receptors and minimal effect on FcRn binding. Likewise, a mutation to reduce FcRn binding (eg, an amino acid modification at position I253, eg, I253A) had the desired effect with minimal impact on Fc-gamma receptor binding. In addition, two mutations (eg, I253 and R292 mutations, such as I253A and R292P) can be combined to achieve reduced binding of the Fc construct to both FcγRIIb and FcRn.

Example 11. Evaluation of the effect of amino acid modifications I253A and R292P on the pharmacokinetics of Fc receptors in mice

The effect of binding-related mutations on pharmacokinetics was initially assessed by comparing construct 4, construct 16, which caused decreased binding to FcγRIIb in all three Fc domains, and construct 6, which caused decreased binding to FcRn in two Fc domains. IVIg was included as a reference sample demonstrating typical IgG behavior. Female C57BL/6 mice (n=12, 8-10 weeks) were treated intravenously (iv) with 0.1 g/kg each of construct 4, construct 6, construct 16, or IVIg. Blood samples (50 μl) were collected from the saphenous veins, from four mice per time point, at alternating time intervals of 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 1 day, 2 days, 3 days, 4 days and 5 days. All mice were bled on days 7, 9, 11, 14, 16, 21, and 23. Serum concentrations of the Fc construct and IVIg were determined using an Fc-specific ELISA with human IgG1. As shown in FIG. 21, both Fc construct 6 and Fc construct 16 exhibited longer persistence in vivo and resulted in a 1.2- to 1.5-fold increase in mean retention time (MRT) (previous increases (e.g., three- to four-fold) measured by area under the curve (AUC)).

To further study the effect of the amino acid modification I253A, which causes a change in FcRn valence, on pharmacokinetics, three compounds with one, two or three domains with reduced affinity for FcRn were compared, as described above. As can be seen in FIG. 22, all mutants persisted longer than the parent compound. The mean retention time (MRT) consistently increased with each Fc domain modified to reduce affinity for FcRn. Drug exposure can also be measured using AUC.

The effect of combining mutations was also studied (Fig. 23). Incorporation of mutations in both receptors increased MRT threefold compared with the twofold increase achieved in this study by reducing binding to FcγRIIb. Drug exposure can also be measured using AUC, such as a sixfold increase.

Example 12. Evaluation of the effect of amino acid modifications I253A and R292P on efficacy in vitro

The impact of binding-affecting mutations on in vitro efficacy was assessed using assays previously shown to be sensitive to Fc-gamma receptor valence. Inhibition of phagocytosis in THP-1 cells was comparable for all Fc constructs (Fig. 24). THP-1 cells were seeded in 96-well plates. Fc constructs were subjected to a 10-fold serial dilution and added to the cells. Incubation was continued for 15 minutes. at 37°C; then latex beads coated with rabbit IgG labeled with FITC (Cayman Chemical) were added to the cells. Incubation was continued for 3 hours at 37°C. Cells were washed twice and resuspended in 100 μl FACS buffer (PBS/2% FBS); Trypan blue was added to quench the FITC signal at the cell surface and samples were read on a BD Canto flow cytometer. Data are presented as the percentage of FITC-positive cells compared to the total cell population in the absence of inhibitor (100% phagocytosis).

The release of IL-8 by plate-bound IgG-stimulated monocytes was inhibited by all Fc constructs with comparable potency (FIG. 25). PBMCs were isolated from buffy coats of healthy human donors (Research Blood Components) by density gradient centrifugation in Ficoll-Paque Plus medium (GE Healthcare Life Sciences). Monocytes were isolated by negative selection using a human monocyte enrichment kit without CD16 depletion (StemCell Technologies). FcγR-mediated cytokine production was stimulated using 96-well plates coated overnight with 50 μl of 100 μg/ml human IgG1 (SouthernBiotech). IVIg or each Fc construct was serially diluted in a culture plate at a concentration twice the final concentration in the assay. Purified monocytes (1.5 x 105 cells/well) were added, incubated for 24 hours, and culture supernatants were assayed for IL-8 concentration (High Concentration IL-8 Human Cytokine Kit, Meso Scale Discovery).

Likewise, ADCC inhibition was comparable for all Fc constructs (FIG. 26). NK cells (Hemacare) were thawed and stored overnight in LGM-3 medium (Lonza). Cultured Raji cells were incubated in the presence of various concentrations of each test compound and rituximab (2 μg/ml) for 30 minutes before the addition of NK cells at an effector:target cell ratio of 50K:10K. NK cells and Raji cells were also seeded in the presence of separate probes. Cells were incubated for 6 hours. Cytotox-Glo was used as a reading agent.

In all analyses, mutations caused a negligible decrease in efficacy.

Example 13: Evaluation of the effect of amino acid modifications I253A and R292P on in vivo efficacy

To further evaluate the effect of mutations affecting receptor binding on the activity of the Fc construct, it was examined in vivo using a collagen antibody-induced arthritis (CAIA) model. Male C57BL/6 mice were injected intraperitoneally with an arthritogenic monoclonal antibody cocktail consisting of four anti-collagen II antibodies (ArthritoMab, MDBiosciences; 8 mg). On day 4, the animals were administered lipopolysaccharide (100 μg) intravenously. At treatment dosing on day 6, animals were randomized based on disease severity into study groups, excluding animals with poor disease induction (score 0 on day of randomization), and administered IV vehicle or test substance. For prophylactic dosing, animals were given an IV dose of vehicle or test compound once per day from day 1 to day 14 (days were numbered without a zero). Clinical scoring of parameters was as follows: 0 = normal, no swelling, redness or deformity; full joint flexibility. 1 = mild arthritis: mild swelling and/or deformity; full joint flexibility. 2 = moderate arthritis: moderate swelling and/or deformity; decreased joint flexibility or grip strength. 3 = severe arthritis: severe swelling and/or deformity; severely reduced joint flexibility or grip strength. 4 = ankylosed joints; no joint flexibility and severely impaired movements; terminal period. Animals were sacrificed after 12 days.

