CH3 domain-based heterodimeric molecules, methods for their preparation and uses thereof

Abstract

This application relates to the field of antibody engineering, and particularly to CH3-based heterodimeric molecules, their preparation methods and uses. By comprehensively considering various intermolecular interactions, such as ionic interactions, hydrophobic interactions and steric interactions, etc., the present invention screens out preferred Fc mutant sequences, which are more inclined to form heterodimers rather than homodimers, thus greatly improving the yield of heterodimeric molecules and creating conditions for the preparation of bispecific molecules and the like.

Description

[0001] This application is a divisional application of a Chinese patent application with an application date of December 16, 2016, an application number of 201680073286.3, and an invention title of "Heterodimer Molecules Based on the CH3 Domain, Their Preparation Methods and Uses". Technical Field

[0002] The invention described in this application belongs to the field of antibody engineering, and specifically relates to heterodimer molecules based on the CH3 domain, their preparation methods and uses. Background Art

[0003] Monoclonal antibody drugs have grown rapidly in the past fifteen years and have become a growth point in the pharmaceutical industry. Since 1996, a total of about 30 monoclonal antibody drugs have been approved for marketing, and nine of them have annual sales exceeding one billion US dollars. In 2010, the total sales of monoclonal antibody drugs exceeded 30 billion US dollars, and the annual growth rate exceeded 10%. Due to the strong target specificity of monoclonal antibodies, they can only inhibit a single target. However, in many diseases, including tumors, autoimmune diseases, etc., it is necessary to inhibit multiple signaling pathways to avoid compensatory effects. For viral infectious diseases, due to the high mutation rate of the virus, it is often necessary to inhibit multiple antigen sites to prevent escape. In addition, bispecific antibodies and proteins are used to specifically activate the human immune system (Wolf, Hofmeister et al. 2005).

[0004] As is well known, the crystallizable fragment (Fc) region of an antibody forms a homodimer, and at the same time, Fc plays a key role in maintaining the in vivo stability of the antibody and Fc fusion protein. Modifying Fc to form a heterodimer is an effective method for generating multifunctional antibodies and proteins and maintaining their in vivo stability.

[0005] A typical example of the application of a heterodimer is a bispecific antibody. A bispecific antibody (BsAbs) is an immunoglobulin molecule containing two different ligand-binding sites. A bispecific antibody can be active against at least two different antigens (Carter 2001). It replaces the form in which the two Fab arms of a classical antibody are the same, but instead adopts the form of Fab arms with different sequences. Therefore, the two arms of the Y shape can bind different antigens. The application of bispecific antibodies in cancer treatment has been reviewed in many literatures (Carter 2001; Chames and Baty 2009; Chames and Baty 2009).

[0006] Bispecific antibodies do not exist in nature and can only be prepared by special methods. In the past, methods for preparing bispecific antibodies included chemical cross-linking, hybrid F(ab')2 molecule methods, and murine hybridoma methods, etc. The heterologous nature of bispecific antibodies produced by chemical cross-linking, the instability of products between batches, and the characteristic that antibody specificity is liable to change due to certain modifications or improper linkages make bispecific antibodies produced by this method unsuitable for in vivo use. The bispecific hybrid molecules produced by digesting fragments F(ab') with sulfhydryl cross-linking protease have relatively homogeneous components, but are time-consuming and laborious, and have very low yields. The bispecific antibodies produced by the hybridoma method have reliable sources, but the random pairing of light and heavy chains will produce various possible antibody forms, making the production and purification of bispecific antibodies very difficult.

[0007] As early as the 1990s, Carter et al. used the "knob into hole" model to modify some amino acids of the heavy chain of antibodies and relatively successfully achieved the preparation of bispecific antibodies (Ridgway, Presta et al. 1996; Carter 2001). The "knob into hole" model was initially proposed by Crick and used to solve the problem of amino acid side chain folding between adjacent α-helices (Crick 1952). Carter et al. created a "knob" (such as T366Y) by mutating an amino acid with a small side chain into an amino acid with a large side chain in the CH3 region of the first heavy chain in the Fc region, and mutated some amino acids on the CH3 of the second heavy chain into amino acids with small side chains to create a "hole" (Y407T, etc.). The principle of the "knob into hole" model is that the interaction of "knob into hole" supports the formation of heterodimers, while the "knob-knob" model and the "hole-hole" model hinder the formation of homodimers. They further introduced disulfide bonds in the CH3 region on the basis of the "knob into hole" mutation to strengthen the binding ability of heterodimers. However, in their research results, the ability of the "knob into hole" model to hinder the formation of homodimers is still insufficient. Subsequently, this research group tried to further increase the content of heterodimers by methods such as random mutagenesis-phage display, but still did not solve the fundamental problem. In order to increase the proportion of heterodimers, some studies formed heterodimers by separately preparing two antibodies and reducing-repairing intermolecular disulfide bonds in vitro, but its preparation process is significantly too complex.

[0008] Therefore, there is still a need in the art to find suitable mutations to further enhance the formation of heterodimer proteins while weakening the formation of homodimer proteins. Summary of the Invention

[0009] By comprehensively considering various interactions between interfacial amino acids, such as ionic interactions, hydrophobic interactions, and steric interactions, etc., the invention described in this application screened beneficial CH3 mutant sequences, which are more inclined to form heterodimers rather than homodimers, thus greatly increasing the yield of heterodimer molecules.

[0010] On the one hand, the invention described in this application relates to a heterodimer molecule, which contains a first polypeptide chain and a second polypeptide chain. The first polypeptide chain contains the first CH3 domain of the antibody heavy chain constant region, and the second polypeptide chain contains the second CH3 domain of the antibody heavy chain constant region. Compared with the corresponding wild-type human antibody heavy chain constant region CH3 domain, the first CH3 domain and the second CH3 domain contain one of the mutations of the amino acids at the positions shown in the following (1) to (3):

[0011] (1) Y349 and T366 in the first CH3 domain are mutated, and D356, T366, L368, and Y407 in the second CH3 domain are mutated, and the first CH3 domain and / or the second CH3 domain also have mutations at 1 to 3 amino acid positions selected from F405, K409, K360, Q347, and L368;

[0012] (2) T366 and K409 in the first CH3 domain are mutated, and T366, L368, Y407, and F405 in the second CH3 domain are mutated, and optionally the first CH3 domain and / or the second CH3 domain also have mutations at the positions of 1 to 2 amino acids selected from K392, D399, Y349, S354, and E357; and

[0013] (3) T366 and F405 in the first CH3 domain are mutated, and T366, L368, Y407, and K409 in the second CH3 domain are mutated, and optionally the first CH3 domain and / or the second CH3 domain also have mutations at the positions of 1 to 2 amino acids selected from K392, D399, Y349, S354, and E357;

[0014] The above-mentioned amino acid positions are determined according to the EU index of the KABAT numbering of the antibody Fc.

[0015] In some embodiments, the first CH3 domain and the second CH3 domain contain the mutations of item (2) or item (3) above and do not contain the mutations Y349C and D356C.

[0016] In some embodiments, the first CH3 domain and / or the second CH3 domain also contain one of the mutations selected from the following:

[0017] 1a) A mutation occurs at F405 in the second CH3 domain;

[0018] 1b) A mutation occurs at F405 in the first CH3 domain;

[0019] 1c) A mutation occurs at K409 in the first CH3 domain and a mutation occurs at F405 in the second CH3 domain;

[0020] 1d) Mutations occur at F405, K360, and Q347 in the first CH3 domain, and a mutation occurs at Q347 in the second CH3 domain;

[0021] 1e) Mutations occur at F405 and Q347 in the first CH3 domain, and mutations occur at K360 and Q347 in the second CH3 domain;

[0022] 1f) Mutations occur at K409, K360, and Q347 in the first CH3 domain, and mutations occur at F405 and Q347 in the second CH3 domain;

[0023] 1g) Mutations occur at K409 and Q347 in the first CH3 domain, and mutations occur at F405, K360, and Q347 in the second CH3 domain; and

[0024] 1h) Mutations occur at K409 and L368 in the first CH3 domain, and a mutation occurs at F405 in the second CH3 domain.

[0025] In certain embodiments, the first CH3 domain and / or the second CH3 domain further optionally contain one of the mutations selected from the following:

[0026] 2a) A mutation occurs at K392 in the first CH3 domain and a mutation occurs at D399 in the second CH3 domain;

[0027] 2b) A mutation occurs at Y349 in the first CH3 domain and a mutation occurs at E357 in the second CH3 domain; and

[0028] 2c) Mutations occur at Y349 and S354 in the first CH3 domain, and a mutation occurs at E357 in the second CH3 domain.

[0029] In certain embodiments, the first CH3 domain and / or the second CH3 domain optionally further contain one of the mutations selected from the following:

[0030] 3a) A mutation occurs at D399 in the first CH3 domain and a mutation occurs at K392 in the second CH3 domain;

[0031] 3b) The Y349 of the first CH3 domain is mutated, and the E357 of the second CH3 domain is mutated; and

[0032] 3c) The Y349 and S354D of the first CH3 domain are mutated, and the E357 of the second CH3 domain is mutated.

[0033] In certain embodiments, each of the mutations is independently selected from the group consisting of mutations from a non-charged amino acid to a charged amino acid, a charged amino acid to a non-charged amino acid, and a charged amino acid to an amino acid with an opposite charge.

[0034] In certain embodiments, the mutations in the first CH3 domain and / or the second CH3 domain include one or more mutations selected from the following: Y349C, Y349D, D356C, T366W, T366S, L368A, L368E, L368G, F405K, Y407V, Y407A, K409E, K409A, K360E, Q347E, Q347R, K392D, D399S, E357A, and S354D. For example, the mutations can be one or more mutations selected from the following: Y349C, Y349D, D356C, T366W, T366S, L368A, L368E, L368G, F405K, Y407V, Y407A, K409E, K409A, K360E, Q347E, Q347R, K392D, D399S, E357A, and S354D.

[0035] In certain embodiments, the first CH3 domain contains one or more (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, or at least 8) mutations selected from the group consisting of Y349, T366, F405, K409, L368, K392, S354, and / or D399.

[0036] In certain embodiments, the second CH3 domain contains one or more (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, or at least 9) mutations selected from the group consisting of D356, T366, L368, Y407, F405, D399, E357, K409, and / or K392.

[0037] In certain embodiments, the first CH3 domain contains one or more (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, or at least 8) mutations selected from the group consisting of: Y349, T366, F405, K409, L368, K392, S354, and / or D399; and the second CH3 domain contains one or more (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, or at least 9) mutations selected from the group consisting of: D356, T366, L368, Y407, F405, D399, E357, K409, and / or K392.

[0038] In certain embodiments, the first CH3 domain contains one or more (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, or at least 9) mutations selected from the group consisting of: Y349C, T366W, F405K, K409A, L368E, K392D, Y349D, S354D, and / or D399S.

[0039] In certain embodiments, the second CH3 domain contains one or more (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or at least 11) mutations selected from the group consisting of: D356C, T366S, L368A, Y407V, F405K, D399S, L368G, Y407A, E357A, K409A, and / or K392D.

[0040] In certain embodiments, the first CH3 domain contains one or more (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, or at least 9) mutations selected from the group consisting of: Y349C, T366W, F405K, K409A, L368E, K392D, Y349D, S354D, and / or D399S; and the second CH3 domain contains one or more (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or at least 11) mutations selected from the group consisting of: D356C, T366S, L368A, Y407V, F405K, D399S, L368G, Y407A, E357A, K409A, and / or K392D.

[0041] In certain embodiments, the first CH3 domain and the second CH3 domain contain mutations selected from the following group:

[0042] 1) The first CH3 domain: Y349C + T366W, the second CH3 domain: D356C + T366S + L368A + Y407V + F405K;

[0043] 2) The first CH3 domain: Y349C + T366W + F405K, the second CH3 domain: D356C + T366S + L368A + Y407V;

[0044] 3) The first CH3 domain: Y349C + T366W + K409E, the second CH3 domain: D356C + T366S + L368A + Y407V + F405K;

[0045] 4) The first CH3 domain: Y349C + T366W + K409A, the second CH3 domain: D356C + T366S + L368A + Y407V + F405K;

[0046] 5) The first CH3 domain: Y349C + T366W + F405K + K360E + Q347E, the second CH3 domain: D356C + T366S + L368A + Y407V + Q347R;

[0047] 6) The first CH3 domain: Y349C + T366W + F405K + Q347R, the second CH3 domain: D356C + T366S + L368A + Y407V + K360E + Q347E;

[0048] 7) The first CH3 domain: Y349C + T366W + K409A + K360E + Q347E, the second CH3 domain: D356C + T366S + L368A + Y407V + F405K + Q347R;

[0049] 8) The first CH3 domain: Y349C + T366W + K409A + Q347R, the second CH3 domain: D356C + T366S + L368A + Y407V + F405K + K360E + Q347E;

[0050] 9) The first CH3 domain: Y349C + T366W + K409A + L368E, the second CH3 domain: D356C + T366S + L368A + Y407V + F405K;

[0051] 10) The first CH3 domain: T366W + K409A + K392D, the second CH3 domain: T366S + L368A + Y407V + D399S + F405K;

[0052] 11) First CH3 domain: T366W + K409A, second CH3 domain: T366S + L368G + Y407A + F405K;

[0053] 12) First CH3 domain: T366W + K409A + Y349D, second CH3 domain: T366S + L368A + Y407V + F405K + E357A;

[0054] 13) First CH3 domain: T366W + K409A + Y349D + S354D, second CH3 domain: T366S + L368A + Y407V + F405K + E357A;

[0055] 14) First CH3 domain: T366W + F405K, second CH3 domain: T366S + L368A + Y407V + K409A;

[0056] 15) First CH3 domain: T366W + F405K + D399S, second CH3 domain: T366S + L368A + Y407V + K409A + K392D;

[0057] 16) First CH3 domain: T366W + F405K, second CH3 domain: T366S + L368G + Y407A + K409A;

[0058] 17) First CH3 domain: T366W + F405K + Y349D, second CH3 domain: T366S + L368A + Y407V + K409A + E357A; and

[0059] 18) First CH3 domain: T366W + F405K + Y349D + S354D, second CH3 domain: T366S + L368A + Y407V + K409A + E357A.

