Heterodimer molecule based on CH3 structural domain and preparation method and application thereof

Abstract

The invention relates to the field of antibody engineering, in particular to a CH3-based heterodimer molecule as well as a preparation method and application thereof. According to the present invention, various intermolecular interactions such as ionic interaction, hydrophobic interaction and spatial interaction are comprehensively considered to screen the optimal Fc mutation sequence, and the optimal Fc mutation sequence is more likely to form the heterodimer instead of the homodimer, such that the yield of the heterodimer molecule is substantially improved, and the application prospect is broad. Conditions are created for preparation of bispecific molecules and the like.

Description

[0001] This application is a divisional application of Chinese patent application No. 202210544074.6, filed on December 16, 2016, entitled "Heterodimer molecule based on CH3 domain, preparation method and use thereof". Technical Field

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

[0003] Monoclonal antibody drugs have experienced rapid growth over the past fifteen years, becoming a growth driver in the pharmaceutical industry. Since 1996, approximately 30 monoclonal antibody drugs have been approved for marketing, nine of which have annual sales exceeding one billion US dollars. In 2010, total sales of monoclonal antibody drugs exceeded 30 billion US dollars, with an annual growth rate exceeding 10%. Due to the high target specificity of monoclonal antibodies, they can only inhibit a single target. However, in many diseases, including tumors and autoimmune diseases, it is necessary to inhibit multiple signaling pathways to avoid compensatory effects. For viral infections, due to the high mutation rate of viruses, it is often necessary to inhibit multiple antigenic sites to prevent escape. In addition, bifunctional antibodies and proteins are used to specifically activate the human immune system (Wolf, Hofmeister et al. 2005).

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

[0005] A typical example of the application of heterodimers is bispecific antibodies (BsAbs), which are immunoglobulin molecules containing two different ligand-binding sites. Bispecific antibodies can be active against at least two different antigens (Carter 2001). They replace the classic antibody format where the two Fab arms are identical, instead employing Fab arms with different sequences, thus allowing the Y-shaped arms to bind to different antigens. The application of bispecific antibodies in cancer therapy has been reviewed in numerous publications (Carter 2001; Chames and Baty 2009; Chames and Baty 2009).

[0006] Bispecific antibodies do not exist naturally and can only be prepared through special methods. Previous methods for preparing bispecific antibodies include chemical cross-linking, hybrid F(ab')2 molecule methods, and mouse hybridoma methods. The heterogeneity of bispecific antibodies produced by chemical cross-linking, the instability between batches, and the susceptibility of antibody specificity to alterations due to modifications or improper linking make these methods unsuitable for in vivo use. While bispecific hybrid molecules produced by digesting the F(ab') fragment with a thiol-crosslinked protease have relatively homogeneous components, they are time-consuming, labor-intensive, and have very low yields. Bispecific antibodies produced by the hybridoma method have reliable sources, but the random pairing of light and heavy chains can generate various possible antibody forms, making the production and purification of bispecific antibodies extremely difficult.

[0007] As early as the 1990s, Carter et al. successfully prepared bispecific antibodies by modifying certain amino acids in the antibody heavy chain using the "knob-to-hole" model (Ridgway, Presta et al. 1996; Carter 2001). The "knob-to-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" (e.g., T366Y) by mutating a small side-chain amino acid to a large side-chain amino acid in the CH3 region of the first heavy chain in the Fc region, and created "holes" (e.g., Y407T) by mutating certain amino acids in the CH3 region of the second heavy chain to small side-chain amino acids. The principle of the "knob-to-hole" model is that the interaction between the "knob" and the "hole" supports the formation of heterodimers, while the "knob-to-knob" model and the "hole-to-hole" model hinder the formation of homodimers. They further introduced disulfide bonds within the CH3 region to strengthen the binding ability of heterodimers, building upon the "handle-hole" mutation. However, their results showed that the "handle-hole" model was still insufficient to prevent homodimer formation. The research group then attempted to increase the heterodimer content in one step through random mutation-phage display, but this did not solve the fundamental problem. To increase the proportion of heterodimers, other studies have prepared two separate antibodies and formed heterodimers in vitro through intermolecular disulfide bond reduction and re-pairing, but this preparation process is significantly overly complex.