As shown in FIG. 27, decreased binding to FcγRIIb (construct 16), FcRn (construct 6), or both (construct 18) had minimal effect on efficacy compared to the parent molecule (construct 4).

Example 14. Evaluation of the effect of amino acid modifications I253A and R292P on the duration of response in vivo

For the CAIA model, mice were prophylactically treated for no more than 14 days before injection of arthritogenic antibodies. As can be seen in FIG. 28, prophylactic treatment on day 1 was more effective with slower clearance Q1 (design 18) than with design 4, likely due to the longer period of drug exposure throughout the multi-day disease induction. Compound 4 loses effectiveness when dosed 3 days before disease induction (FIG. 29), whereas Q1 (design 18) provides protection even when dosed 7 days before disease induction (FIG. 30). Both designs were ineffective when dosed 10 days before disease induction (FIG. 31). These results indicate that the pharmacokinetic profile of construct 18, which covers a longer period of time, provides greater durability of response compared to construct 4.

Prophylactic dosing in the CAIA model was performed using 100 mg/kg of construct 4 (parent molecule), construct 18 (I253A in two domains and R292P in three domains) (Q1), or construct 19 (I253A and R292P in all three domains) (Q2) on day 1 (Fig. 32), day 3 (Fig. 33), day 7 (Fig. 34) or day 10 (Fig. 35). Vehicle control (saline) was administered on day 1 only.

In fig. Figure 32 shows a graph comparing the effectiveness of Design 4 (AA: black squares, solid line), Design 18 (Q1: black triangles, dashed line), Design 19 (Q2: black diamonds, dotted line) or saline (gray circles, dashed line). at a dose of 100 mg/kg on day 1 in the collagen antibody-induced arthritis (CAIA) model. In fig. 33 is a graph comparing Design 4 (AA: black squares, solid line), Design 18 (Q1: black triangles, dashed line), or Design 19 (Q2: black diamonds, dotted line) administered at 100 mg/kg per day. 3. In FIG. 34 is a graph comparing Design 4 (AA: black squares, solid line), Design 18 (Q1: black triangles, dashed line), or Design 19 (Q2: black diamonds, dotted line) administered at 100 mg/kg per day. 7. In FIG. 35 is a graph comparing Design 4 (AA: black squares, solid line), Design 18 (Q1: black triangles, dashed line), or Design 19 (Q2: black diamonds, dotted line) administered at 100 mg/kg per day. 10. In each case, an equivalent volume of saline (gray circles, dashed line) was administered on day 1. The mean and standard error of the mean are shown for each time point.

Example 15. Assessment of the effect of amino acid modifications I253A and R292P on Fc multimers

Fc multimers were prepared as described in (Strome et al, US 2010/0239633 A1; Jain et al, Arthritis Res. Ther. 14, R192 (2012)). Specifically, the IgG1 Fc was fused at the C-terminus to the IgG2 hinge sequence. DNA constructs were generated using the wild-type IgG1 Fc sequence (construct X1) and using the double mutant I253A/R292P (construct X2). Plasmid DNA-based constructs were transfected via liposomes into HEK293 cells. After seven days of culture, the cells were purified by centrifugation.

Expressed proteins were purified from cell culture supernatant by protein A binding affinity column chromatography using a Poros MabCapture A column. Captured constructs were washed with phosphate-buffered saline (low salt wash) and eluted using 100 mM glycine pH 3. The eluate was quickly neutralized by adding 1 M TRIS pH 7.4 and filtered under sterile conditions through a 0.2 µm filter.

The proteins were then fractionated by ion exchange chromatography using Poros XS resin. The column was pre-equilibrated with 50 mM MES, pH 6 (buffer A) and the sample was diluted in equilibration buffer before loading. The sample was eluted using a multi-step gradient with 50 mM MES, 400 mM sodium chloride, pH 6 (buffer B) as elution buffer. The gradient steps included changing the B concentration from 0 to 40% for a run of 2 column volumes (CV) to remove low molecular weight species, a hold step at 40% B (4 CV), then changing the B concentration from 40 to 80% (4 CV) to isolation of target molecules and then linear increase to 100% B. All protein-containing fractions were screened using analytical size exclusion chromatography and components were quantified by absorbance at 280 nm. Fractions with total Fc content (approximately 50 kDa) together with Fc dimers (approximately 100 kDa) greater than 8% were excluded. In the case of design X1, all remaining fractions were combined. Due to the shift in molecular weight distribution between constructs X1 and X2, fractions of construct X2 were chosen to mimic the molecular weight distribution of construct X1.

After ion exchange, the target fraction buffer was replaced with PBS buffer using a polyethersulfone (PES) membrane cartridge with a cutoff limit of 30 kDa on a tangential flow filtration system. Samples were concentrated to approximately 30 mg/mL and filtered under sterile conditions through a 0.2 μm filter.