[0060] In certain embodiments, the first CH3 domain and the second CH3 domain contain mutations selected from the following group:

[0061] 2) First CH3 domain: Y349C + T366W + F405K, second CH3 domain: D356C + T366S + L368A + Y407V;

[0062] 4) First CH3 domain: Y349C + T366W + K409A, second CH3 domain: D356C + T366S + L368A + Y407V + F405K;

[0063] 9) First CH3 domain: Y349C + T366W + K409A + L368E, second CH3 domain: D356C + T366S + L368A + Y407V + F405K;

[0064] 10) First CH3 domain: T366W + K409A + K392D, second CH3 domain: T366S + L368A + Y407V + D399S + F405K;

[0065] 11) First CH3 domain: T366W + K409A, second CH3 domain: T366S + L368G + Y407A + F405K;

[0066] 13) First CH3 domain: T366W + K409A + Y349D + S354D, second CH3 domain: T366S + L368A + Y407V + F405K + E357A;

[0067] 15) First CH3 domain: T366W + F405K + D399S, second CH3 domain: T366S + L368A + Y407V + K409A + K392D;

[0068] 16) First CH3 domain: T366W + F405K, second CH3 domain: T366S + L368G + Y407A + K409A; and

[0069] 18) First CH3 domain: T366W + F405K + Y349D + S354D, second CH3 domain: T366S + L368A + Y407V + K409A + E357A.

[0070] In certain embodiments, the first polypeptide chain and the second polypeptide chain further each contain a CH2 domain of the antibody heavy chain constant region. In certain embodiments, the CH2 domain is located at the N-terminus of the CH3 domain and is directly connected to the N-terminus of the CH3 domain or connected by a linker peptide.

[0071] In certain embodiments, the first polypeptide chain and the second polypeptide chain further each contain a hinge region or a part of the hinge region of the antibody heavy chain constant region. In certain embodiments, a part of the hinge region is D221 - P230.

[0072] In certain embodiments, the hinge region or a part of the hinge region is located at the N-terminus of the CH3 domain. When a CH2 domain is present, the hinge region or a part of the hinge region is further located at the N-terminus of the CH2 domain and is directly connected to the CH2 or CH3 domain or connected by a linker peptide.

[0073] In certain embodiments, the wild-type human antibody heavy chain constant region CH3 domain is selected from the human IgG (such as IgG1, IgG2, IgG3 or IgG4) heavy chain constant region CH3 domain, the human IgA (such as IgA1, IgA2) heavy chain constant region CH3 domain, the human IgD heavy chain constant region CH3 domain, the human IgE heavy chain constant region CH3 domain, and the human IgM heavy chain constant region CH3 domain.

[0074] In certain embodiments, the wild-type human antibody heavy chain constant region CH3 domain is the human IgG1 heavy chain constant region CH3 domain.

[0075] In certain embodiments, the first and / or second polypeptide chain further contains a molecular binding region, such as an antigen binding region, a receptor binding region, or an enzyme binding region. In certain embodiments, the antigen binding region contains an antibody variable region.

[0076] In certain embodiments, the heterodimeric molecule is a bispecific antibody, a bispecific fusion protein, or an antibody-fusion protein chimera.

[0077] In another aspect, the present application relates to a composition (e.g., a pharmaceutical composition) comprising any one of the heterodimeric molecules described in the present application, and optionally a pharmaceutically acceptable carrier or excipient.

[0078] The present application also relates to a nucleic acid molecule encoding the first polypeptide chain or the second polypeptide chain of the heterodimeric molecule described in the present application, or encoding the first polypeptide chain and the second polypeptide chain of the heterodimeric molecule described in the present application.

[0079] The present application also relates to a vector containing the nucleic acid molecule described in the present application.

[0080] The present application also relates to a host cell containing the vector described in the present application.

[0081] The present application also relates to the use of the heterodimeric molecule, composition, nucleic acid molecule, vector, or host cell in the preparation of bispecific antibodies, bispecific fusion proteins, and antibody-fusion protein chimeras.

[0082] The present application also relates to a method for preparing a heterodimeric molecule, which includes the step of expressing the heterodimeric molecule using the host cell described in the present application.

[0083] In certain embodiments of the method for preparing a heterodimeric molecule, the host cell simultaneously contains a vector encoding the first polypeptide chain and the second polypeptide chain in the heterodimeric molecule, and the method includes using the host cell to express, recover, and obtain the heterodimeric molecule.

[0084] In certain embodiments of the method for preparing heterodimeric molecules, the host cell comprises a first group of cells and a second group of cells, and each of the first group of cells and the second group of cells contains a vector encoding the first polypeptide chain and the second polypeptide chain of the heterodimeric molecule, respectively. The method comprises: expressing the first polypeptide chain and the second polypeptide chain in the first group of cells and the second group of cells respectively to form the first polypeptide chain homodimer and the second polypeptide chain homodimer, and then mixing the first polypeptide chain homodimer and the second polypeptide chain homodimer under suitable conditions and preparing the heterodimeric molecule. In certain embodiments, the method further comprises reducing and dissociating the first polypeptide chain homodimer and the second polypeptide chain homodimer into monomers, mixing, oxidizing, and then purifying to prepare the heterodimeric molecule. In certain embodiments, the host cells contain vectors encoding the first polypeptide chain and the second polypeptide chain in the heterodimeric molecule respectively. The first polypeptide chain and the second polypeptide chain are expressed in two host cells respectively to form the first polypeptide chain homodimer and the second polypeptide chain homodimer, and then the first polypeptide chain homodimer and the second polypeptide chain homodimer are reduced, mixed, oxidized under suitable conditions, and then purified to prepare the heterodimeric molecule.

[0085] In certain embodiments of the method for preparing heterodimeric molecules, a construct or vector containing the first polypeptide chain or the second polypeptide chain is transfected into the first group of cells and the second group of cells respectively. The transfection can be transient transfection. In the transfection, the molar ratio of the construct or vector containing the first polypeptide chain to the construct or vector containing the second polypeptide chain can be from 1:4 to 4:1, such as from 1:2 to 2:1, such as about 1:1.

[0086] Those skilled in the art can easily insight into other aspects and advantages of the present disclosure from the following detailed description. Only exemplary embodiments of the present disclosure are shown and described in the following detailed description. As those skilled in the art will recognize, the content of the present disclosure enables those skilled in the art to make changes to the disclosed specific embodiments without departing from the spirit and scope of the invention involved in the present application. Accordingly, the descriptions in the drawings and the specification of the present application are merely exemplary and not restrictive. Description of the Drawings

[0087] Figure 1Electrophoresis analysis results showing the transient expression of ScFv-Fc / Fc heterodimers were presented. 4%-12% SDS-PAGE protein gel electrophoresis was used. The lanes from 1 to 7 were, in sequence: protein molecular weight standard, mutant combination KH, mutant combination 1, mutant combination 2, mutant combination 3, mutant combination 4, and wild-type negative control combination. The homodimers and heterodimers contained in each group of products had different migration distances in the gel electrophoresis due to the molecular weight differences. The positions of different homodimers or heterodimer proteins were marked in the figure.

[0088] Figure 2 Electrophoresis analysis results showing the transient expression of ScFv-Fc / Fc heterodimers were presented. 12% SDS-PAGE protein gel electrophoresis was used. The lanes from 1 to 9 were, in sequence: mutant combination 9, mutant combination 8, mutant combination 7, mutant combination 4, mutant combination 6, mutant combination 5, mutant combination 2, blank control (cell supernatant), and protein molecular weight standard. The homodimers and heterodimers contained in each group of products had different migration distances in the gel electrophoresis due to the molecular weight differences. Similar to Figure 1 the above, from top to bottom were ScFv-Fc / ScFv-Fc homodimer, ScFv-Fc / Fc heterodimer, and Fc / Fc homodimer.

[0089] Figure 3 A partial crystal structure diagram of the CH3-CH3 interface on the Fc of the heterodimer in mutant combination 4 was shown. The mutated amino acid residues were represented by short sticks. The specific pairs of mutated amino acid residues in contact were as follows: T366W / A chain - T366S, L368A, Y407V / B chain, K409A / A chain - F405K / B chain, S354C / A chain - Y349C / B chain. The A chain (the lighter-colored chain on the left) was represented in green, and the B chain (the darker-colored chain on the right) was represented in light blue.

[0090] Figure 4 It was shown that introducing a new pair of mutations D399S-K392D near the pair of mutated amino acid residues F405K-K409A could further enhance the mutual attraction between heterodimers and at the same time increase the mutual repulsion force between homodimers. Figure 4 A shows the interaction of interface amino acids near the mutation pair F405K-K409A when introducing the new mutation. Figure 4 B shows the change in the interaction brought about after introducing the new mutation. Detailed implementation manners

[0091] The following specific examples illustrate the implementation manners of the present invention. Those skilled in the art can understand other advantages and effects of the present invention through the content disclosed in this specification.

[0092] In the present application, both the first polypeptide chain and the second polypeptide chain contain the CH3 region of the antibody Fc fragment, and the two polypeptide chains interact with each other through the CH3 region or the Fc fragment containing the CH3 region to form a dimer, especially a heterodimer. The two polypeptide chains of the heterodimer can be different combinations. For example, the first polypeptide chain is an antibody, the second polypeptide chain is a fusion protein, or both polypeptide chains are fusion proteins, or both polypeptide chains are antibodies (for example, antibodies targeting different antigens or epitopes). When the fusion protein contains the Fc segment of an antibody and the extracellular region of a cell adhesion molecule, it is also called an immunoadhesin. The cell adhesion molecule mainly refers to a molecule that can recognize a cell surface receptor of a specific ligand cell, such as including cadherin, selectin, immunoglobulin superfamily, integrin, and hyaluronectin.

[0093] In the present application, the CH3 region is derived from the antibody Fc fragment, for example, from the Fc fragment of a human antibody (for example, the Fc fragment of the human antibody heavy chain constant region). In certain embodiments, the CH3 region is derived from the Fc fragment of the human immunoglobulin (Ig) heavy chain constant region, for example, from the Fc fragment of IgM, IgG (such as IgG1, IgG2, IgG3, IgG4), IgA (such as IgA1, IgA2), IgE, and / or IgD heavy chain constant regions. In certain embodiments, the CH3 region described in the present application (such as the wild-type human antibody heavy chain constant region CH3 domain) is derived from wild-type human IgG1, for example, from the CH3 domain of the wild-type human IgG1 antibody heavy chain constant region. Generally, the CH3 region of the human antibody Fc fragment is derived from the corresponding wild-type human antibody Fc fragment. The wild-type human antibody Fc fragment refers to the antibody Fc fragment in the natural human population, for example, the human antibody Fc fragment without artificial mutagenesis or artificial modification. In certain embodiments, the human antibody Fc fragment described in the present application also includes changes in individual amino acids of the corresponding wild-type human antibody Fc sequence, such as including certain amino acids mutated at glycosylation sites, or other nonsense mutations, and also includes changes in individual amino acids mutated according to the "knob-hole" model. For example, for the CH3 and CH2 domains, in addition to the mutations mentioned in the present application, there may also be other mutations that do not affect the function of the antibody (especially the Fc segment).

[0094] In the present application, when the first polypeptide chain and / or the second polypeptide chain contains a hinge region, the hinge region is connected between the two polypeptide segments as a flexible fragment to ensure the function of each polypeptide segment; those skilled in the art can select the length of the hinge region according to needs, for example, a full-length sequence or a partial sequence thereof can be selected.

[0095] In the present application, the numbering of amino acid positions in the Fc or its CH2, CH3 domains or hinge region is determined according to the positions in the Kabat EU numbering index. Those skilled in the art will appreciate that even if the amino acid sequence is altered due to insertions, deletions or other mutations in the above regions, the position numbers of the respective amino acids corresponding to the standard sequence determined according to the Kabat EU numbering index remain unchanged.

[0096] In the present application, the human antibody heavy chain constant region may comprise a combination of two or more domains of heavy chain CH1, CH2, CH3, CH4 and the antibody hinge region. In certain embodiments, the human antibody Fc fragment comprises at least one antibody hinge region, one CH2 domain and one CH3 domain. In certain embodiments, the CH2 domain is the CH2 domain of the human IgG1 heavy chain constant region, which corresponds to amino acids 228 - 340 according to the EU numbering system. In certain embodiments, the CH2 domain corresponds to the corresponding region of any other antibody isotype described in the present application. In certain embodiments, the CH3 domain is the CH3 domain of the human IgG1 heavy chain constant region, which corresponds to amino acids 341 - 447 according to the EU numbering system. In certain embodiments, the CH3 domain corresponds to the corresponding region of any other antibody isotype described in the present application.

[0097] In the present application, the charged amino acids include arginine, lysine, aspartic acid and glutamic acid.

[0098] In the present application, the heterodimeric molecule can be purified from the host cell by standard experimental means. For example, when the heterodimeric protein contains an antibody Fc fragment, it can be purified using Protein A. The purification methods include but are not limited to chromatographic techniques such as size exclusion, ion exchange, affinity chromatography and ultrafiltration, or a suitable combination of the above methods.

[0099] In the present application, the EU index is described, for example, in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD. (1991).

[0100] By comprehensively considering various interactions between interface amino acids, such as ionic interactions, hydrophobic interactions, and steric interactions, etc., the present application has screened out a preferred CH3 mutation sequence, which is more inclined to form heterodimers rather than homodimers, thus greatly increasing the yield of heterodimer molecules. At the same time, in some embodiments of the present application, heterodimer protein crystals containing Fc fragments are prepared. Through the analysis of crystal structures and three-dimensional structure modeling, a further understanding of the direct interactions between interface amino acids is obtained. Meanwhile, the view that stable disulfide bonds will necessarily form between the two cysteines of Y349C and D356C, which was previously considered, is discarded. On this basis, the mutation combinations are more conducive to the formation of heterodimers and less conducive to the formation of homodimers, greatly increasing the proportion of heterodimers while greatly reducing the proportion of homodimers.

[0101] The embodiments of the invention described in the present application will be described in detail below in conjunction with examples. However, those skilled in the art will understand that the following examples are only used to illustrate the invention described in the present application by way of example and should not be construed as limiting the scope of the invention described in the present application. For those not specified in the examples, they are carried out under conventional conditions or conditions recommended by the manufacturer. For reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0102] Embodiments

[0103] 1. A heterodimer molecule, which comprises a first polypeptide chain and a second polypeptide chain. The first polypeptide chain comprises a first CH3 domain of an antibody heavy chain constant region, and the second polypeptide chain comprises a second CH3 domain of an antibody heavy chain constant region. Compared with the corresponding wild-type human antibody heavy chain constant region CH3 domain, the first CH3 domain and the second CH3 domain contain one of the mutations of amino acids at the positions selected from the following (1) to (3):

[0104] (1) Y349 and T366 of the first CH3 domain are mutated, and D356, T366, L368, and Y407 of the second CH3 domain are mutated, and the first CH3 domain and / or the second CH3 domain also have mutations at the positions of 1 to 3 amino acids selected from F405, K409, K360, Q347, and L368;

[0105] (2) T366 and K409 of the first CH3 domain are mutated, and T366, L368, Y407, and F405 of the second CH3 domain are mutated, and optionally the first CH3 domain and / or the second CH3 domain also have mutations at the positions of 1 to 2 amino acids selected from K392, D399, Y349, S354, and E357; and

[0106] (3) Mutations occur at T366 and F405 in the first CH3 domain, and at T366, L368, Y407 and K409 in the second CH3 domain, and optionally the first CH3 domain and / or the second CH3 domain further have mutations at the positions of 1 to 2 amino acids selected from K392, D399, Y349, S354 and E357;

[0107] The positions of the above-mentioned amino acids are determined according to the EU index of the KABAT numbering of the antibody Fc.