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

[0009] The invention described in this application, by comprehensively considering various interactions between amino acids at the interface, such as ionic interactions, hydrophobic interactions, and steric interactions, screens out beneficial CH3 mutant sequences that are more likely 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 heterodimeric molecule containing a first polypeptide chain and a second polypeptide chain, the first polypeptide chain containing a first CH3 domain of the antibody heavy chain constant region, and the second polypeptide chain containing a second CH3 domain of the antibody heavy chain constant region, wherein, compared with the corresponding wild-type human antibody heavy chain constant region CH3 domain, the first CH3 domain and the second CH3 domain contain a mutation of an amino acid selected from the positions shown in (1) to (3) below:

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

[0012] (2) Mutations occur at T366 and K409 of the first CH3 domain, and mutations occur at T366, L368, Y407, and F405 of the second CH3 domain. Optionally, the first CH3 domain and / or the second CH3 domain also have mutations at one or two amino acid positions selected from K392, D399, Y349, S354, and E357; and

[0013] (3) Mutations occur at T366 and F405 of the first CH3 domain, and mutations occur at T366, L368, Y407 and K409 of the second CH3 domain, 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 amino acid positions mentioned above were determined based on the EU index of the KABAT number of the antibody Fc.

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

[0016] In some embodiments, the first CH3 domain and / or the second CH3 domain further comprises 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 at F405 in the second CH3 domain.

[0025] In some embodiments, the first CH3 domain and / or the second CH3 domain may 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 at E357 in the second CH3 domain.

[0029] In some embodiments, the first CH3 domain and / or the second CH3 domain optionally further comprises 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) A mutation occurs at Y349 in the first CH3 domain, and a mutation occurs at E357 in the second CH3 domain; and

[0032] 3c) Mutations occur at Y349 and S354D in the first CH3 domain, and at E357 in the second CH3 domain.

[0033] In some embodiments, each mutation is independently selected from non-charged amino acid mutations to charged amino acids, charged amino acid mutations to non-charged amino acids, and charged amino acid mutations to amino acids with opposite charges.

[0034] In some 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 mutation may be selected from one or more of the following mutations: Y349C, Y349D, D356C, T366W, T366S, L368A, L368E, L368G, F405K, Y407V, Y407A, K409E, K409A, K360E, Q347E, Q347R, K392D, D399S, E357A, and S354D.

[0035] In some implementations, 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.

[0036] In some embodiments, 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.

[0037] In some embodiments, 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.

[0038] In some implementations, the first CH3 domain contains one or more mutations selected from the group consisting of (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): Y349C, T366W, F405K, K409A, L368E, K392D, Y349D, S354D, and / or D399S.

[0039] In some embodiments, the second CH3 domain contains one or more mutations selected from the group consisting of (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): D356C, T366S, L368A, Y407V, F405K, D399S, L368G, Y407A, E357A, K409A, and / or K392D.

[0040] In some embodiments, the first CH3 domain contains one or more mutations selected from the group consisting of (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): 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 (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): D356C, T366S, L368A, Y407V, F405K, D399S, L368G, Y407A, E357A, K409A, and / or K392D).

[0041] In some embodiments, the first CH3 domain and the second CH3 domain contain mutations selected from the group consisting of:

[0042] 1) First CH3 domain: Y349C+T366W, Second CH3 domain: D356C+T366S+L368A+Y407V+F405K;

[0043] 2) First CH3 domain: Y349C+T366W+F405K, Second CH3 domain: D356C+T366S+L368A+Y407V;

[0044] 3) First CH3 domain: Y349C+T366W+K409E, Second CH3 domain: D356C+T366S+L368A+Y407V+F405K;

[0045] 4) First CH3 structural domain: Y349C+T366W+K409A, Second CH3 structural domain: D356C+T366S+L368A+Y407V+F405K;

[0046] 5) First CH3 domain: Y349C+T366W+F405K+K360E+Q347E, Second CH3 domain: D356C+T366S+L368A+Y407V+Q347R;

[0047] 6) First CH3 domain: Y349C+T366W+F405K+Q347R, Second CH3 domain: D356C+T366S+L368A+Y407V+K360E+Q347E;

[0048] 7) First CH3 structural domain: Y349C+T366W+K409A+K360E+Q347E, Second CH3 structural domain: D356C+T366S+L368A+Y407V+F405K+Q347R;

[0049] 8) First CH3 domain: Y349C+T366W+K409A+Q347R, Second CH3 domain: D356C+T366S+L368A+Y407V+F405K+K360E+Q347E;

[0050] 9) First CH3 domain: Y349C+T366W+K409A+L368E, Second CH3 domain: D356C+T366S+L368A+Y407V+F405K;

[0051] 10) First CH3 domain: T366W+K409A+K392D, 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 some embodiments, the first CH3 domain and the second CH3 domain contain mutations selected from the group consisting of:

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

[0062] 4) First CH3 structural domain: Y349C+T366W+K409A, Second CH3 structural 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 some embodiments, the first and second polypeptide chains further contain a CH2 domain of the antibody heavy chain constant region. In some 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 via a linker peptide.