The molecular weight distributions for construct 4 and construct X1 were compared using analytical size exclusion chromatography (FIG. 36) (purified constructs X1 (gray) and X2 (black) normalized to peak maxima) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (FIG. 37) (purified construct X1 (right), purified construct X2 (center), and molecular weight standards (right). Equal masses of the two constructs were loaded). Constructs X1 and X2 consist of different molecules ranging from ~100 kDa (two Fc domains), with a large number of spots located above the 250 kDa value, and neither component is predominant. The size distribution for construct X2 is similar to that for construct X1, but is slightly wider with more components of both the highest and lowest molecular weight.

Female C57BL/6 mice (n=15, 8 weeks) were injected intravenously (iv) with 0.1 g/kg of each of construct X1 or construct 4. Blood (25 μl) was collected from the submandibular vein and processed to obtain serum. Five mice from each group were bled at different time points during day 5, while at all other time points blood was collected from all fifteen mice in each group. Time points at which sampling was performed included 15 and 30 minutes; 1, 2, 4, 6, 8, 24 hours; 2 days. Serum concentrations of Fc multimers were determined by anti-human IgG ELISA using an Fc-specific identifying antibody.

As shown in FIG. 38, at each time point, serum levels of construct X1 are lower than levels of construct X2. Introduction of the I253A and R292P mutations into construct X2 delayed the clearance of Fc multimers compared with the corresponding wild-type material (construct X1).

Example 16. Evaluation of the effect of amino acid modifications I253A and R292P on the pharmacokinetics of Fc receptors in cynomolgus macaques

The effect of binding-related mutations on pharmacokinetics in cynomolgus monkeys was assessed by comparing constructs 6, 16, and 18. Historical data for construct 4 come from a study in cynomolgus monkeys performed at different dose levels, as opposed to a study in which comparison of constructs 6, 16 and 18. Male cynomolgus macaques (N=3) were administered 10 or 30 mg/kg of each of constructs 6, 16 and 18 intravenously. Blood samples were collected over 44 days. Fc construct concentrations were determined by ELISA using antibodies specific for human IgG1-based Fc constructs, including constructs 4, 6, 16, and 18.

As shown in FIG. 39, construct 16, which was administered at a dose of 10 mg/kg, persists longer in vivo than construct 4, which was administered at a dose of 20 mg/kg. Construct 18, which was administered at a dose of 10 mg/kg, has the same persistence in vivo as construct 4, which was administered at a dose of 20 mg/kg. Construct 6, which was administered at a dose of 10 mg/kg, did not persist as long as construct 4, which was administered at a dose of 20 mg/kg.

As shown in FIG. 40, construct 16, which was administered at a dose of 30 mg/kg, persists in vivo longer than construct 4, which was administered at a dose of 20 or 50 mg/kg. The persistence value of construct 18, which was administered at a dose of 30 mg/kg, is intermediate between the persistence value of construct 4, which was administered at a dose of 20 or 50 mg/kg. Construct 6, which was administered at a dose of 30 mg/kg, did not persist in vivo as long as construct 4, which was administered at a dose of 20 or 50 mg/kg.

It is noteworthy that the pharmacokinetic properties in the case of cynomolgus monkeys differ from those in the case of mice. In mice, decreasing the binding of both FcRn and Fc-gamma-RIIb led to increased persistence, and the combination of both led to a further increase in persistence. In cynomolgus monkeys, a decrease in FcRn resulted in decreased persistence, a decrease in Fc-gamma-RIIb resulted in increased persistence, and their combination provided a small net change in persistence compared to the parent molecule (design 4).

Other embodiments

All publications, patents and patent applications mentioned in the present invention are incorporated herein by reference to the same extent as if each individual publication or patent application were specifically and separately identified as being incorporated by reference.

Although the present invention has been described in connection with specific embodiments, it will be understood that further modifications are possible, and this application is intended to cover any variations, uses, or adaptations of the present disclosure generally following the teachings of the present disclosure. and including such departures from the present disclosure as are known or customary practice within the field of technology to which the present invention belongs and which may be applied to the essential features set forth herein above.

Other embodiments are within the scope of the claims.

--->

LIST OF SEQUENCES

<110> Momenta Pharmaceuticals, Inc.

<120> COMPOSITIONS AND METHODS RELATED TO CONSTRUCTED

Fc-CONSTRUCTIONS

<130> 14131-0150WO1

<150>US 62/589,473

<151> 2017-11-21

<150> US 62/510,228

<151> 2017-05-23

<150> US 62/443,495

<151> 2017-01-06

<160> 80

<170> PatentIn version 3.5

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Gly Gly Gly Gly Ser

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Gly Gly Ser Gly

1

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Ser Gly Gly Gly

1

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Gly Ser Gly Ser

1

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Gly Ser Gly Ser Gly Ser

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Gly Ser Gly Ser Gly Ser Gly Ser

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Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser

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Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser

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Gly Gly Ser Gly Gly Ser

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Gly Gly Ser Gly Gly Ser Gly Gly Ser

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Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser

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Gly Gly Ser Gly Gly Gly Ser Gly

15

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Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly

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Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly

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Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly

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Gly Gly Ser Gly

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Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

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Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

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Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly

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Ser Gly Gly Gly

20

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Gly Gly Gly Gly

1

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Gly Gly Gly Gly Gly Gly Gly Gly

15

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Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly

1 5 10

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Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly

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Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly

1 5 10 15

Gly Gly Gly Gly

20

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Gly Gly Gly Gly Gly

15

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Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly

1 5 10

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<400> 26

Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly

1 5 10 15

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Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly

1 5 10 15

Gly Gly Gly Gly

20

<210> 28

<211> 10

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Gly Glu Asn Leu Tyr Phe Gln Ser Gly Gly

1 5 10

<210> 29

<211> 8

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<400> 29

Ser Ala Cys Tyr Cys Glu Leu Ser

15

<210> 30

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<400> 30

Arg Ser Ile Ala Thr

15

<210> 31

<211> 17

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Arg Pro Ala Cys Lys Ile Pro Asn Asp Leu Lys Gln Lys Val Met Asn

1 5 10 15

His

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<211> 36

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<400> 32

Gly Gly Ser Ala Gly Gly Ser Gly Ser Gly Ser Ser Gly Gly Ser Ser

1 5 10 15

Gly Ala Ser Gly Thr Gly Thr Ala Gly Gly Thr Gly Ser Gly Ser Gly

20 25 30

Thr Gly Ser Gly

35

<210> 33

<211> 17

<212> PROTEIN

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<400> 33

Ala Ala Ala Asn Ser Ser Ile Asp Leu Ile Ser Val Pro Val Asp Ser

1 5 10 15

Arg

<210> 34

<211> 36

<212> PROTEIN

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<400> 34

Gly Gly Ser Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly

1 5 10 15

Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser

20 25 30

Gly Gly Gly Ser

35

<210> 35

<211> 12

<212> PROTEIN

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<400> 35

Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser

1 5 10

<210> 36

<211> 18

<212> PROTEIN

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<400> 36

Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly

1 5 10 15

Gly Ser

<210> 37

<211> 11

<212> PROTEIN

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<220>

<223> Albumin-binding peptide

<400> 37

Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp

1 5 10

<210> 38

<211> 6

<212> PROTEIN

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<220>

<223> Hexahistidine peptide

<400> 38

His His His His His

15

<210> 39

<211> 8

<212> PROTEIN

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<220>

<223> FLAG peptide

<400> 39

Asp Tyr Lys Asp Asp Asp Asp Lys

15

<210> 40

<211> 10

<212> PROTEIN

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<220>

<223> Myc Peptide

<400> 40

Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu

1 5 10

<210> 41

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Peptide HA

<400> 41

Tyr Pro Tyr Asp Val Pro Asp Tyr Ala

15

<210> 42

<211> 227

<212> PROTEIN

<213> Homo sapiens

<400> 42

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

1 5 10 15

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Lys

225

<210> 43

<211> 474

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Long chain polypeptide

<400> 43

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

1 5 10 15

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Lys Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser

225 230 235 240

Gly Gly Gly Ser Gly Gly Gly Asp Lys Thr His Thr Cys Pro Pro Cys

245 250 255

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

260 265 270

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

275 280 285

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

290 295 300

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

305 310 315 320

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

325 330 335

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

340 345 350

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

355 360 365

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

370 375 380

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

385 390 395 400

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

405 410 415

Asn Tyr Lys Thr Thr Pro Pro Val Leu Lys Ser Asp Gly Ser Phe Phe

420 425 430

Leu Tyr Ser Asp Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

435 440 445

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

450 455 460

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

465 470

<210> 44

<211> 227

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Fc domain monomer, short chain polypeptide

<400> 44

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

1 5 10 15

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Lys

225

<210> 45

<211> 473

<212> PROTEIN

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<220>

<223> Long chain polypeptide

<400> 45

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

1 5 10 15

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Lys Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser

225 230 235 240

Gly Gly Gly Ser Gly Gly Gly Asp Lys Thr His Thr Cys Pro Pro Cys

245 250 255

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

260 265 270

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

275 280 285

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

290 295 300

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

305 310 315 320

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

325 330 335

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

340 345 350

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

355 360 365

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

370 375 380

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

385 390 395 400

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

405 410 415

Asn Tyr Lys Thr Thr Pro Pro Val Leu Lys Ser Asp Gly Ser Phe Phe

420 425 430

Leu Tyr Ser Asp Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

435 440 445

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

450 455 460

Gln Lys Ser Leu Ser Leu Ser Pro Gly

465 470

<210> 46

<211> 226

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Fc domain monomer, short chain polypeptide

<400> 46

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

1 5 10 15

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly

225

<210> 47

<211> 468

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Long chain polypeptide

<400> 47

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

1 5 10 15

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Cys Arg Asp Lys Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Lys Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly

225 230 235 240

Gly Gly Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu

245 250 255

Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

260 265 270

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

275 280 285

Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val

290 295 300

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser

305 310 315 320

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

325 330 335

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala

340 345 350

Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

355 360 365

Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln

370 375 380

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

385 390 395 400

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

405 410 415

Pro Pro Val Leu Lys Ser Asp Gly Ser Phe Phe Leu Tyr Ser Asp Leu

420 425 430

Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser

435 440 445

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

450 455 460

Leu Ser Pro Gly

465

<210> 48

<211> 226

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Fc domain monomer, short chain polypeptide

<400> 48

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

1 5 10 15

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Ser Cys Ala Val Asp Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly

225

<210> 49

<211> 473

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Long polypeptide, design 4/AA

<400> 49

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

1 5 10 15

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Cys Arg Asp Lys Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Lys Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly

225 230 235 240

Gly Gly Gly Gly Gly Gly Gly Asp Lys Thr His Thr Cys Pro Pro Cys

245 250 255

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

260 265 270

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

275 280 285

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

290 295 300

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

305 310 315 320

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

325 330 335

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

340 345 350

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

355 360 365

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

370 375 380

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

385 390 395 400

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

405 410 415

Asn Tyr Lys Thr Thr Pro Pro Val Leu Lys Ser Asp Gly Ser Phe Phe

420 425 430

Leu Tyr Ser Asp Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

435 440 445

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

450 455 460

Gln Lys Ser Leu Ser Leu Ser Pro Gly

465 470

<210> 50

<211> 227

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Fc domain monomer, short chain polypeptide

<400> 50

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

1 5 10 15

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Lys

225

<210> 51

<211> 227

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Fc domain monomer, short chain polypeptide

<400> 51

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

1 5 10 15

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Lys Ser Asp Gly Ser Phe Phe Leu Tyr Ser Asp Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Lys

225

<210> 52

<211> 225

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Fc domain monomer, short chain polypeptide

<400> 52

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

1 5 10 15

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Lys Ser Asp Gly Ser Phe Phe Leu Tyr Ser Asp Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro

225

<210> 53

<211> 227

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Fc domain monomer, short chain polypeptide

<400> 53

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

1 5 10 15

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Cys Arg Asp Lys Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Lys

225

<210> 54

<211> 474

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Fc-construct of a long polypeptide with an N-terminal Asp substitution

on Gln

<400> 54

Gln Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

1 5 10 15

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Lys Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser

225 230 235 240

Gly Gly Gly Ser Gly Gly Gly Asp Lys Thr His Thr Cys Pro Pro Cys

245 250 255

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

260 265 270

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

275 280 285

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

290 295 300

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

305 310 315 320

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

325 330 335

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

340 345 350

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

355 360 365

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

370 375 380

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

385 390 395 400

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

405 410 415

Asn Tyr Lys Thr Thr Pro Pro Val Leu Lys Ser Asp Gly Ser Phe Phe

420 425 430

Leu Tyr Ser Asp Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

435 440 445

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

450 455 460

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

465 470

<210> 55

<211> 227

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Fc-construct of a short polypeptide with an N-terminal Asp substitution

on Gln

<400> 55

Gln Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

1 5 10 15

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Lys

225

<210> 56

<211> 473

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Fc-construct of a long polypeptide with an N-terminal Asp substitution

on Gln

<400> 56

Gln Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

1 5 10 15

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Lys Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser

225 230 235 240

Gly Gly Gly Ser Gly Gly Gly Asp Lys Thr His Thr Cys Pro Pro Cys

245 250 255

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

260 265 270

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

275 280 285

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

290 295 300

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

305 310 315 320

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

325 330 335

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

340 345 350

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

355 360 365

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

370 375 380

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

385 390 395 400

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

405 410 415

Asn Tyr Lys Thr Thr Pro Pro Val Leu Lys Ser Asp Gly Ser Phe Phe

420 425 430

Leu Tyr Ser Asp Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

435 440 445

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

450 455 460

Gln Lys Ser Leu Ser Leu Ser Pro Gly

465 470

<210> 57

<211> 226

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Fc-construct of a short polypeptide with an N-terminal Asp substitution

on Gln

<400> 57

Gln Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

1 5 10 15

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly

225

<210> 58

<211> 468

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Fc-construct of a long polypeptide with an N-terminal Asp substitution

on Gln

<400> 58

Gln Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

1 5 10 15

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Cys Arg Asp Lys Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Lys Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly

225 230 235 240

Gly Gly Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu

245 250 255

Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

260 265 270

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

275 280 285

Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val

290 295 300

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser

305 310 315 320

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

325 330 335

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala

340 345 350

Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

355 360 365

Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln

370 375 380

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

385 390 395 400

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

405 410 415

Pro Pro Val Leu Lys Ser Asp Gly Ser Phe Phe Leu Tyr Ser Asp Leu

420 425 430

Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser

435 440 445

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

450 455 460

Leu Ser Pro Gly

465

<210> 59

<211> 226

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Fc-construct of a short polypeptide with an N-terminal Asp substitution

on Gln

<400> 59

Gln Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

1 5 10 15

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Ser Cys Ala Val Asp Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly

225

<210> 60

<211> 473

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Fc-construct of a long polypeptide with an N-terminal Asp substitution

on Gln

<400> 60

Gln Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

1 5 10 15

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Cys Arg Asp Lys Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Lys Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly

225 230 235 240

Gly Gly Gly Gly Gly Gly Gly Asp Lys Thr His Thr Cys Pro Pro Cys

245 250 255

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

260 265 270

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

275 280 285

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

290 295 300

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

305 310 315 320

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

325 330 335

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

340 345 350

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

355 360 365

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

370 375 380

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

385 390 395 400

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

405 410 415

Asn Tyr Lys Thr Thr Pro Pro Val Leu Lys Ser Asp Gly Ser Phe Phe

420 425 430

Leu Tyr Ser Asp Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

435 440 445

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

450 455 460

Gln Lys Ser Leu Ser Leu Ser Pro Gly

465 470

<210> 61

<211> 226

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Short polypeptide

<400> 61

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

1 5 10 15

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Ser Cys Ala Val Asp Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly

225

<210> 62

<211> 473

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Long polypeptide

<400> 62

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

1 5 10 15

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Cys Arg Asp Lys Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Lys Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly

225 230 235 240

Gly Gly Gly Gly Gly Gly Gly Asp Lys Thr His Thr Cys Pro Pro Cys

245 250 255

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

260 265 270

Lys Pro Lys Asp Thr Leu Met Ala Ser Arg Thr Pro Glu Val Thr Cys

275 280 285

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

290 295 300

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

305 310 315 320

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

325 330 335

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

340 345 350

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

355 360 365

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

370 375 380

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

385 390 395 400

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

405 410 415

Asn Tyr Lys Thr Thr Pro Pro Val Leu Lys Ser Asp Gly Ser Phe Phe

420 425 430

Leu Tyr Ser Asp Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

435 440 445

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

450 455 460

Gln Lys Ser Leu Ser Leu Ser Pro Gly

465 470

<210> 63

<211> 226

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Short polypeptide, design 6/A1

<400> 63

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

1 5 10 15

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ala Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Ser Cys Ala Val Asp Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly

225

<210> 64

<211> 473

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Long peptide, design 6/A1

<400> 64

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

1 5 10 15

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ala Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Cys Arg Asp Lys Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Lys Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly

225 230 235 240

Gly Gly Gly Gly Gly Gly Gly Asp Lys Thr His Thr Cys Pro Pro Cys

245 250 255

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

260 265 270

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

275 280 285

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

290 295 300

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

305 310 315 320

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

325 330 335

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

340 345 350

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

355 360 365

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

370 375 380

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

385 390 395 400

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

405 410 415

Asn Tyr Lys Thr Thr Pro Pro Val Leu Lys Ser Asp Gly Ser Phe Phe

420 425 430

Leu Tyr Ser Asp Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

435 440 445

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

450 455 460

Gln Lys Ser Leu Ser Leu Ser Pro Gly

465 470

<210> 65

<211> 473

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Long polypeptide

<400> 65

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

1 5 10 15

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ala Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Cys Arg Asp Lys Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Lys Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly

225 230 235 240

Gly Gly Gly Gly Gly Gly Gly Asp Lys Thr His Thr Cys Pro Pro Cys

245 250 255

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

260 265 270

Lys Pro Lys Asp Thr Leu Met Ala Ser Arg Thr Pro Glu Val Thr Cys

275 280 285

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

290 295 300

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

305 310 315 320

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

325 330 335

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

340 345 350

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

355 360 365

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

370 375 380

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

385 390 395 400

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

405 410 415

Asn Tyr Lys Thr Thr Pro Pro Val Leu Lys Ser Asp Gly Ser Phe Phe

420 425 430

Leu Tyr Ser Asp Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

435 440 445

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

450 455 460

Gln Lys Ser Leu Ser Leu Ser Pro Gly

465 470

<210> 66

<211> 473

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Long polypeptide

<400> 66

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

1 5 10 15

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Cys Arg Asp Lys Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Lys Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly

225 230 235 240

Gly Gly Gly Gly Gly Gly Gly Asp Lys Thr His Thr Cys Pro Pro Cys

245 250 255

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

260 265 270

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

275 280 285

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

290 295 300

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Pro Glu

305 310 315 320

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

325 330 335

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

340 345 350

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

355 360 365

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

370 375 380

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

385 390 395 400

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

405 410 415

Asn Tyr Lys Thr Thr Pro Pro Val Leu Lys Ser Asp Gly Ser Phe Phe

420 425 430

Leu Tyr Ser Asp Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

435 440 445

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

450 455 460

Gln Lys Ser Leu Ser Leu Ser Pro Gly

465 470

<210> 67

<211> 473

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Long polypeptide

<400> 67

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

1 5 10 15

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Cys Arg Asp Lys Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Lys Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly

225 230 235 240

Gly Gly Gly Gly Gly Gly Gly Asp Lys Thr His Thr Cys Pro Pro Cys

245 250 255

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

260 265 270

Lys Pro Lys Asp Thr Leu Met Ala Ser Arg Thr Pro Glu Val Thr Cys

275 280 285

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

290 295 300

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Pro Glu

305 310 315 320

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

325 330 335

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

340 345 350

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

355 360 365

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

370 375 380

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

385 390 395 400

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

405 410 415

Asn Tyr Lys Thr Thr Pro Pro Val Leu Lys Ser Asp Gly Ser Phe Phe

420 425 430

Leu Tyr Ser Asp Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

435 440 445

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

450 455 460

Gln Lys Ser Leu Ser Leu Ser Pro Gly

465 470

<210> 68

<211> 473

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Long protein

<400> 68

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

1 5 10 15

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ala Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Cys Arg Asp Lys Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Lys Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly

225 230 235 240

Gly Gly Gly Gly Gly Gly Gly Asp Lys Thr His Thr Cys Pro Pro Cys

245 250 255

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

260 265 270

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

275 280 285

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

290 295 300

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Pro Glu

305 310 315 320

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

325 330 335

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

340 345 350

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

355 360 365

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

370 375 380

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

385 390 395 400

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

405 410 415

Asn Tyr Lys Thr Thr Pro Pro Val Leu Lys Ser Asp Gly Ser Phe Phe

420 425 430

Leu Tyr Ser Asp Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

435 440 445

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

450 455 460

Gln Lys Ser Leu Ser Leu Ser Pro Gly

465 470

<210> 69

<211> 473

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Long polypeptide

<400> 69

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

1 5 10 15

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ala Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Cys Arg Asp Lys Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Lys Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly

225 230 235 240

Gly Gly Gly Gly Gly Gly Gly Asp Lys Thr His Thr Cys Pro Pro Cys

245 250 255

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

260 265 270

Lys Pro Lys Asp Thr Leu Met Ala Ser Arg Thr Pro Glu Val Thr Cys

275 280 285

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

290 295 300

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Pro Glu

305 310 315 320

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

325 330 335

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

340 345 350

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

355 360 365

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

370 375 380

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

385 390 395 400

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

405 410 415

Asn Tyr Lys Thr Thr Pro Pro Val Leu Lys Ser Asp Gly Ser Phe Phe

420 425 430

Leu Tyr Ser Asp Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

435 440 445

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

450 455 460

Gln Lys Ser Leu Ser Leu Ser Pro Gly

465 470

<210> 70

<211> 226

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Short polypeptide, design 16/CC

<400> 70

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

1 5 10 15

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Pro Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Ser Cys Ala Val Asp Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly

225

<210> 71

<211> 473

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Long protein

<400> 71

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

1 5 10 15

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Pro Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Cys Arg Asp Lys Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Lys Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly

225 230 235 240

Gly Gly Gly Gly Gly Gly Gly Asp Lys Thr His Thr Cys Pro Pro Cys

245 250 255

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

260 265 270

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

275 280 285

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

290 295 300

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

305 310 315 320

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

325 330 335

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

340 345 350

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

355 360 365

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

370 375 380

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

385 390 395 400

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

405 410 415

Asn Tyr Lys Thr Thr Pro Pro Val Leu Lys Ser Asp Gly Ser Phe Phe

420 425 430

Leu Tyr Ser Asp Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

435 440 445

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

450 455 460

Gln Lys Ser Leu Ser Leu Ser Pro Gly

465 470

<210> 72

<211> 473

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Long protein

<400> 72

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

1 5 10 15

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Pro Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Cys Arg Asp Lys Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Lys Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly

225 230 235 240

Gly Gly Gly Gly Gly Gly Gly Asp Lys Thr His Thr Cys Pro Pro Cys

245 250 255

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

260 265 270

Lys Pro Lys Asp Thr Leu Met Ala Ser Arg Thr Pro Glu Val Thr Cys

275 280 285

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

290 295 300

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

305 310 315 320

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

325 330 335

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

340 345 350

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

355 360 365

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

370 375 380

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

385 390 395 400

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

405 410 415

Asn Tyr Lys Thr Thr Pro Pro Val Leu Lys Ser Asp Gly Ser Phe Phe

420 425 430

Leu Tyr Ser Asp Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

435 440 445

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

450 455 460

Gln Lys Ser Leu Ser Leu Ser Pro Gly

465 470

<210> 73

<211> 226

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Short polypeptide, design 18/Q1

<400> 73

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

1 5 10 15

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ala Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Pro Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Ser Cys Ala Val Asp Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly

225

<210> 74

<211> 473

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Long polypeptide

<400> 74

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

1 5 10 15

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ala Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Pro Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Cys Arg Asp Lys Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Lys Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly

225 230 235 240

Gly Gly Gly Gly Gly Gly Gly Asp Lys Thr His Thr Cys Pro Pro Cys

245 250 255

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

260 265 270

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

275 280 285

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

290 295 300

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

305 310 315 320

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

325 330 335

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

340 345 350

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

355 360 365

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

370 375 380

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

385 390 395 400

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

405 410 415

Asn Tyr Lys Thr Thr Pro Pro Val Leu Lys Ser Asp Gly Ser Phe Phe

420 425 430

Leu Tyr Ser Asp Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

435 440 445

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

450 455 460

Gln Lys Ser Leu Ser Leu Ser Pro Gly

465 470

<210> 75

<211> 473

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Long polypeptide

<400> 75

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

1 5 10 15

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ala Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Pro Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Cys Arg Asp Lys Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Lys Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly

225 230 235 240

Gly Gly Gly Gly Gly Gly Gly Asp Lys Thr His Thr Cys Pro Pro Cys

245 250 255

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

260 265 270

Lys Pro Lys Asp Thr Leu Met Ala Ser Arg Thr Pro Glu Val Thr Cys

275 280 285

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

290 295 300

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

305 310 315 320

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

325 330 335

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

340 345 350

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

355 360 365

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

370 375 380

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

385 390 395 400

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

405 410 415

Asn Tyr Lys Thr Thr Pro Pro Val Leu Lys Ser Asp Gly Ser Phe Phe

420 425 430

Leu Tyr Ser Asp Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

435 440 445

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

450 455 460

Gln Lys Ser Leu Ser Leu Ser Pro Gly

465 470

<210> 76

<211> 473

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Long polypeptide

<400> 76

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

1 5 10 15

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Pro Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Cys Arg Asp Lys Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Lys Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly

225 230 235 240

Gly Gly Gly Gly Gly Gly Gly Asp Lys Thr His Thr Cys Pro Pro Cys

245 250 255

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

260 265 270

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

275 280 285

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

290 295 300

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Pro Glu

305 310 315 320

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

325 330 335

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

340 345 350

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

355 360 365

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

370 375 380

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

385 390 395 400

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

405 410 415

Asn Tyr Lys Thr Thr Pro Pro Val Leu Lys Ser Asp Gly Ser Phe Phe

420 425 430

Leu Tyr Ser Asp Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

435 440 445

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

450 455 460

Gln Lys Ser Leu Ser Leu Ser Pro Gly

465 470

<210> 77

<211> 473

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Long polypeptide

<400> 77

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

1 5 10 15

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Pro Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Cys Arg Asp Lys Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Lys Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly

225 230 235 240

Gly Gly Gly Gly Gly Gly Gly Asp Lys Thr His Thr Cys Pro Pro Cys

245 250 255

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

260 265 270

Lys Pro Lys Asp Thr Leu Met Ala Ser Arg Thr Pro Glu Val Thr Cys

275 280 285

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

290 295 300

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Pro Glu

305 310 315 320

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

325 330 335

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

340 345 350

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

355 360 365

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

370 375 380

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

385 390 395 400

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

405 410 415

Asn Tyr Lys Thr Thr Pro Pro Val Leu Lys Ser Asp Gly Ser Phe Phe

420 425 430

Leu Tyr Ser Asp Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

435 440 445

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

450 455 460

Gln Lys Ser Leu Ser Leu Ser Pro Gly

465 470

<210> 78

<211> 473

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Long polypeptide Design 18/Q1

<400> 78

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

1 5 10 15

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ala Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Pro Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Cys Arg Asp Lys Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Lys Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly

225 230 235 240

Gly Gly Gly Gly Gly Gly Gly Asp Lys Thr His Thr Cys Pro Pro Cys

245 250 255

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

260 265 270

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

275 280 285

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

290 295 300

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Pro Glu

305 310 315 320

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

325 330 335

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

340 345 350

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

355 360 365

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

370 375 380

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

385 390 395 400

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

405 410 415

Asn Tyr Lys Thr Thr Pro Pro Val Leu Lys Ser Asp Gly Ser Phe Phe

420 425 430

Leu Tyr Ser Asp Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

435 440 445

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

450 455 460

Gln Lys Ser Leu Ser Leu Ser Pro Gly

465 470

<210> 79

<211> 473

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Long polypeptide design 19/Q2

<400> 79

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

1 5 10 15

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ala Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Pro Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Cys Arg Asp Lys Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Lys Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly

225 230 235 240

Gly Gly Gly Gly Gly Gly Gly Asp Lys Thr His Thr Cys Pro Pro Cys

245 250 255

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

260 265 270

Lys Pro Lys Asp Thr Leu Met Ala Ser Arg Thr Pro Glu Val Thr Cys

275 280 285

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

290 295 300

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Pro Glu

305 310 315 320

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

325 330 335

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

340 345 350

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

355 360 365

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

370 375 380

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

385 390 395 400

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

405 410 415

Asn Tyr Lys Thr Thr Pro Pro Val Leu Lys Ser Asp Gly Ser Phe Phe

420 425 430

Leu Tyr Ser Asp Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

435 440 445

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

450 455 460

Gln Lys Ser Leu Ser Leu Ser Pro Gly

465 470

<210> 80

<211> 32

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Linker sequence

<400> 80

Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly Gly

1 5 10 15

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

20 25 30

<---

Claims (24)

1. An Fc construct for reducing an immune response in a subject, comprising: a) the first polypeptide containing i) the first monomer of the Fc domain, ii) a second monomer of the Fc domain and iii) a linker connecting the first Fc domain monomer to the second Fc domain monomer; b) a second polypeptide containing i) third monomer of the Fc domain, ii) a fourth monomer of the Fc domain and iii) a linker connecting a third Fc domain monomer to a fourth Fc domain monomer; c) a third polypeptide containing a fifth monomer of the Fc domain; And d) a fourth polypeptide containing a sixth monomer of the Fc domain; wherein the first Fc domain monomer and the fifth Fc domain monomer combine to form a first Fc domain; a second Fc domain monomer and a fourth Fc domain monomer combine to form a second Fc domain; and a third Fc domain monomer and a sixth Fc domain monomer combine to form a third Fc domain; wherein each of the first and second polypeptides contains the sequence and each of the third and fourth polypeptides contains the sequence 2. Fc-construct according to claim 1, in which each of the first and second polypeptides consists of the sequence and each of the third and fourth polypeptides consists of the sequence 3. Use of an Fc construct according to claim 1 or 2 to reduce the immune response in a subject in need thereof. 4. Use of the Fc construct according to claim 3 for reducing the immune response in the treatment of an inflammatory, autoimmune or immune disease selected from rheumatoid arthritis (RA); systemic lupus erythematosus (SLE); ANCA-associated vasculitis; antiphospholipid antibody syndrome; autoimmune hemolytic anemia; chronic inflammatory demyelinating neuropathy; organ transplantation; GVHD; dermatomyositis; Goodpasture's syndrome; type II organ system hypersensitivity syndromes mediated by antibody-dependent cell-mediated cytotoxicity, eg Guillain-Barré syndrome, CIDP, Felty's syndrome, antibody-mediated rejection, autoimmune thyroid disease, ulcerative colitis, autoimmune liver disease; idiopathic thrombocytopenic purpura; myasthenia gravis, neuromyelitis optica; pemphigus and other autoimmune disorders accompanied by the formation of blisters; Sjögren's syndrome; autoimmune cytopenias and other disorders mediated by antibody-dependent phagocytosis; heparin-induced thrombocytopenia (HIT); myositis; polymyositis; antibody-dependent enhancement; muscle stiffness syndrome; Kawasaki disease; myositis with inclusion bodies; systemic sclerosis; IgA nephropathy; IgG4 related disease; Graves' disease; autoimmune inner ear disease (AIED); antiphospholipid syndrome (APS); pemphigus vulgaris; exfoliative pemphigus; pemphigus of pregnant women; paraneoplastic pemphigus; optic neuritis, Parry-Romberg syndrome; FcR-dependent inflammatory syndromes; synovitis; glomerulitis and vasculitis.

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