[0108] 2. The heterodimeric molecule according to embodiment 1, wherein the first CH3 domain and / or the second CH3 domain further comprises one of the following mutations:

[0109] 1a) Mutation occurs at F405 in the second CH3 domain;

[0110] 1b) Mutation occurs at F405 in the first CH3 domain;

[0111] 1c) Mutation occurs at K409 in the first CH3 domain and at F405 in the second CH3 domain;

[0112] 1d) Mutations occur at F405, K360 and Q347 in the first CH3 domain, and at Q347 in the second CH3 domain;

[0113] 1e) Mutations occur at F405 and Q347 in the first CH3 domain, and at K360 and Q347 in the second CH3 domain;

[0114] 1f) Mutations occur at K409, K360 and Q347 in the first CH3 domain, and at F405 and Q347 in the second CH3 domain;

[0115] 1g) Mutations occur at K409 and Q347 in the first CH3 domain, and at F405, K360 and Q347 in the second CH3 domain; and

[0116] 1h) Mutations occur at K409 and L368 in the first CH3 domain, and at F405 in the second CH3 domain.

[0117] 3. The heterodimeric molecule according to any one of embodiments 1-2, wherein the first CH3 domain and / or the second CH3 domain further optionally comprises one of the following mutations:

[0118] 2a) Mutation occurs at K392 in the first CH3 domain and at D399 in the second CH3 domain;

[0119] 2b) The Y349 of the first CH3 domain is mutated, and the E357 of the second CH3 domain is mutated; and

[0120] 2c) The Y349 and S354 of the first CH3 domain are mutated, and the E357 of the second CH3 domain is mutated.

[0121] 4. The heterodimer molecule according to any one of embodiments 1-3, wherein the first CH3 domain and / or the second CH3 domain optionally further comprises one of the mutations selected from the following:

[0122] 3a) The D399 of the first CH3 domain is mutated, and the K392 of the second CH3 domain is mutated;

[0123] 3b) The Y349 of the first CH3 domain is mutated, and the E357 of the second CH3 domain is mutated; and

[0124] 3c) The Y349 and S354D of the first CH3 domain are mutated, and the E357 of the second CH3 domain is mutated.

[0125] 5. The heterodimer molecule according to any one of embodiments 1-4, wherein each of the mutations is independently selected from a mutation of a non-charged amino acid to a charged amino acid, a charged amino acid to a non-charged amino acid, or a charged amino acid to an amino acid with an opposite charge.

[0126] 6. The heterodimer molecule according to any one of embodiments 1-5, wherein the mutations in the first CH3 domain and / or the second CH3 domain comprise one or several mutations selected from the following: Y349C, Y349D, D356C, T366W, T366S, L368A, L368E, L368G, F405K, Y407V, Y407A, K409E, K409A, K360E, Q347E, Q347R, K392D, D399S, E357A and S354D.

[0127] 7. The heterodimer molecule according to any one of embodiments 1-6, wherein the first CH3 domain comprises one or more mutations selected from the following group: Y349, T366, F405, K409, L368, K392, S354 and / or D399.

[0128] 8. The heterodimer molecule according to any one of embodiments 1-7, wherein the second CH3 domain comprises one or more mutations selected from the following group: D356, T366, L368, Y407, F405, D399, E357, K409 and / or K392.

[0129] 9. The heterodimeric molecule according to any one of embodiments 1-8, wherein the first CH3 domain contains one or more mutations selected from the group consisting of: Y349, T366, F405, K409, L368, K392, S354, and / or D399; and the second CH3 domain contains one or more mutations selected from the group consisting of: D356, T366, L368, Y407, F405, D399, E357, K409, and / or K392.

[0130] 10. The heterodimeric molecule according to any one of embodiments 1-9, wherein the first CH3 domain contains one or more mutations selected from the group consisting of: Y349C, T366W, F405K, K409A, L368E, K392D, Y349D, S354D, and / or D399S.

[0131] 11. The heterodimeric molecule according to any one of embodiments 1-10, wherein the second CH3 domain contains one or more mutations selected from the group consisting of: D356C, T366S, L368A, Y407V, F405K, D399S, L368G, Y407A, E357A, K409A, and / or K392D.

[0132] 12. The heterodimeric molecule according to any one of embodiments 1-11, wherein the first CH3 domain contains one or more mutations selected from the group consisting of: Y349C, T366W, F405K, K409A, L368E, K392D, Y349D, S354D, and / or D399S; and the second CH3 domain contains one or more mutations selected from the group consisting of: D356C, T366S, L368A, Y407V, F405K, D399S, L368G, Y407A, E357A, K409A, and / or K392D.

[0133] 13. The heterodimeric molecule according to any one of embodiments 1-12, wherein the first CH3 domain and the second CH3 domain contain mutations selected from the following group:

[0134] 1) First CH3 domain: Y349C + T366W, second CH3 domain: D356C + T366S + L368A + Y407V + F405K;

[0135] 2) First CH3 domain: Y349C + T366W + F405K, second CH3 domain: D356C + T366S + L368A + Y407V;

[0136] 3) First CH3 domain: Y349C + T366W + K409E, second CH3 domain: D356C + T366S + L368A + Y407V + F405K;

[0137] 4) First CH3 domain: Y349C + T366W + K409A, second CH3 domain: D356C + T366S + L368A + Y407V + F405K;

[0138] 5) First CH3 domain: Y349C + T366W + F405K + K360E + Q347E, second CH3 domain: D356C + T366S + L368A + Y407V + Q347R;

[0139] 6) First CH3 domain: Y349C + T366W + F405K + Q347R, second CH3 domain: D356C + T366S + L368A + Y407V + K360E + Q347E;

[0140] 7) First CH3 domain: Y349C + T366W + K409A + K360E + Q347E, second CH3 domain: D356C + T366S + L368A + Y407V + F405K + Q347R;

[0141] 8) First CH3 domain: Y349C + T366W + K409A + Q347R, second CH3 domain: D356C + T366S + L368A + Y407V + F405K + K360E + Q347E;

[0142] 9) First CH3 domain: Y349C + T366W + K409A + L368E, second CH3 domain: D356C + T366S + L368A + Y407V + F405K;

[0143] 10) First CH3 domain: T366W + K409A + K392D, second CH3 domain: T366S + L368A + Y407V + D399S + F405K;

[0144] 11) First CH3 domain: T366W + K409A, second CH3 domain: T366S + L368G + Y407A + F405K;

[0145] 12) First CH3 domain: T366W + K409A + Y349D, second CH3 domain: T366S + L368A + Y407V + F405K + E357A;

[0146] 13) First CH3 domain: T366W + K409A + Y349D + S354D, second CH3 domain: T366S + L368A + Y407V + F405K + E357A;

[0147] 14) First CH3 domain: T366W + F405K, second CH3 domain: T366S + L368A + Y407V + K409A;

[0148] 15) First CH3 domain: T366W + F405K + D399S, second CH3 domain: T366S + L368A + Y407V + K409A + K392D;

[0149] 16) First CH3 domain: T366W + F405K, second CH3 domain: T366S + L368G + Y407A + K409A;

[0150] 17) First CH3 domain: T366W + F405K + Y349D, second CH3 domain: T366S + L368A + Y407V + K409A + E357A;

[0151] and

[0152] 18) First CH3 domain: T366W + F405K + Y349D + S354D, second CH3 domain: T366S + L368A + Y407V + K409A + E357A.

[0153] 14. The heterodimeric molecule according to any one of embodiments 1 - 13, wherein the first CH3 domain and the second CH3 domain contain mutations selected from the following group:

[0154] 2) First CH3 domain: Y349C + T366W + F405K, second CH3 domain: D356C + T366S + L368A + Y407V;

[0155] 4) First CH3 domain: Y349C + T366W + K409A, second CH3 domain: D356C + T366S + L368A + Y407V + F405K;

[0156] 9) First CH3 domain: Y349C + T366W + K409A + L368E, second CH3 domain: D356C + T366S + L368A + Y407V + F405K;

[0157] 10) First CH3 domain: T366W+K409A+K392D, second CH3 domain: T366S+L368A+Y407V+D399S+F405K;

[0158] 11) First CH3 domain: T366W+K409A, second CH3 domain: T366S+L368G+Y407A+F405K;

[0159] 13) First CH3 domain: T366W+K409A+Y349D+S354D, second CH3 domain: T366S+L368A+Y407V+F405K+E357A;

[0160] 15) First CH3 domain: T366W+F405K+D399S, second CH3 domain: T366S+L368A+Y407V+K409A+K392D;

[0161] 16) First CH3 domain: T366W+F405K, second CH3 domain: T366S+L368G+Y407A+K409A; and

[0162] 18) First CH3 domain: T366W+F405K+Y349D+S354D, second CH3 domain: T366S+L368A+Y407V+K409A+E357A.

[0163] 15. The heterodimeric molecule according to any one of embodiments 1-14, wherein the first polypeptide chain and the second polypeptide chain further each contain a CH2 domain of the heavy chain constant region of an antibody.

[0164] 16. The heterodimeric molecule according to any one of embodiments 1-15, wherein the first polypeptide chain and the second polypeptide chain further each contain a hinge region or a part of the hinge region of the heavy chain constant region of an antibody.

[0165] 17. The heterodimeric molecule according to any one of embodiments 1-16, wherein the wild-type human heavy chain constant region CH3 domain is selected from the human IgG heavy chain constant region CH3 domain, the human IgA heavy chain constant region CH3 domain, the human IgD heavy chain constant region CH3 domain, the human IgE heavy chain constant region CH3 domain, and the human IgM heavy chain constant region CH3 domain.

[0166] 18. The heterodimeric molecule according to any one of embodiments 1-17, wherein the wild-type human heavy chain constant region CH3 domain is the human IgG1 heavy chain constant region CH3 domain.

[0167] 19. The heterodimeric molecule according to any one of embodiments 1-18, wherein the first and / or second polypeptide chain further contains a molecular binding region, and the molecular binding domain is selected from an antigen-binding region, a receptor-binding region, and an enzyme-binding region.

[0168] 20. The heterodimeric molecule according to embodiment 19, wherein the antigen-binding region contains an antibody variable region.

[0169] 21. The heterodimeric molecule according to any one of embodiments 1-20, which is a bispecific antibody, a bispecific fusion protein, or an antibody-fusion protein chimera.

[0170] 22. A composition comprising the heterodimeric molecule according to any one of embodiments 1-21, and optionally a pharmaceutically acceptable carrier or excipient.

[0171] 23. A nucleic acid molecule encoding the first polypeptide chain and / or the second polypeptide chain of the heterodimeric molecule according to any one of embodiments 1-21.

[0172] 24. A vector containing the nucleic acid molecule according to embodiment 23.

[0173] 25. A host cell containing the vector according to embodiment 24.

[0174] 26. Use of the heterodimeric molecule according to any one of embodiments 1-21, the composition according to embodiment 22, the nucleic acid molecule according to embodiment 23, the vector according to embodiment 24, or the host cell according to embodiment 25 in the preparation of a bispecific antibody, a bispecific fusion protein, or an antibody-fusion protein chimera.

[0175] 27. A method for preparing a heterodimeric molecule, which comprises the step of expressing the heterodimeric molecule using the host cell according to embodiment 25.

[0176] 28. The method according to embodiment 27, wherein the host cell simultaneously contains a vector encoding the first polypeptide chain and the second polypeptide chain in the heterodimeric molecule, and the method comprises using the host cell to express, recover, and obtain the heterodimeric molecule.

[0177] 29. The method according to embodiment 28, wherein the host cell comprises a first group of cells and a second group of cells, and the first group of cells and the second group of cells each contain a vector encoding a first polypeptide chain and a second polypeptide chain of the heterodimeric molecule respectively. The method comprises: expressing the first polypeptide chain and the second polypeptide chain in the first group of cells and the second group of cells respectively to form the first polypeptide chain homodimer and the second polypeptide chain homodimer, and then mixing the first polypeptide chain homodimer and the second polypeptide chain homodimer under suitable conditions to obtain the heterodimeric molecule.

[0178] Example 1: Obtaining the first-round mutant candidate combination sequences

[0179] 1. Fc structure modeling and obtaining interface amino acids

[0180] A total of 48 human IgG1 antibody crystal structures containing the Fc region were obtained from the Protein Data Bank (PDB, www.pdb.org). Through the structure similarity search algorithm (Reference: Yuzhen Ye and Adam Godzik. FATCAT: a webserver for flexible structure comparison and structure similarity searching. Nucleic Acids Res., 2004, 32(Web Server issue): W582-585.), it was found that the Fc segments of these 48 antibodies were from 1DN2 (PDB ID).

[0181] Using the protein contact amino acid recognition software CMA (website: http: / / ligin.weizmann.ac.il / cma / ), the amino acid contacts between CH3-CH3 in the antibody (PDB ID: 1DN2) were screened and identified according to the distance of amino acid interaction. According to the amino acid contact rule, interface amino acids refer to some amino acids whose distance between the heavy atoms of the side chain and the heavy atoms of any amino acid in another chain is less than a threshold. The threshold in this example was selected as It can also be selected as (For example, in the literature: B. Erman, I. Bahar and R. L. Jernigan. Equilibrium states of rigid bodies with multiple interaction sites. Application to protein helices. J. Chem. Phys. 1997, 107: 2046 - 2059.) The conservation of the amino acid contact interface between human and murine IgG subtypes can be obtained through sequence multiple alignment. Table 1 shows 34 interfacial amino acids of antibody 1DN2 screened by amino acid contact (i.e., the amino acid distance is less than ). Among them, chain A and chain B represent the first chain and the second chain of antibody 1DN2 respectively. The following amino acid positions are named according to the EU index of the KABAT numbering of the antibody Fc.