[0071] In some embodiments, the first and second polypeptide chains further include a hinge region or a portion of a hinge region of the antibody heavy chain constant region. In some embodiments, a portion of the hinge region is D221-P230.

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

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

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

[0075] In some 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 some embodiments, the antigen-binding region contains an antibody variable region.

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

[0077] On the other hand, this application relates to a composition (e.g., a pharmaceutical composition) containing any of the heterodimeric molecules described in this application, and optionally a pharmaceutically acceptable carrier or excipient.

[0078] This application also relates to nucleic acid molecules that encode a first polypeptide chain or a second polypeptide chain of the heterodimer molecule described in this application, or that encode a first polypeptide chain or a second polypeptide chain of the heterodimer molecule described in this application.

[0079] This application also relates to vectors containing the nucleic acid molecules described in this application.

[0080] This application also relates to host cells containing the vector described in this application.

[0081] This application also relates to the use of the aforementioned heterodimer molecules, compositions, nucleic acid molecules, vectors, or host cells in the preparation of bispecific antibodies, bispecific fusion proteins, and antibody-fusion protein chimeras.

[0082] This application also relates to a method for preparing heterodimer molecules, which includes the step of expressing the heterodimer molecules using the host cells described in this application.

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

[0084] In some embodiments of the method for preparing heterodimeric molecules, the host cells include a first group of cells and a second group of cells, each group containing a vector encoding a first polypeptide chain and a second polypeptide chain of the heterodimeric molecule. The method includes: 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 a first polypeptide chain homodimer and a second polypeptide chain homodimer; then mixing the first polypeptide chain homodimer and the second polypeptide chain homodimer under suitable conditions to prepare the heterodimeric molecule. In some embodiments, the method further includes reducing and dissociating the first polypeptide chain homodimer and the second polypeptide chain homodimer into monomers, mixing and oxidizing them, and then purifying them to prepare the heterodimeric molecule. In some embodiments, the host cells contain vectors encoding the first and second polypeptide chains of the heterodimer molecule, respectively. After the first and second polypeptide chains are expressed in the two host cells, they form a first polypeptide chain homodimer and a second polypeptide chain homodimer, respectively. Then, the first polypeptide chain homodimer and the second polypeptide chain homodimer are reduced, mixed, and oxidized under appropriate conditions, and then purified to obtain the heterodimer molecule.

[0085] In some embodiments of the method for preparing heterodimer 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 may be transient. During transfection, the molar ratio of the construct or vector containing the first polypeptide chain to the construct or vector containing the second polypeptide chain may be 1:4 to 4:1, for example, 1:2 to 2:1, for example, about 1:1.

[0086] Other aspects and advantages of this disclosure will readily become apparent to those skilled in the art from the detailed description below. Only exemplary embodiments of this disclosure are shown and described in the following detailed description. As will be appreciated by those skilled in the art, the content of this disclosure enables them to make modifications to the specific embodiments disclosed without departing from the spirit and scope of the invention to which this application pertains. Accordingly, the descriptions in the accompanying drawings and specification of this application are merely exemplary and not restrictive. Attached Figure Description

[0087] Figure 1This image shows the electrophoretic analysis results of transient expression of ScFv-Fc / Fc heterodimers. Protein gel electrophoresis was performed using 4% to 12% SDS-PAGE. Lanes 1 to 7 are: 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 product group migrated at different distances during gel electrophoresis due to their molecular weight differences. The positions of different homodimers or heterodimers are marked on the image.

[0088] Figure 2 Electrophoretic analysis results of transient expression of ScFv-Fc / Fc heterodimers are shown. 12% SDS-PAGE protein gel electrophoresis was used. Lanes 1 to 9 are: 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 standards. The homodimers and heterodimers contained in each product migrated different distances during gel electrophoresis due to their molecular weight differences. Figure 1 Similarly, from top to bottom, they are 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 heterodimer Fc in mutant combination 4 is shown. Mutated amino acid residues are indicated by short sticks, and the specific contacting mutant amino acid residue pairs are as follows: T366W / A chain - T366S, L368A, Y407V / B chain, K409A / A chain - F405K / B chain, S354C / A chain - Y349C / B chain. Chain A (the lighter-colored chain on the left) is shown in green, and chain B (the darker-colored chain on the right) is shown in light blue.