[0182] Table 1. List of amino acids at the CH3 - CH3 interface of antibody 1DN2

[0183]

[0184]

[0185] 2. Mutate amino acids to cause changes in ionic interactions

[0186] According to the results in Table 1, select the amino acid pairs containing charged amino groups in the contact amino acid pairs, and mutate an amino group on one of the chains (changing a non - charged amino acid to a charged amino acid, or a charged amino acid to a non - charged amino acid, or changing the charge property of the charged amino acid), so that the ionic interaction between Fc chain A and Fc chain B is unbalanced, and the probability of homodimer formation is reduced and / or the probability of heterodimer formation is increased.

[0187] As an example, mutate Phe405 on chain A to Phe405Lys (also written as F405K), while keeping chain B unchanged. Since there are two Lys, both positively charged amino acids, among the contact amino acid residues on chain B around the 405th amino acid residue, when chain A pairs with chain A, the positive charges carried by the F405K mutations on the two chains will introduce a great repulsive force; when chain A pairs with chain B, only one chain (A) has the repulsive force introduced by the F405K mutation, while the other chain (B) remains Phe405 without introducing repulsive force. In this case, the mutual repulsion between the two chains in AA is very significant, much greater than that in AB or BB, so the formation of AA homodimers can be effectively reduced.

[0188] If, while introducing the F405K mutation into Chain A, the contact amino acid residue Lys409 corresponding to the F405K mutant residue of Chain A on Chain B is mutated to K409E or K409A, then when Chain A pairs with Chain A, the positive charges introduced by the F405K mutation on the two Chains A will still introduce a great repulsive force; while when Chain A pairs with Chain B, there is an F405K mutation on Chain A, which interacts with the K409E or K409A mutation on Chain B, without repulsive force, and even has an attractive force (K409E); and when Chain B pairs with Chain B, neither repulsive force nor attractive force is introduced. In this case, the mutual repulsion between the two chains of AA is very significant, the repulsive force between AB is reduced or an attractive force is introduced, so the formation of AA homodimers can be effectively reduced, and the formation of AB heterodimers can be promoted at the same time.

[0189] And so on, the mutant combinations obtained in this example are shown in the following table:

[0190] Table 2: List of heterodimer mutant combinations

[0191]

[0192] Example 2: Preparation and investigation of ScFv-Fc / Fc heterodimers

[0193] 1. Construct recombinant vectors expressing the Fc fragment of mutant human IgG1 and the ScFv-Fc fusion protein

[0194] According to the amino acid sequence of the constant region of human immunoglobulin gamma1 (IgG1) (P01857) in the protein database Uniprot, the amino acid sequence of the human IgG1-Fc region (SEQ ID NO: 1) was obtained. Through reverse transcription PCR, a nucleic acid fragment encoding human IgG1-Fc (SEQ ID NO: 2, named Fc gene) was obtained from total RNA of human PBMC. The coding sequence of the mouse kappaIII signal peptide (as shown in SEQ ID NO: 3) was added to its 5' end by overlapping PCR, and then subcloned into the vector pcDNA4 (Invitrogen, Cat V86220) to obtain a recombinant expression vector for expressing human IgG1-Fc (abbreviated as Fc) protein in mammalian cells.

[0195] The coding gene of the ScFv-Fc fusion protein as shown in SEQ ID NO: 5 was obtained by artificial synthesis (where ScFv refers to a single-chain antibody against HER2). The amino acid sequence of the ScFv-Fc fusion protein encoded by this gene is shown in SEQ ID: 4, and then subcloned into the mammalian cell expression vector pcDNA4 (Invitrogen, Cat V86220) to obtain a recombinant expression vector for expressing the ScFv-Fc fusion protein in mammalian cells.

[0196] According to Table 2 of Example 1, combinatorial mutations were performed on the scFV-Fc and Fc encoding genes using the overlap PCR method, where the mutation for the A chain was located on the scFV-Fc fusion protein, and the mutation for the B chain was located on the Fc protein. The mutated genes were subcloned into pcDNA4 (Invitrogen, Cat V86220), and finally, recombinant expression vectors for expressing the mutated scFV-Fc fusion protein and the mutated Fc protein in mammalian cells were obtained respectively.

[0197] 2. Transiently express the ScFv-Fc / Fc heterodimer and detect the effect of different mutation combinations on the content of the heterodimer

[0198] The expression vectors corresponding to the 4 mutation combinations in step 1, the KH combination (as the reference group), and an additional wild-type combination (i.e., the unmutated ScFv-Fc fusion protein and Fc protein, as the negative control group) were transfected into suspension-cultured 293H cells (ATCC CRL-1573) using PEI. Each mutation combination included the co-transfection of the recombinant expression vectors of its corresponding A chain (referring to the scFV-Fc fusion protein chain) and B chain (referring to the Fc protein chain), and the co-transfection ratio of the recombinant expression vectors of the A chain and B chain was 1:1. After culturing for 5 to 6 days, the transient expression culture supernatant was collected, and through Protein A affinity chromatography, the transient transfection products of the 4 mutation combinations, the KH mutation combination, and the wild-type negative control group were obtained after preliminary purification. These transient transfection products all contained different proportions of homodimer proteins (ScFv-Fc / ScFv-Fc, Fc / Fc) and heterodimer proteins (ScFv-Fc / Fc). Since the molecular weights of these three proteins (ScFv-Fc / ScFv-Fc, Fc / Fc, and ScFv-Fc / Fc) are different, the composition of the homodimer proteins (ScFv-Fc / ScFv-Fc, Fc / Fc) and heterodimer proteins (ScFv-Fc / Fc) in each group of products can be detected by SDS-PAGE electrophoresis under non-reducing conditions, and at the same time, the proportion of the homodimer proteins (ScFv-Fc / ScFv-Fc, Fc / Fc) and heterodimer proteins (ScFv-Fc / Fc) was analyzed using the professional image analysis software ImageLab launched by BioRad. The electrophoresis detection results are as Figure 1 and Table 3 show.

[0199] Table 3. Proportions of homodimers and heterodimers in the transient transfection products of each mutation combination

[0200]

[0201] The ratios of the four groups of candidate mutant combinations and the heterodimers (ScFv-Fc / Fc) in the KH combination all increased significantly compared with the wild-type negative control combination. At the same time, on the basis of KH, after introducing new mutations, the ratios of heterodimers also changed. Some increased significantly (such as combinations 2 and 4), while some increased moderately (such as combinations 1 and 3). It should be noted here that since these new mutant combinations involve the adjustment of two main interactions, namely the steric effect and the ionic effect of the side-chain groups on the contact surface, the influence of these mutations on the content of heterodimers cannot be simply considered as the superposition of the two effects. For example, although the introduction of the F405K mutation increased the repulsion between homodimers, the effect of increasing heterodimers in mutant combination 2 was much higher than that in mutant combination 1 (the content of heterodimers in mutant combination 2 was about 70%, while that in 1 was about 58%). In addition, for the mutations introduced at the K409 site, the increase in the content of heterodimers (77%) caused by the uncharged mutation in mutant combination 4 was much better than that caused by the opposite-charge mutation in mutant combination 3 (57%); theoretically, if only considering the superposition of the two interactions, the effects of these two mutations should be similar.

[0202] To further investigate the effect of the co - transfection ratio of the recombinant expression vectors of chain A and chain B on the ratios of homodimers and heterodimers, the two superior mutant combinations (2 and 4), and the co - transfection expression vectors used in the KH combination were transfected into suspension - cultured 293H cells (ATCC CRL - 1573) at ratios of 4:1 and 1:4 respectively using PEI. After culturing for 5 - 6 days, the cell supernatants were collected. Through Protein A affinity chromatography, the respective transient transfection products were obtained. The compositions of homodimer proteins (ScFv - Fc / ScFv - Fc, Fc / Fc) and heterodimer proteins (ScFv - Fc / Fc) were detected by SDS - PAGE electrophoresis under non - reducing conditions. The specific results are shown in Table 4. It can be seen from the results that: the co - transfection ratio of the recombinant expression vectors has a relatively obvious impact on the ratios of homodimers and heterodimers in the product. Whether the co - transfection ratio is 4:1 or 1:4, the content of heterodimers in the product is significantly reduced. This result indicates that although these three combinations can greatly increase the ratio of heterodimers and reduce the ratio of homodimers in the product when the expression of chain A and chain B is relatively balanced, when the expression of chain A and chain B in the product is unbalanced, resulting in an excess of chain A or chain B, the ratio of the excess product forming homodimers will increase, while the content of heterodimers will relatively decrease. Among them, in the KH combination, regardless of which chain is in excess, it will cause a significant reduction in the content of heterodimers; in mutant combination 2, the excess of chain B (Fc) has a greater impact; in mutant combination 4, the excess of chain A (ScFv - Fc) has a greater impact. However, even when chain B or chain A in mutant combination 2 or mutant combination 4 is in excess, the ratio of heterodimer formation is still significantly higher than that of the control KH combination. Further analysis of this result shows that among these three mutant combinations, although the interaction between chain A and chain B has been greatly enhanced, the weakening amplitude of the interaction between chain A and chain A or between chain B and chain B is still insufficient. This further causes the balance of homodimer and heterodimer formation to be broken and more homodimers to be produced when one of the components is overexpressed. Among them, the new mutant combinations 2 and 4 have relatively obvious optimization in preventing homodimer formation compared with the KH combination.

[0203] Table 4. Effect of different co - transfection ratios on the ratios of homodimers and heterodimers

[0204]

[0205] Example 3: Obtaining the sequences of the second - round mutant candidate combinations

[0206] Based on the preferred Fc mutation combinations (mutation combination 2 and mutation combination 4) mentioned in Examples 1 and 2, and according to the publicly available three-dimensional crystal structure of wild-type Fc, interfacial amino acid mutations were further introduced with the aim of further reducing the mutual attraction between chain A-chain A and chain B-chain B, and inhibiting the formation of homodimer proteins.

[0207] According to the results in Table 1, paired amino acids containing charged amino acids among the contacting amino acids near the mutation sites in mutation combination 2 and mutation combination 4 were further selected, and one amino acid on one of the chains was mutated (changing a non-charged amino acid to a charged amino acid, or a charged amino acid to a non-charged amino acid, or changing the charge property of the charged amino acid), so as to further increase the imbalance of ionic interactions between chain A and chain B, and reduce the probability of homodimer formation or simultaneously increase the probability of heterodimer formation.

[0208] For example, the pair of contacting amino acids Lys360 on chain A and Gln347 on chain B were mutated to change the ionic interaction between them. On one of the chains (such as chain A), both of these two amino acid residues were mutated to negatively charged amino acid residues, such as introducing two mutations K360E and Q347E; while on the other chain (such as chain B), the non-charged amino acid residue was mutated to a positively charged amino acid residue, such as introducing the mutation Q347R. At this time, when chain A-chain A interacts, the negative charges carried at positions 360 and 347 will repel each other; when chain B-chain B interacts, the positive charges at these two positions will repel each other; only when chain A and chain B interact, the positive and negative charges of each will attract each other. This mutation is expected to increase the mutual repulsion between the two chains of AA and BB, and at the same time increase the mutual attraction between the two chains of AB.

[0209] At the same time, the amino acid residue Leu368 was investigated. There are two charged amino acid residues, Glu357 and Lys409, around this residue. Considering that in the previous mutation combination 4, we had introduced the K409A mutation, in this case, on the same Fc chain where the K409A mutation had been introduced (designated as chain A according to Example 2), we further mutated Leu368 to a negatively charged amino acid residue (such as 368E). At this time, when chain A pairs with chain A, the negative charges carried by L368E on the two chains will interact with the negative charge on E357, introducing a repulsive force; when chain A pairs with chain B, the negative charge carried by L368E on chain A repels the negative charge on E357 on chain B, but at the same time attracts K409 on chain B. Overall, not too much repulsive or attractive force is introduced. This mutation is expected to increase the mutual repulsion between the two chains of AA, but does not affect the interaction between the two chains of AB or BB.

[0210] Based on the preferred Fc mutation combinations (mutation combination 2 and mutation combination 4) mentioned in Examples 1 and 2, plus the newly introduced mutation combinations, the resulting mutation combinations are shown in Table 5:

[0211] Table 5: List of Heterodimer Mutation Combinations - 2

[0212]

[0213] Example 4: Preparation and Investigation of a New Round of ScFv-Fc / Fc Heterodimer Mutation Combinations

[0214] 1. Construction of Recombinant Vectors Expressing Mutated Fc Fragments of Human IgG1 and ScFv-Fc Fusion Proteins

[0215] Using the recombinant vector for expressing wild-type scFV-Fc and Fc proteins constructed in Example 2 as a template, according to Table 5 in Example 3, the coding genes of scFV-Fc and Fc were subjected to combinatorial mutagenesis by overlap PCR. The mutation for the A chain is located on the scFV-Fc fusion protein, and the mutation for the B chain is located on the Fc protein. The mutated genes were subcloned into pcDNA4 (Invitrogen, Cat V86220), and finally, a recombinant expression vector was obtained for expressing a new round of mutated scFV-Fc fusion proteins and mutated Fc proteins in mammalian cells.

[0216] 2. Transient Expression of ScFv-Fc / Fc Heterodimers and Detection of the Effects of Different Mutation Combinations on the Content of Heterodimers

[0217] According to the method described in Example 2-2, five new mutant combinations (5 to 9), as well as the preferred mutant combinations (2 and 4) of the first round, were transiently expressed using 293H cells (ATCC CRL-1573). The co-transfection ratio of the recombinant expression vectors of chain A and chain B was 1:1. After culturing for 5 to 6 days, the transient expression culture supernatant was collected, and through Protein A affinity chromatography, the transient transfection products of 5 groups of new mutant combinations and 2 groups of preferred combinations in the first round were obtained. These transient transfection products all contained different proportions of homodimer proteins (ScFv-Fc / ScFv-Fc, Fc / Fc) and heterodimer proteins (ScFv-Fc / Fc). Since the molecular weights of these three proteins (ScFv-Fc / ScFv-Fc, Fc / Fc, and ScFv-Fc / Fc) are different, the composition of homodimer proteins (ScFv-Fc / ScFv-Fc, Fc / Fc) and heterodimer proteins (ScFv-Fc / Fc) in each group of products can be detected by SDS-PAGE electrophoresis under non-reducing conditions. At the same time, the proportions of homodimer proteins (ScFv-Fc / ScFv-Fc, Fc / Fc) and heterodimer proteins (ScFv-Fc / Fc) were analyzed using the professional image analysis software ImageLab launched by BioRad. The electrophoresis detection results are as Figure 2 shown in Table 6.

[0218] Table 6. Proportions of Homodimers and Heterodimers in Transient Transfection Products of Each Mutant Combination - 2

[0219]

[0220] Compared with the preferred combinations of the first-round mutations, some of the newly introduced mutations slightly increased the proportion of heterodimer formation, such as combination 5 compared with combination 2; several groups had little change, such as combination 6 compared with combination 2, and combinations 7 and 8 compared with combination 4; in addition, after introducing new mutations in combination 9, the proportion of heterodimer formation decreased significantly instead. It is speculated that the negative charge carried by the newly introduced L368E on chain A repels the negative charge on E357 on chain B more than it attracts K409 on chain B, making the heterodimer unstable. Generally speaking, although several of the newly introduced mutations are helpful for the formation of heterodimers, they do not bring a significant increase.

[0221] To further investigate the effects of the newly introduced mutations on the A-chain / A-chain and B-chain / B-chain homodimers, we transiently expressed the A-chain protein or B-chain protein alone and examined the trend of homodimer formation by comparing the expression levels of the homodimer proteins under the same transient transfection conditions. The recombinant expression vectors were transfected into suspension-cultured 293H cells (ATCC CRL-1573) using PEI respectively. After culturing for 5-6 days, the cell supernatants were collected. The respective transient transfection products were obtained by Protein A affinity chromatography, and their expression levels were detected using OD280. The results are shown in Table 7. From the perspective of the expression levels, some of the mutations introduced into chain A in combination 4 (combinations 8 and 9) could reduce the trend of its homodimer formation; some of the mutations introduced into chain B in combination 2 (combination 5) could reduce the trend of its homodimer formation; the remaining new mutations had little effect on the formation of homodimers. At the same time, it can also be seen from these results that compared with combination 4 and the mutant combinations derived from combination 4 (7, 8, 9), the trend of chain A to form homodimers in combination 2 and the mutant combinations derived from combination 2 (5, 6) was smaller; while the trend of chain B in the latter to form homodimers was relatively smaller than that in the former. This result is consistent with the result obtained in Example 2 and further proves the feasibility of preliminarily investigating the trend of homodimer formation by this method. In addition, from the perspective of the expression levels, all of chain B were much lower than chain A. We also performed separate transient expressions of wild-type chain A and wild-type chain B and found that in the absence of any mutations, the expression level of the wild-type B-chain homodimer was lower than that of the wild-type A-chain (the former was about half of the latter). It can be inferred that the fusion of the ScFv sequence to the N-terminus of the Fc sequence in chain A contributed to improving its expression level; and the difference in the expression levels between chain A and chain B could not directly reflect the difference in the trend of homodimer formation between chain A and chain B respectively.

[0222] Table 7. Comparison of the expression levels of homodimers after separate transient transfection of chain A or chain B in each mutant combination

[0223]

[0224] Example 5: Obtaining the sequences of the third-round mutant candidate combinations

[0225] Based on the crystal structure of mutant combination 4 and in combination with structural modeling, new candidate sequences of amino acid mutations at the contact interface were identified, with the expectation of further inhibiting the formation of homodimer proteins or promoting the formation of heterodimer proteins on the basis of the original mutant combinations (such as mutant combination 2 or 4).

[0226] Crystal structure analysis of the heterodimer protein of mutant combination 4

[0227] Select mutation combination 4, transiently express and purify the heterodimeric protein of mutation combination 4 in 293H cells (ATCC CRL-1573), and perform crystal structure analysis. Here, we inserted a His-tag sequence at the C-terminus of chain B of mutation combination 4 using molecular cloning, so that after Protein A affinity chromatography, a relatively pure chain A-chain B heterodimeric protein can be obtained by the IMAC method for crystallization.

[0228] The crystal structure analysis process is as follows:

[0229] Heterodimeric Fc crystals were formed under the following conditions: 2 μL of crystallization buffer (15% PEG3350, 1 M LiCl, 0.1 M MES, pH 6.0) was mixed with 2 μL of protein solution (10 mg / mL target protein, 10 mM Tris, 150 mM NaCl, pH 7.4), and crystallized in a sitting drop at 22 °C. Crystals grew out after about 3 days. Then the crystals were placed in the following solution: 17% PEG3350, 1 M LiCl, 0.1 M MES, pH 6.0 and 20% glycerol; subsequently, they were quickly infiltrated and frozen in liquid nitrogen. X-ray diffraction data were collected at SSRF BL17U. The structure was solved by molecular replacement using the structure of wild-type Fc (PDB accession number: 3AVE) as a template.

[0230] The crystal structure shows that the overall structure of the mutant Fc heterodimer is similar to that of wild-type Fc, but it is changed due to the interaction of different side-chain groups at the CH3 interface where the mutation was introduced. The specific crystal structure of the CH3 interface is shown in Figure 3 .

[0231] 2. Obtaining new mutant candidate combinations

[0232] Based on the results of the crystal structure of mutation combination 4, further screen for new candidate mutations.

[0233] First, we found through the three-dimensional crystal structure that Y349C on chain A and D356C on chain B cannot form a disulfide bond due to the orientation of the two Cys side-chain groups, but become a pair of free sulfhydryl groups. According to this result, we will cancel this pair of mutations in the third round of mutation to restore it to the wild-type amino acid sequence before mutation.

[0234] Second, through three-dimensional structure modeling comparison, further mutations were introduced near the pair of mutant amino acid residues F405K-K409A to change ionic bonds and hydrogen bonds. Assume that K409A is on chain A and F405K is on chain B. Then introduce the K392D mutation on chain A and the D399S mutation on chain B. As Figure 4As shown, for the interaction between chain A and chain B, the newly introduced mutations add a pair of ionic bonds, K392D-F405K, and a pair of hydrogen bonds, K392D-D399S, which are expected to effectively increase the tendency of heterodimer formation. In the A-chain / A-chain interaction, an electrostatic repulsion between K329D and D399 is introduced, inhibiting the formation of A-chain homodimers. In the B-chain / B-chain interaction, the original ionic bond between K409 and D399 disappears due to the introduction of the D399S mutation, reducing the tendency of B-chain homodimer formation.

[0235] Third, comparing the crystal structure of mutant combination 4 with that of the wild-type Fc protein, it was found that chain A in mutant combination 4 shifted outward (i.e., away from chain B). It is speculated that this might be due to the larger side-chain group in the T366W mutation of chain A, which brings a certain steric hindrance. On this basis, we further mutated the amino acid residues on chain B that contact the T366W residue in chain A to amino acid residues with smaller side-chain groups. For example, on chain B, the original Y407V and L368A mutations were replaced with two mutations, Y407A and L386G, to leave enough space for the T366W mutation, which might further stabilize the heterodimer structure.

[0236] Fourth, outside the pair of mutant amino acid residues F405K-K409A, other contacting interface amino acid pairs were mutated to change the interface electrostatic interaction. Here, the contacting amino acid pair Y349 and E357 was investigated. Mutation Y349D was introduced into chain A, and E357A was introduced into chain B. Then, the electrostatic repulsion introduced between Y349D and E357A in A-A will inhibit the formation of A-A homodimers; no new interactions were introduced between A-B and B-B. On this basis, mutation S354D was further introduced into chain A to strengthen the electrostatic repulsion between it and E357A, further inhibiting the formation of A-A homodimers.

[0237] First, based on mutant combination 4, the above mutations were introduced, and the resulting mutant combinations are shown in Table 8:

[0238] Table 8: List of Heterodimer Mutant Combinations - 3

[0239]

[0240] Subsequently, based on mutant combination 2, the above mutations were introduced while referring to mutant combination 4, and the resulting mutant combinations are shown in Table 9:

[0241] Table 9: List of Heterodimer Mutant Combinations - 4

[0242]

[0243] Example 6: Preparation and investigation of the third round of ScFv-Fc / VhH-Fc heterodimer mutant combinations

[0244] 1. Construction of recombinant vectors expressing the Fc fragment of mutant human IgG1 and the ScFv-Fc fusion protein

[0245] Considering that the expression level of the simple Fc fragment is lower than that of scFv-Fc, in order to better control the expression ratio of the two chains, we fused a variable region sequence of a camel single-domain antibody (labeled as VhH) to the N-terminus of the original B chain (simple Fc chain). The coding gene of the VhH-Fc fusion protein shown in SEQ ID NO: 36 was obtained by artificial synthesis. The amino acid sequence of the VhH-Fc fusion protein encoded by this gene is shown in SEQ ID: 35. Then it was subcloned into the mammalian cell expression vector pcDNA4 (Invitrogen, CatV86220) to obtain a recombinant expression vector for expressing the VhH-Fc fusion protein in mammalian cells.

[0246] Using the recombinant expression vector of the wild-type scFV-Fc protein constructed in Example 2 and the recombinant expression vector of the above VhH-Fc fusion protein as templates, according to Table 8 of Example 5, the coding genes of scFV-Fc and VhH-Fc (SEQ ID NO: 5 and SEQ ID NO: 36) were combinatorially mutated by the overlap PCR method. The mutation of the A chain was located on the scFV-Fc fusion protein, and the mutation of the B chain was located on the VhH-Fc protein. The mutated genes were subcloned into pcDNA4 (Invitrogen, Cat V86220), and finally a recombinant expression vector for expressing the third-round mutant scFV-Fc fusion protein and mutant VhH-Fc protein (SEQ ID NO: 4 to SEQ ID NO: 35) in mammalian cells was obtained.

[0247] 2. Transient expression of the ScFv-Fc / VhH-Fc heterodimer and detection of the effect of different mutant combinations on the heterodimer content

[0248] According to the method described in Example 2-2, four mutant combinations (10 to 13) in Table 8, as well as mutant combination 4, were transiently expressed using 293H cells (ATCC CRL-1573). The co-transfection ratio of the recombinant expression vectors of chain A and chain B was 4:1, 1:1, and 1:4. After culturing for 5 to 6 days, the transient expression culture supernatant was collected, and four newly mutant combinations and the transient transfection products of mutant combination 4 were obtained through Protein A affinity chromatography. These transient transfection products all contained different proportions of homodimer proteins (ScFv-Fc / ScFv-Fc, VhH-Fc / VhH-Fc) and heterodimer proteins (ScFv-Fc / VhH-Fc). Since the molecular weights of these three proteins (ScFv-Fc / ScFv-Fc, VhH-Fc / VhH-Fc, and ScFv-Fc / VhH-Fc) were different, the composition of homodimer proteins (ScFv-Fc / ScFv-Fc, VhH-Fc / VhH-Fc) and heterodimer proteins (ScFv-Fc / VhH-Fc) in each group of products could be detected by SDS-PAGE electrophoresis under non-reducing conditions. At the same time, the proportion of homodimer proteins (ScFv-Fc / ScFv-Fc, VhH-Fc / VhH-Fc) and heterodimer proteins (ScFv-Fc / VhH-Fc) was analyzed using the professional image analysis software ImageLab launched by BioRad. The electrophoresis detection results are shown in Table 10.

[0249] Table 10. Proportions of Homodimers and Heterodimers in Transient Transfection Products of Each Mutant Combination - 3

[0250]

[0251] To further investigate the effect of the newly introduced mutations on the A-chain-A-chain and B-chain-B-chain homodimers, we transiently expressed the A-chain protein or B-chain protein alone, and by comparing the expression levels of homodimer proteins under the same transient transfection conditions, we investigated the trend of homodimer formation. The recombinant expression vectors were transfected into suspension-cultured 293H cells (ATCC CRL-1573) using PEI respectively. After culturing for 5 - 6 days, the cell supernatant was collected. Through Protein A affinity chromatography, the respective transient transfection products were obtained, and their expression levels were detected using OD280. The results are shown in Table 11.

[0252] Table 11. Comparison of Homodimer Expression Levels of Chain A or Chain B in Each Mutant Combination - 2

[0253]

[0254] Based on the above results, it can be seen that after introducing the third round of mutations to mutant combination 4, although no significant effect was shown in inhibiting the formation of strand A homodimers, the formation of strand B homodimers was significantly inhibited and the formation of heterodimers was effectively promoted. When the expression of the two strands was close to equilibrium (1:1), the content of heterodimers in several new mutant combinations could reach over 80%, showing a significant improvement compared to mutant combination 4. Among them, in mutant combination 11, the new mutations targeting strand B could basically completely inhibit the formation of strand B homodimers. It can be seen that even at a transient transfection ratio of 1:4 (A:B), no strand B homodimers were observed, and the content of heterodimers reached 89%.

[0255] Based on the results of combinations 10 to 13, we further selected mutant combinations 15, 16, and 18 and examined their effects on promoting heterodimer formation through transient expression.

[0256] According to the method described in Example 2-2, three mutation combinations (15, 16, 18) in Table 9 and mutation combination 2 were transiently expressed using 293H cells (ATCC CRL-1573). The co-transfection ratio of the recombinant expression vectors of chain A and chain B was 4:1, 1:1, and 1:4. After culturing for 5 to 6 days, the transient expression culture supernatant was collected, and three newly mutated combinations and the transient transfection products of mutation combination 2 were preliminarily purified by Protein A affinity chromatography. These transient transfection products all contained different proportions of homodimer proteins (ScFv-Fc / ScFv-Fc, VhH-Fc / VhH-Fc) and heterodimer proteins (ScFv-Fc / VhH-Fc). Since the molecular weights of these three proteins (ScFv-Fc / ScFv-Fc, VhH-Fc / VhH-Fc, and ScFv-Fc / VhH-Fc) are different, the composition of homodimer proteins (ScFv-Fc / ScFv-Fc, VhH-Fc / VhH-Fc) and heterodimer proteins (ScFv-Fc / VhH-Fc) in each group of products can be detected by SDS-PAGE electrophoresis under non-reducing conditions. At the same time, the proportion of homodimer proteins (ScFv-Fc / ScFv-Fc, VhH-Fc / VhH-Fc) and heterodimer proteins (ScFv-Fc / VhH-Fc) was analyzed using the professional image analysis software ImageLab launched by BioRad. The electrophoresis detection results are shown in Table 12. It can be seen that after introducing the third round of mutations into mutation combination 2, it also showed a more significant effect in inhibiting the formation of homodimers of chain B and effectively promoted the formation of heterodimers. When the expression of the two chains was close to equilibrium (1:1), the content of heterodimers in several newly mutated combinations could reach more than 80%, which was significantly higher than that of mutation combination 4. Among them, the proportion of heterodimers in mutation combinations 16 and 18 still reached more than 80% when the transient transfection vector ratio was appropriately changed (excess plasmid of chain B or balance of the two plasmids).

[0257] Table 12. Proportions of Homodimers and Heterodimers in Transient Transfection Products of Each Mutation Combination - 4

[0258]

[0259] Example 7: Assessment of Other Indicators of Heterodimers

[0260] 1. Accelerated Stability Detection of Heterodimers

[0261] We selected the heterodimers of mutation combinations 4, 11, and 16 for the accelerated stability experiment. The experimental period was 31 days, the temperature was 45 °C, and the buffer was PBS. Non-reducing CE-SDS was detected on day 0, day 8, day 18, and day 31, and compared with the corresponding wild-type Fc protein. The 31-day accelerated stability SDS-PAGE results showed that for the three mutant samples and the wild-type control sample, the decrease in the main peak content did not exceed 2% until day 31. It can be considered that the heterodimer has the same thermal stability as the wild-type.

[0262] Although the specific embodiments of the invention described in this application have been described in detail, those skilled in the art will understand that various modifications and substitutions can be made to those details based on all the teachings that have been disclosed, and these changes are within the protection scope of the invention described in this application. The full scope of the invention described in this application is given by the appended claims and any equivalents thereof. Sequence Listing <110> Jiangsu Conning Pharmaceutical Co., Ltd.; Suzhou Conning Pharmaceutical Technology Co., Ltd. <120> CH3 Domain-Based Heterodimer Molecules, Their Preparation Methods and Uses <130> 0041-PA-002CN.DIV1 <160> 36 <170> PatentIn version 3.5 <210> 1 <211> 227 <212> PRT <213> Homo sapiens <400> 1 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> 2 <211> 681 <212> DNA <213> Homo sapiens <400> 2 gacaagaccc acacctgccc cccctgcccc gcccccgagc tgctgggcgg ccccagcgtg 60 ttcctgttcc cccccaagcc caaggacacc ctgatgatca gccgcacccc cgaggtgacc 120 tgcgtggtgg tggacgtgag ccacgagaac cccgaggtga agttcaactg gtacgtggac 180 ggcgtggagg tgcacaacgc caagaccaag ccccgcgagg agcagtacaa cagcacctac 240 cgcgtggtga gcgtgctgac cgtgctgcac caggactggc tgaacggcaa ggagtacaag 300 tgcaaggtga gcaacaaggc cctgcccgcc cccatcgaga agaccatcag caaggccaag 360 ggccagcccc gcgagcccca ggtgtacacc ctgcccccca gccgcgacga gctgaccaag 420 aaccaggtga gcctgacctg cctggtgaag ggcttctacc ccagcgacat cgccgtggag 480 tgggagagca acggccagcc cgagaacaac tacaagacca ccccccccgt gctggacagc 540 gacggcagct tcttcctgta cagcaagctg accgtggaca agagccgctg gcagcagggc 600 aacgtgttca gctgcagcgt gatgcacgag gccctgcaca accactacac ccagaagagc 660 ctgagcctga gccccggcaa g 681 <210> 3 <211> 741 <212> DNA <213> Artificial Sequence <220> <223> kappaIII Signal Peptide <400> 3 atggagaccg acaccctgct gctgtgggtg ctgctgctgt gggtgcccgg cagcaccggc 60 gacaagaccc acacctgccc cccctgcccc gcccccgagc tgctgggcgg ccccagcgtg 120 ttcctgttcc cccccaagcc caaggacacc ctgatgatca gccgcacccc cgaggtgacc 180 tgcgtggtgg tggacgtgag ccacgagaac cccgaggtga agttcaactg gtacgtggac 240 ggcgtggagg tgcacaacgc caagaccaag ccccgcgagg agcagtacaa cagcacctac 300 cgcgtggtga gcgtgctgac cgtgctgcac caggactggc tgaacggcaa ggagtacaag 360 tgcaaggtga gcaacaaggc cctgcccgcc cccatcgaga agaccatcag caaggccaag 420 ggccagcccc gcgagcccca ggtgtacacc ctgcccccca gccgcgacga gctgaccaag 480 aaccaggtga gcctgacctg cctggtgaag ggcttctacc ccagcgacat cgccgtggag 540 tgggagagca acggccagcc cgagaacaac tacaagacca ccccccccgt gctggacagc 600 gacggcagct tcttcctgta cagcaagctg accgtggaca agagccgctg gcagcagggc 660 aacgtgttca gctgcagcgt gatgcacgag gccctgcaca accactacac ccagaagagc 720 ctgagcctga gccccggcaa g 741 <210> 4 <211> 471 <212> PRT <213> Artificial sequence <220> <223> ScFv-Fc fusion protein <400> 4 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ile Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Pro Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Ser Ile Ser Tyr Asp Gly Ser Tyr Lys Tyr Lys Ala Asp Ser Met 50 55 60 Lys Gly Arg Leu Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Glu Met Asn Ser Leu Thr Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Thr Ala Phe Phe Asn Ala Tyr Asp Phe Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Gly Gly 115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Met Thr Gln Ser Pro 130 135 140 Ala Thr Leu Ser Val Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg 145 150 155 160 Ala Ser Gln Ser Val Arg Ser Asn Leu Ala Trp Tyr Gln Gln Lys Pro 165 170 175 Gly Gln Ala Pro Arg Leu Leu Ile Tyr Ala Ala Ser Thr Arg Ala Thr 180 185 190 Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr 195 200 205 Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp Phe Ala Val Tyr Tyr Cys 210 215 220 Gln Gln Tyr Asn Glu Trp Phe Arg Thr Ser Gly Gln Gly Thr Lys Val 225 230 235 240 Glu Ile Lys Arg Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 245 250 255 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 260 265 270 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 275 280 285 Asp Val Ser His Glu Asn Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 290 295 300 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 305 310 315 320 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 325 330 335 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 340 345 350 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 355 360 365 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 370 375 380 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 385 390 395 400 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 405 410 415 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 420 425 430 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 435 440 445 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 450 455 460 Leu Ser Leu Ser Pro Gly Lys 465 470 <210> 5 <211> 1473 <212> DNA <213> artificial sequence <220> <223> ScFv-Fc fusion protein coding gene <400> 5 atggagaccg acaccctgct gctgtgggtg ctgctgctgt gggtgcccgg cagcaccggc 60 gaggtgcagc tgctggagag cggcggcggc gtggtgcagc ccggccgcag cctgcgcctg 120 agctgcatcg ccagcggctt caccttcagc agctacccca tgacctgggt gcgccaggcc 180 agctgcatcg ccagcggctt caccttcagc agctacccca tgacctgggt gcgccaggcc 180 cccggcaagg gcctggagtg ggtggccagc atcagctacg acggcagcta caagtacaag 240 cccggcaagg gcctggagtg ggtggccagc atcagctacg acggcagcta caagtacaag 240 gccgacagca tgaagggccg cctgaccatc agccgcgaca acagcaagaa caccctgtac 300 gccgacagca tgaagggccg cctgaccatc agccgcgaca acagcaagaa caccctgtac 300 ctggagatga acagcctgac cgccgaggac accgccgtgt actactgcgc ccgcaccgcc 360 ctggagatga acagcctgac cgccgaggac accgccgtgt actactgcgc ccgcaccgcc 360 ttcttcaacg cctacgactt ctggggccag ggcaccctgg tgaccgtgag cagcgccagc 420 ttcttcaacg cctacgactt ctggggccag ggcaccctgg tgaccgtgag cagcgccagc 420 accaagggcc ccagcgtggg cggcggcggc agcggcggcg gcggcagcga gatcgtgatg 480 accaagggcc ccagcgtggg cggcggcggc agcggcggcg gcggcagcga gatcgtgatg 480 acccagagcc ccgccaccct gagcgtgagc cccggcgagc gcgccaccct gagctgccgc 540 acccagagcc ccgccaccct gagcgtgagc cccggcgagc gcgccaccct gagctgccgc 540 gccagccaga gcgtgcgcag caacctggcc tggtaccagc agaagcccgg ccaggccccc 600 gccagccaga gcgtgcgcag caacctggcc tggtaccagc agaagcccgg ccaggccccc 600 cgcctgctga tctacgccgc cagcacccgc gccaccggca tccccgcccg cttcagcggc 660 cgcctgctga tctacgccgc cagcacccgc gccaccggca tccccgcccg cttcagcggc 660 agcggcagcg gcaccgagtt caccctgacc atcagcagcc tgcagagcga ggacttcgcc 720 agcggcagcg gcaccgagtt caccctgacc atcagcagcc tgcagagcga ggacttcgcc 720 gtgtactact gccagcagta caacgagtgg ttccgcacca gcggccaggg caccaaggtg 780 gtgtactact gccagcagta caacgagtgg ttccgcacca gcggccaggg caccaaggtg 780 gagatcaagc gcgacaagac ccacacctgc cccccctgcc ccgcccccga gctgctgggc 840 gagatcaagc gcgacaagac ccacacctgc cccccctgcc ccgcccccga gctgctgggc 840 ggccccagcg tgttcctgtt cccccccaag cccaaggaca ccctgatgat cagccgcacc 900 cccgaggtga cctgcgtggt ggtggacgtg agccacgaga accccgaggt gaagttcaac 960 tggtacgtgg acggcgtgga ggtgcacaac gccaagacca agccccgcga ggagcagtac 1020 aacagcacct accgcgtggt gagcgtgctg accgtgctgc accaggactg gctgaacggc 1080 aaggagtaca agtgcaaggt gagcaacaag gccctgcccg cccccatcga gaagaccatc 1140 agcaaggcca agggccagcc ccgcgagccc caggtgtaca ccctgccccc cagccgcgac 1200 gagctgacca agaaccaggt gagcctgacc tgcctggtga agggcttcta ccccagcgac 1260 atcgccgtgg agtgggagag caacggccag cccgagaaca actacaagac cacccccccc 1320 gtgctggaca gcgacggcag cttcttcctg tacagcaagc tgaccgtgga caagagccgc 1380 tggcagcagg gcaacgtgtt cagctgcagc gtgatgcacg aggccctgca caaccactac 1440 acccagaaga gcctgagcct gagccccggc aag 1473 <210> 6 <211> 471 <212> PRT <213> Artificial Sequence <220> <223> Y349C+T366W <400> 6 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ile Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Pro Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Ser Ile Ser Tyr Asp Gly Ser Tyr Lys Tyr Lys Ala Asp Ser Met 50 55 60 Lys Gly Arg Leu Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Glu Met Asn Ser Leu Thr Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Thr Ala Phe Phe Asn Ala Tyr Asp Phe Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Gly Gly 115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Met Thr Gln Ser Pro 130 135 140 Ala Thr Leu Ser Val Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg 145 150 155 160 Ala Ser Gln Ser Val Arg Ser Asn Leu Ala Trp Tyr Gln Gln Lys Pro 165 170 175 Gly Gln Ala Pro Arg Leu Leu Ile Tyr Ala Ala Ser Thr Arg Ala Thr 180 185 190 Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr 195 200 205 Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp Phe Ala Val Tyr Tyr Cys 210 215 220 Gln Gln Tyr Asn Glu Trp Phe Arg Thr Ser Gly Gln Gly Thr Lys Val 225 230 235 240 Glu Ile Lys Arg Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 245 250 255 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 260 265 270 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 275 280 285 Asp Val Ser His Glu Asn Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 290 295 300 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 305 310 315 320 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 325 330 335 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 340 345 350 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 355 360 365 Glu Pro Gln Val Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 370 375 380 Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 385 390 395 400 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 405 410 415 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 420 425 430 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 435 440 445 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 450 455 460 Leu Ser Leu Ser Pro Gly Lys 465 470 <210> 7 <211> 227 <212> PRT <213> artificial sequence <220> <223> D356C+T366S+L368A+Y407V <400> 7 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 Cys 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> 8 <211> 227 <212> PRT <213> artificial sequence <220> <223> D356C+T366S+L368A+Y407V+F405K <400> 8 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 Cys 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 Lys 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> 9 <211> 471 <212> PRT <213> artificial sequence <220> <223> Y349C+T366W+F405K <400> 9 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ile Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Pro Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Ser Ile Ser Tyr Asp Gly Ser Tyr Lys Tyr Lys Ala Asp Ser Met 50 55 60 Lys Gly Arg Leu Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Glu Met Asn Ser Leu Thr Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Thr Ala Phe Phe Asn Ala Tyr Asp Phe Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Gly Gly 115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Met Thr Gln Ser Pro 130 135 140 Ala Thr Leu Ser Val Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg 145 150 155 160 Ala Ser Gln Ser Val Arg Ser Asn Leu Ala Trp Tyr Gln Gln Lys Pro 165 170 175 Gly Gln Ala Pro Arg Leu Leu Ile Tyr Ala Ala Ser Thr Arg Ala Thr 180 185 190 Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr 195 200 205 Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp Phe Ala Val Tyr Tyr Cys 210 215 220 Gln Gln Tyr Asn Glu Trp Phe Arg Thr Ser Gly Gln Gly Thr Lys Val 225 230 235 240 Glu Ile Lys Arg Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 245 250 255 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 260 265 270 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 275 280 285 Asp Val Ser His Glu Asn Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 290 295 300 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 305 310 315 320 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 325 330 335 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 340 345 350 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 355 360 365 Glu Pro Gln Val Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 370 375 380 Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 385 390 395 400 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 405 410 415 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Lys Leu Tyr Ser 420 425 430 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 435 440 445 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 450 455 460 Leu Ser Leu Ser Pro Gly Lys 465 470 <210> 10 <211> 471 <212> PRT <213> artificial sequence <220> <223> Y349C+T366W+K409E <400> 10 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ile Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Pro Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Ser Ile Ser Tyr Asp Gly Ser Tyr Lys Tyr Lys Ala Asp Ser Met 50 55 60 Lys Gly Arg Leu Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Glu Met Asn Ser Leu Thr Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Thr Ala Phe Phe Asn Ala Tyr Asp Phe Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Gly Gly 115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Met Thr Gln Ser Pro 130 135 140 Ala Thr Leu Ser Val Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg 145 150 155 160 Ala Ser Gln Ser Val Arg Ser Asn Leu Ala Trp Tyr Gln Gln Lys Pro 165 170 175 Gly Gln Ala Pro Arg Leu Leu Ile Tyr Ala Ala Ser Thr Arg Ala Thr 180 185 190 Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr 195 200 205 Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp Phe Ala Val Tyr Tyr Cys 210 215 220 Gln Gln Tyr Asn Glu Trp Phe Arg Thr Ser Gly Gln Gly Thr Lys Val 225 230 235 240 Glu Ile Lys Arg Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 245 250 255 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 260 265 270 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 275 280 285 Asp Val Ser His Glu Asn Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 290 295 300 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 305 310 315 320 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 325 330 335 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 340 345 350 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 355 360 365 Glu Pro Gln Val Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 370 375 380 Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 385 390 395 400 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 405 410 415 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 420 425 430 Glu Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 435 440 445 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 450 455 460 Leu Ser Leu Ser Pro Gly Lys 465 470 <210> 11 <211> 471 <212> PRT <213> artificial sequence <220> <223> Y349C+T366W+K409A <400> 11 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ile Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Pro Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Ser Ile Ser Tyr Asp Gly Ser Tyr Lys Tyr Lys Ala Asp Ser Met 50 55 60 Lys Gly Arg Leu Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Glu Met Asn Ser Leu Thr Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Thr Ala Phe Phe Asn Ala Tyr Asp Phe Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Gly Gly 115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Met Thr Gln Ser Pro 130 135 140 Ala Thr Leu Ser Val Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg 145 150 155 160 Ala Ser Gln Ser Val Arg Ser Asn Leu Ala Trp Tyr Gln Gln Lys Pro 165 170 175 Gly Gln Ala Pro Arg Leu Leu Ile Tyr Ala Ala Ser Thr Arg Ala Thr 180 185 190 Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr 195 200 205 Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp Phe Ala Val Tyr Tyr Cys 210 215 220 Gln Gln Tyr Asn Glu Trp Phe Arg Thr Ser Gly Gln Gly Thr Lys Val 225 230 235 240 Glu Ile Lys Arg Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 245 250 255 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 260 265 270 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 275 280 285 Asp Val Ser His Glu Asn Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 290 295 300 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 305 310 315 320 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 325 330 335 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 340 345 350 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 355 360 365 Glu Pro Gln Val Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 370 375 380 Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 385 390 395 400 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 405 410 415 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 420 425 430 Ala Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 435 440 445 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 450 455 460 Leu Ser Leu Ser Pro Gly Lys 465 470 <210> 12 <211> 471 <212> PRT <213> Artificial Sequence <220> <223> Y349C+T366W+F405K+K360E+Q347E <400> 12 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ile Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Pro Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Ser Ile Ser Tyr Asp Gly Ser Tyr Lys Tyr Lys Ala Asp Ser Met 50 55 60 Lys Gly Arg Leu Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Glu Met Asn Ser Leu Thr Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Thr Ala Phe Phe Asn Ala Tyr Asp Phe Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Gly Gly 115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Met Thr Gln Ser Pro 130 135 140 Ala Thr Leu Ser Val Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg 145 150 155 160 Ala Ser Gln Ser Val Arg Ser Asn Leu Ala Trp Tyr Gln Gln Lys Pro 165 170 175 Gly Gln Ala Pro Arg Leu Leu Ile Tyr Ala Ala Ser Thr Arg Ala Thr 180 185 190 Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr 195 200 205 Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp Phe Ala Val Tyr Tyr Cys 210 215 220 Gln Gln Tyr Asn Glu Trp Phe Arg Thr Ser Gly Gln Gly Thr Lys Val 225 230 235 240 Glu Ile Lys Arg Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 245 250 255 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 260 265 270 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 275 280 285 Asp Val Ser His Glu Asn Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 290 295 300 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 305 310 315 320 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 325 330 335 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 340 345 350 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 355 360 365 Glu Pro Glu Val Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Glu 370 375 380 Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 385 390 395 400 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 405 410 415 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Lys Leu Tyr Ser 420 425 430 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 435 440 445 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 450 455 460 Leu Ser Leu Ser Pro Gly Lys 465 470 <210> 13 <211> 227 <212> PRT <213> artificial sequence <220> <223> D356C+T366S+L368A+Y407V+Q347R <400> 13 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 Arg Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Cys 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> 14 <211> 471 <212> PRT <213> artificial sequence <220> <223> Y349C+T366W+F405K+Q347R <400> 14 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ile Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Pro Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Ser Ile Ser Tyr Asp Gly Ser Tyr Lys Tyr Lys Ala Asp Ser Met 50 55 60 Lys Gly Arg Leu Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Glu Met Asn Ser Leu Thr Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Thr Ala Phe Phe Asn Ala Tyr Asp Phe Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Gly Gly 115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Met Thr Gln Ser Pro 130 135 140 Ala Thr Leu Ser Val Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg 145 150 155 160 Ala Ser Gln Ser Val Arg Ser Asn Leu Ala Trp Tyr Gln Gln Lys Pro 165 170 175 Gly Gln Ala Pro Arg Leu Leu Ile Tyr Ala Ala Ser Thr Arg Ala Thr 180 185 190 Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr 195 200 205 Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp Phe Ala Val Tyr Tyr Cys 210 215 220 Gln Gln Tyr Asn Glu Trp Phe Arg Thr Ser Gly Gln Gly Thr Lys Val 225 230 235 240 Glu Ile Lys Arg Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 245 250 255 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 260 265 270 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 275 280 285 Asp Val Ser His Glu Asn Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 290 295 300 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 305 310 315 320 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 325 330 335 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 340 345 350 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 355 360 365 Glu Pro Arg Val Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 370 375 380 Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 385 390 395 400 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 405 410 415 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Lys Leu Tyr Ser 420 425 430 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 435 440 445 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 450 455 460 Leu Ser Leu Ser Pro Gly Lys 465 470 <210> 15 <211> 227 <212> PRT <213> Artificial Sequence <220> <223> D356C+T366S+L368A+Y407V+K360E+Q347E <400> 15 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 Glu Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Cys Glu Leu Thr Glu 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> 16 <211> 471 <212> PRT <213> artificial sequence <220> <223> Y349C+T366W+K409A+K360E+Q347E <400> 16 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ile Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Pro Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Ser Ile Ser Tyr Asp Gly Ser Tyr Lys Tyr Lys Ala Asp Ser Met 50 55 60 Lys Gly Arg Leu Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Glu Met Asn Ser Leu Thr Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Thr Ala Phe Phe Asn Ala Tyr Asp Phe Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Gly Gly 115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Met Thr Gln Ser Pro 130 135 140 Ala Thr Leu Ser Val Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg 145 150 155 160 Ala Ser Gln Ser Val Arg Ser Asn Leu Ala Trp Tyr Gln Gln Lys Pro 165 170 175 Gly Gln Ala Pro Arg Leu Leu Ile Tyr Ala Ala Ser Thr Arg Ala Thr 180 185 190 Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr 195 200 205 Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp Phe Ala Val Tyr Tyr Cys 210 215 220 Gln Gln Tyr Asn Glu Trp Phe Arg Thr Ser Gly Gln Gly Thr Lys Val 225 230 235 240 Glu Ile Lys Arg Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 245 250 255 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 260 265 270 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 275 280 285 Asp Val Ser His Glu Asn Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 290 295 300 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 305 310 315 320 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 325 330 335 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 340 345 350 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 355 360 365 Glu Pro Glu Val Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Glu 370 375 380 Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 385 390 395 400 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 405 410 415 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 420 425 430 Ala Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 435 440 445 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 450 455 460 Leu Ser Leu Ser Pro Gly Lys 465 470 <210> 17 <211> 227 <212> PRT <213> Artificial Sequence <220> <223> D356C+T366S+L368A+Y407V+F405K+Q347R <400> 17 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 Arg Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Cys 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 Lys 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> 18 <211> 471 <212> PRT <213> artificial sequence <220> <223> Y349C+T366W+K409A+Q347R <400> 18 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ile Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Pro Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Ser Ile Ser Tyr Asp Gly Ser Tyr Lys Tyr Lys Ala Asp Ser Met 50 55 60 Lys Gly Arg Leu Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Glu Met Asn Ser Leu Thr Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Thr Ala Phe Phe Asn Ala Tyr Asp Phe Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Gly Gly 115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Met Thr Gln Ser Pro 130 135 140 Ala Thr Leu Ser Val Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg 145 150 155 160 Ala Ser Gln Ser Val Arg Ser Asn Leu Ala Trp Tyr Gln Gln Lys Pro 165 170 175 Gly Gln Ala Pro Arg Leu Leu Ile Tyr Ala Ala Ser Thr Arg Ala Thr 180 185 190 Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr 195 200 205 Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp Phe Ala Val Tyr Tyr Cys 210 215 220 Gln Gln Tyr Asn Glu Trp Phe Arg Thr Ser Gly Gln Gly Thr Lys Val 225 230 235 240 Glu Ile Lys Arg Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 245 250 255 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 260 265 270 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 275 280 285 Asp Val Ser His Glu Asn Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 290 295 300 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 305 310 315 320 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 325 330 335 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 340 345 350 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 355 360 365 Glu Pro Arg Val Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 370 375 380 Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 385 390 395 400 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 405 410 415 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 420 425 430 Ala Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 435 440 445 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 450 455 460 Leu Ser Leu Ser Pro Gly Lys 465 470 <210> 19 <211> 227 <212> PRT <213> artificial sequence <220> <223> D356C+T366S+L368A+Y407V+F405K+K360E+Q347E <400> 19 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 Glu Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Cys Glu Leu Thr Glu 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 Lys 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> 20 <211> 471 <212> PRT <213> artificial sequence <220> <223> Y349C+T366W+K409A+L368E <400> 20 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ile Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Pro Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Ser Ile Ser Tyr Asp Gly Ser Tyr Lys Tyr Lys Ala Asp Ser Met 50 55 60 Lys Gly Arg Leu Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Glu Met Asn Ser Leu Thr Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Thr Ala Phe Phe Asn Ala Tyr Asp Phe Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Gly Gly 115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Met Thr Gln Ser Pro 130 135 140 Ala Thr Leu Ser Val Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg 145 150 155 160 Ala Ser Gln Ser Val Arg Ser Asn Leu Ala Trp Tyr Gln Gln Lys Pro 165 170 175 Gly Gln Ala Pro Arg Leu Leu Ile Tyr Ala Ala Ser Thr Arg Ala Thr 180 185 190 Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr 195 200 205 Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp Phe Ala Val Tyr Tyr Cys 210 215 220 Gln Gln Tyr Asn Glu Trp Phe Arg Thr Ser Gly Gln Gly Thr Lys Val 225 230 235 240 Glu Ile Lys Arg Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 245 250 255 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 260 265 270 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 275 280 285 Asp Val Ser His Glu Asn Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 290 295 300 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 305 310 315 320 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 325 330 335 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 340 345 350 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 355 360 365 Glu Pro Gln Val Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 370 375 380 Asn Gln Val Ser Leu Trp Cys Glu Val Lys Gly Phe Tyr Pro Ser Asp 385 390 395 400 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 405 410 415 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 420 425 430 Ala Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 435 440 445 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 450 455 460 Leu Ser Leu Ser Pro Gly Lys 465 470 <210> 21 <211> 471 <212> PRT <213> artificial sequence <220> <223> T366W+K409A+K392D <400> 21 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ile Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Pro Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Ser Ile Ser Tyr Asp Gly Ser Tyr Lys Tyr Lys Ala Asp Ser Met 50 55 60 Lys Gly Arg Leu Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Glu Met Asn Ser Leu Thr Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Thr Ala Phe Phe Asn Ala Tyr Asp Phe Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Gly Gly 115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Met Thr Gln Ser Pro 130 135 140 Ala Thr Leu Ser Val Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg 145 150 155 160 Ala Ser Gln Ser Val Arg Ser Asn Leu Ala Trp Tyr Gln Gln Lys Pro 165 170 175 Gly Gln Ala Pro Arg Leu Leu Ile Tyr Ala Ala Ser Thr Arg Ala Thr 180 185 190 Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr 195 200 205 Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp Phe Ala Val Tyr Tyr Cys 210 215 220 Gln Gln Tyr Asn Glu Trp Phe Arg Thr Ser Gly Gln Gly Thr Lys Val 225 230 235 240 Glu Ile Lys Arg Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 245 250 255 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 260 265 270 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 275 280 285 Asp Val Ser His Glu Asn Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 290 295 300 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 305 310 315 320 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 325 330 335 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 340 345 350 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 355 360 365 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 370 375 380 Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 385 390 395 400 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Asp 405 410 415 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 420 425 430 Ala Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 435 440 445 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 450 455 460 Leu Ser Leu Ser Pro Gly Lys 465 470 <210> 22 <211> 227 <212> PRT <213> artificial sequence <220> <223> T366S+L368A+Y407V+D399S+F405K <400> 22 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 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 Ser Ser Asp Gly Ser Phe Lys 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> 23 <211> 471 <212> PRT <213> artificial sequence <220> <223> T366W+K409A <400> 23 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ile Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Pro Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Ser Ile Ser Tyr Asp Gly Ser Tyr Lys Tyr Lys Ala Asp Ser Met 50 55 60 Lys Gly Arg Leu Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Glu Met Asn Ser Leu Thr Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Thr Ala Phe Phe Asn Ala Tyr Asp Phe Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Gly Gly 115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Met Thr Gln Ser Pro 130 135 140 Ala Thr Leu Ser Val Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg 145 150 155 160 Ala Ser Gln Ser Val Arg Ser Asn Leu Ala Trp Tyr Gln Gln Lys Pro 165 170 175 Gly Gln Ala Pro Arg Leu Leu Ile Tyr Ala Ala Ser Thr Arg Ala Thr 180 185 190 Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr 195 200 205 Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp Phe Ala Val Tyr Tyr Cys 210 215 220 Gln Gln Tyr Asn Glu Trp Phe Arg Thr Ser Gly Gln Gly Thr Lys Val 225 230 235 240 Glu Ile Lys Arg Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 245 250 255 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 260 265 270 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 275 280 285 Asp Val Ser His Glu Asn Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 290 295 300 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 305 310 315 320 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 325 330 335 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 340 345 350 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 355 360 365 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 370 375 380 Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 385 390 395 400 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 405 410 415 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 420 425 430 Ala Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 435 440 445 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 450 455 460 Leu Ser Leu Ser Pro Gly Lys 465 470 <210> 24 <211> 227 <212> PRT <213> artificial sequence <220> <223> T366S+L368G+Y407A+F405K <400> 24 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 Ser Cys Gly 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 Lys Leu Ala 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> 25 <211> 227 <212> PRT <213> artificial sequence <220> <223> T366S+L368A+Y407V+F405K+E357A <400> 25 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 Ala 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 Lys 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> 26 <211> 471 <212> PRT <213> artificial sequence <220> <223> T366W+K409A+Y349D+S354D <400> 26 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ile Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Pro Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Ser Ile Ser Tyr Asp Gly Ser Tyr Lys Tyr Lys Ala Asp Ser Met 50 55 60 Lys Gly Arg Leu Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Glu Met Asn Ser Leu Thr Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Thr Ala Phe Phe Asn Ala Tyr Asp Phe Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Gly Gly 115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Met Thr Gln Ser Pro 130 135 140 Ala Thr Leu Ser Val Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg 145 150 155 160 Ala Ser Gln Ser Val Arg Ser Asn Leu Ala Trp Tyr Gln Gln Lys Pro 165 170 175 Gly Gln Ala Pro Arg Leu Leu Ile Tyr Ala Ala Ser Thr Arg Ala Thr 180 185 190 Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr 195 200 205 Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp Phe Ala Val Tyr Tyr Cys 210 215 220 Gln Gln Tyr Asn Glu Trp Phe Arg Thr Ser Gly Gln Gly Thr Lys Val 225 230 235 240 Glu Ile Lys Arg Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 245 250 255 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 260 265 270 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 275 280 285 Asp Val Ser His Glu Asn Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 290 295 300 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 305 310 315 320 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 325 330 335 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 340 345 350 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 355 360 365 Glu Pro Gln Val Asp Thr Leu Pro Pro Asp Arg Asp Glu Leu Thr Lys 370 375 380 Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 385 390 395 400 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 405 410 415 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 420 425 430 Ala Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 435 440 445 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 450 455 460 Leu Ser Leu Ser Pro Gly Lys 465 470 <210> 27 <211> 471 <212> PRT <213> Artificial Sequence <220> <223> T366W+F405K <400> 27 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ile Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Pro Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Ser Ile Ser Tyr Asp Gly Ser Tyr Lys Tyr Lys Ala Asp Ser Met 50 55 60 Lys Gly Arg Leu Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Glu Met Asn Ser Leu Thr Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Thr Ala Phe Phe Asn Ala Tyr Asp Phe Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Gly Gly 115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Met Thr Gln Ser Pro 130 135 140 Ala Thr Leu Ser Val Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg 145 150 155 160 Ala Ser Gln Ser Val Arg Ser Asn Leu Ala Trp Tyr Gln Gln Lys Pro 165 170 175 Gly Gln Ala Pro Arg Leu Leu Ile Tyr Ala Ala Ser Thr Arg Ala Thr 180 185 190 Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr 195 200 205 Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp Phe Ala Val Tyr Tyr Cys 210 215 220 Gln Gln Tyr Asn Glu Trp Phe Arg Thr Ser Gly Gln Gly Thr Lys Val 225 230 235 240 Glu Ile Lys Arg Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 245 250 255 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 260 265 270 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 275 280 285 Asp Val Ser His Glu Asn Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 290 295 300 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 305 310 315 320 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 325 330 335 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 340 345 350 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 355 360 365 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 370 375 380 Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 385 390 395 400 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 405 410 415 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Lys Leu Tyr Ser 420 425 430 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 435 440 445 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 450 455 460 Leu Ser Leu Ser Pro Gly Lys 465 470 <210> 28 <211> 227 <212> PRT <213> artificial sequence <220> <223> T366S+L368A+Y407V+K409A <400> 28 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 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 Ala 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> 29 <211> 471 <212> PRT <213> artificial sequence <220> <223> T366W+F405K+D399S <400> 29 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ile Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Pro Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Ser Ile Ser Tyr Asp Gly Ser Tyr Lys Tyr Lys Ala Asp Ser Met 50 55 60 Lys Gly Arg Leu Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Glu Met Asn Ser Leu Thr Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Thr Ala Phe Phe Asn Ala Tyr Asp Phe Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Gly Gly 115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Met Thr Gln Ser Pro 130 135 140 Ala Thr Leu Ser Val Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg 145 150 155 160 Ala Ser Gln Ser Val Arg Ser Asn Leu Ala Trp Tyr Gln Gln Lys Pro 165 170 175 Gly Gln Ala Pro Arg Leu Leu Ile Tyr Ala Ala Ser Thr Arg Ala Thr 180 185 190 Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr 195 200 205 Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp Phe Ala Val Tyr Tyr Cys 210 215 220 Gln Gln Tyr Asn Glu Trp Phe Arg Thr Ser Gly Gln Gly Thr Lys Val 225 230 235 240 Glu Ile Lys Arg Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 245 250 255 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 260 265 270 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 275 280 285 Asp Val Ser His Glu Asn Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 290 295 300 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 305 310 315 320 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 325 330 335 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 340 345 350 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 355 360 365 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 370 375 380 Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 385 390 395 400 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 405 410 415 Thr Thr Pro Pro Val Leu Ser Ser Asp Gly Ser Phe Lys Leu Tyr Ser 420 425 430 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 435 440 445 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 450 455 460 Leu Ser Leu Ser Pro Gly Lys 465 470 <210> 30 <211> 227 <212> PRT <213> Artificial sequence <220> <223> T366S+L368A+Y407V+K409A+K392D <400> 30 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 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 Asp Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Ala 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> 31 <211> 227 <212> PRT <213> artificial sequence <220> <223> T366S+L368G+Y407A+K409A <400> 31 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 Ser Cys Gly 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 Ala Ser Ala 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> 32 <211> 471 <212> PRT <213> artificial sequence <220> <223> T366W +F405K +Y349D <400> 32 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ile Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Pro Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Ser Ile Ser Tyr Asp Gly Ser Tyr Lys Tyr Lys Ala Asp Ser Met 50 55 60 Lys Gly Arg Leu Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Glu Met Asn Ser Leu Thr Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Thr Ala Phe Phe Asn Ala Tyr Asp Phe Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Gly Gly 115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Met Thr Gln Ser Pro 130 135 140 Ala Thr Leu Ser Val Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg 145 150 155 160 Ala Ser Gln Ser Val Arg Ser Asn Leu Ala Trp Tyr Gln Gln Lys Pro 165 170 175 Gly Gln Ala Pro Arg Leu Leu Ile Tyr Ala Ala Ser Thr Arg Ala Thr 180 185 190 Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr 195 200 205 Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp Phe Ala Val Tyr Tyr Cys 210 215 220 Gln Gln Tyr Asn Glu Trp Phe Arg Thr Ser Gly Gln Gly Thr Lys Val 225 230 235 240 Glu Ile Lys Arg Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 245 250 255 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 260 265 270 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 275 280 285 Asp Val Ser His Glu Asn Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 290 295 300 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 305 310 315 320 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 325 330 335 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 340 345 350 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 355 360 365 Glu Pro Gln Val Asp Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 370 375 380 Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 385 390 395 400 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 405 410 415 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Lys Leu Tyr Ser 420 425 430 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 435 440 445 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 450 455 460 Leu Ser Leu Ser Pro Gly Lys 465 470 <210> 33 <211> 227 <212> PRT <213> artificial sequence <220> <223> T366S+L368A+Y407V +K409A +E357A <400> 33 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 Ala 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 Ala 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> 34 <211> 471 <212> PRT <213> artificial sequence <220> <223> T366W+F405K+Y349D+S354D <400> 34 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ile Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Pro Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Ser Ile Ser Tyr Asp Gly Ser Tyr Lys Tyr Lys Ala Asp Ser Met 50 55 60 Lys Gly Arg Leu Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Glu Met Asn Ser Leu Thr Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Thr Ala Phe Phe Asn Ala Tyr Asp Phe Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Gly Gly 115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Met Thr Gln Ser Pro 130 135 140 Ala Thr Leu Ser Val Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg 145 150 155 160 Ala Ser Gln Ser Val Arg Ser Asn Leu Ala Trp Tyr Gln Gln Lys Pro 165 170 175 Gly Gln Ala Pro Arg Leu Leu Ile Tyr Ala Ala Ser Thr Arg Ala Thr 180 185 190 Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr 195 200 205 Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp Phe Ala Val Tyr Tyr Cys 210 215 220 Gln Gln Tyr Asn Glu Trp Phe Arg Thr Ser Gly Gln Gly Thr Lys Val 225 230 235 240 Glu Ile Lys Arg Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 245 250 255 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 260 265 270 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 275 280 285 Asp Val Ser His Glu Asn Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 290 295 300 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 305 310 315 320 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 325 330 335 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 340 345 350 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 355 360 365 Glu Pro Gln Val Asp Thr Leu Pro Pro Asp Arg Asp Glu Leu Thr Lys 370 375 380 Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 385 390 395 400 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 405 410 415 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Lys Leu Tyr Ser 420 425 430 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 435 440 445 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 450 455 460 Leu Ser Leu Ser Pro Gly Lys 465 470 <210> 35 <211> 354 <212> PRT <213> artificial sequence <220> <223> VhH-Fc fusion protein <400> 35 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Glu Tyr Ile Tyr Ser Ser Tyr 20 25 30 Cys Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ala Val Ile Gly Ser Asp Gly Ser Thr Ser Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Lys Asp Asn Ala Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala 85 90 95 Ala Ile Gly Gly Tyr Cys Tyr Gln Pro Pro Tyr Glu Tyr Gln Tyr Trp 100 105 110 Gly Gln Gly Thr Gln Val Thr Val Ser Ser Glu Pro Lys Ser Ser Asp 115 120 125 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 130 135 140 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 145 150 155 160 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 165 170 175 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 180 185 190 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 195 200 205 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 210 215 220 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 225 230 235 240 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 245 250 255 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 260 265 270 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 275 280 285 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 290 295 300 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 305 310 315 320 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 325 330 335 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 340 345 350 Gly Lys <210> 36 <211> 1121 <212> DNA <213> artificial sequence <220> <223> VhH-Fc encoding gene <400> 36 atggagaccg acaccctgct gctgtgggtg ctgctgctgt gggtgcccgg cagcaccggc 60 caggtgcagc tgcaggagtc tgggggaggc tcggtgcagg ctggagggtc tctgagactc 120 tcctgtgcag cctctgaata catctacagt agctactgca tggcctggtt ccgccaggct 180 ccagggaagg agcgcgaggg ggtcgcagtt attgggagtg atggtagcac aagctacgca 240 gactccgtga aaggccgatt caccatctcc aaagacaacg ccaagaacac tctgtatctg 300 caaatgaaca gcctgaaacc tgaggacact gccatgtact actgtgcggc catcggtggt 360 tactgctacc aaccacccta tgagtaccag tactggggcc aggggaccca ggtcaccgtc 420 tcccagaacc gaaaagcagc gacaagaccc acacctgccc cccctgcccc gcccccgagc 480 tgctgggcgg ccccagcgtg ttcctgttcc cccccaagcc caaggacacc ctgatgatca 540 gccgcacccc cgaggtgacc tgcgtggtgg tggacgtgag ccacgagaac cccgaggtga 600 agttcaactg gtacgtggac ggcgtggagg tgcacaacgc caagaccaag ccccgcgagg 660 agcagtacaa cagcacctac cgcgtggtga gcgtgctgac cgtgctgcac caggactggc 720 tgaacggcaa ggagtacaag tgcaaggtga gcaacaaggc cctgcccgcc cccatcgaga 780 agaccatcag caaggccaag ggccagcccc gcgagcccca ggtgtacacc ctgcccccca 840 gccgcgacga gctgaccaag aaccaggtga gcctgacctg cctggtgaag ggcttctacc 900 ccagcgacat cgccgtggag tgggagagca acggccagcc cgagaacaac tacaagacca 960 ccccccccgt gctggacagc gacggcagct tcttcctgta cagcaagctg accgtggaca 1020 agagccgctg gcagcagggc aacgtgttca gctgcagcgt gatgcacgag gccctgcaca 1080 accactacac ccagaagagc ctgagcctga gccccggcaa g 1121

Claims

1. An heterodimeric molecule comprising a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises a first CH3 domain of an antibody heavy chain constant region, and the second polypeptide chain comprises a second CH3 domain of an antibody heavy chain constant region, and the first CH3 domain and the second CH3 domain are mutated compared to the corresponding wild-type human antibody heavy chain constant region CH3 domain, and the mutation is selected from one of the following groups: 1) First CH3 domain: T366W + F405K + D399S, second CH3 domain: T366S + L368A + Y407V + K409A + K392D; 2) First CH3 domain: T366W + F405K, second CH3 domain: T366S + L368G + Y407A + K409A; and 3) First CH3 domain: T366W + F405K + Y349D + S354D, second CH3 domain: T366S + L368A + Y407V + K409A + E357A, The positions of the above-mentioned amino acids are determined according to the EU index of the KABAT numbering of the antibody Fc, wherein the wild-type human antibody heavy chain constant region CH3 domain is the human IgG1 heavy chain constant region CH3 domain.

2. The heterodimeric molecule according to claim 1, wherein the first polypeptide chain and the second polypeptide chain further contain a CH2 domain of an antibody heavy chain constant region, respectively.

3. The heterodimeric molecule according to claim 1, wherein the first polypeptide chain and the second polypeptide chain further contain a hinge region or a part of the hinge region of an antibody heavy chain constant region, respectively.

4. The heterodimeric molecule according to claim 1, wherein the first and / or the second polypeptide chain further contains a molecular binding region, and the molecular binding region is selected from an antigen binding region, a receptor binding region, and an enzyme binding region.

5. The heterodimeric molecule according to claim 4, wherein the antigen binding region contains an antibody variable region.

6. The heterodimeric molecule according to claim 1, which is a bispecific antibody, a bispecific fusion protein, or an antibody-fusion protein chimera.

7. A composition comprising the heterodimeric molecule according to any one of claims 1-6, and optionally a pharmaceutically acceptable excipient.

8. A composition comprising the heterodimeric molecule according to any one of claims 1-6, and optionally a pharmaceutically acceptable carrier.

9. A nucleic acid molecule encoding the first polypeptide chain and the second polypeptide chain of the heterodimeric molecule according to any one of claims 1-6.

10. A vector containing the nucleic acid molecule according to claim 9.

11. A host cell containing the vector according to claim 10.

12. Use of the heterodimeric molecule according to any one of claims 1-6, the composition according to any one of claims 7-8, the nucleic acid molecule according to claim 9, the vector according to claim 10, or the host cell according to claim 11 in the preparation of a bispecific antibody, a bispecific fusion protein, or an antibody-fusion protein chimera.

13. A method for preparing a heterodimeric molecule, which comprises the step of expressing the heterodimeric molecule using the host cell according to claim 11.

14. The method according to claim 13, wherein the host cell simultaneously contains vectors encoding the first polypeptide chain and the second polypeptide chain in the heterodimeric molecule, and the method comprises using the host cell to express, recover and obtain the heterodimeric molecule.

15. The method according to claim 13, wherein the host cell comprises a first group of cells and a second group of cells, and the first group of cells and the second group of cells each separately contain a vector encoding a first polypeptide chain and a second polypeptide chain of the heterodimeric molecule, and the method comprises: The first polypeptide chain homodimer and the second polypeptide chain homodimer are formed after the first polypeptide chain and the second polypeptide chain are respectively expressed in the first group of cells and the second group of cells, and then the first polypeptide chain homodimer and the second polypeptide chain homodimer are mixed under suitable conditions and the heterodimeric molecule is prepared.

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