[0090] Figure 4 The study showed that introducing a new mutation pair, D399S-K392D, near the F405K-K409A mutant amino acid residue pair could further enhance the attraction between heterodimers and increase the repulsion between homodimers. Figure 4 A shows the interaction of the F405K-K409A mutation with nearby interfacial amino acids when the new mutation is introduced. Figure 4 B shows the changes in interaction resulting from the introduction of the new mutation. Detailed Implementation

[0091] The following specific examples illustrate the implementation 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 this application, both the first and second polypeptide chains contain the CH3 region of the antibody Fc fragment. The two polypeptide chains interact through the CH3 region or the Fc fragment containing the CH3 region to form a dimer, particularly a heterodimer. The two polypeptide chains of the heterodimer can be different combinations; for example, the first polypeptide chain may be an antibody and the second polypeptide chain may be a fusion protein, or both polypeptide chains may be fusion proteins, or both polypeptide chains may be antibodies (e.g., antibodies targeting different antigens or epitopes). When a 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 molecules that can recognize specific ligand cell surface receptors, such as cadherins, selectins, immunoglobulin superfamily, integrins, and hyaluronic acid mucin.

[0093] In this application, the CH3 region is derived from an antibody Fc fragment, such as a human antibody Fc fragment (e.g., a human antibody heavy chain constant region Fc fragment). In some embodiments, the CH3 region is derived from an Fc fragment of the human immunoglobulin (Ig) heavy chain constant region, for example, from the heavy chain constant region Fc fragments of IgM, IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgA (e.g., IgA1, IgA2), IgE, and / or IgD. In some embodiments, the CH3 region described in this application (e.g., the CH3 domain of the wild-type human antibody heavy chain constant region) is derived from wild-type human IgG1, for example, from the CH3 domain of the antibody heavy chain constant region of wild-type human IgG1. Generally, the CH3 region of the human antibody Fc fragment is derived from the corresponding wild-type human antibody Fc fragment. A wild-type human antibody Fc fragment refers to an antibody Fc fragment from a natural human population, for example, a human antibody Fc fragment that has not been artificially mutagenized or modified. In some embodiments, the human antibody Fc fragment described in this application also includes changes to individual amino acids of the corresponding wild-type human antibody Fc sequence, such as certain amino acid mutations at glycosylation sites, or other nonsense mutations, as well as changes to individual amino acids mutated according to the "handle-hole" model. For example, for the CH3 and CH2 domains, in addition to the mutations mentioned in this application, there may be other mutations that do not affect the function of the antibody (especially the Fc fragment).

[0094] In this application, when the first polypeptide chain and / or the second polypeptide chain contains a hinge region, the hinge region acts as a flexible fragment connecting the two polypeptide segments to ensure the function of each polypeptide chain; those skilled in the art can select the length of the hinge region as needed, for example, the full-length sequence or a portion thereof.

[0095] In this application, the amino acid positions in the Fc or its CH2, CH3 domains or hinge regions are numbered according to the Kabat EU numbering index. Those skilled in the art will understand that even if the amino acid sequence changes due to insertion, deletion, or other mutations in the aforementioned regions, the position numbers of each amino acid corresponding to the standard sequence, determined according to the Kabat EU numbering index, remain unchanged.

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

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

[0098] In this application, heterodimeric molecules can be purified from host cells using standard experimental methods. For example, when a heterodimeric protein contains an antibody Fc fragment, it can be purified using protein A. Purification methods include, but are not limited to, chromatographic techniques such as size exclusion, ion exchange, affinity chromatography, and ultrafiltration, or appropriate combinations of the above methods.

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

[0100] The invention described in this application, by comprehensively considering various interactions between interfacial amino acids, such as ionic interactions, hydrophobic interactions, and steric interactions, screened for preferred CH3 mutant sequences, which are more likely to form heterodimers than homodimers, thus greatly increasing the yield of heterodimer molecules. Furthermore, in some embodiments of this application, heterodimer protein crystals containing the Fc fragment were prepared. Through crystal structure analysis and three-dimensional structural modeling, the direct interactions between interfacial amino acids were further understood. The previous view that a stable disulfide bond must form between the two cysteine ​​residues of Y349C and D356C was abandoned. Based on this, the mutant combinations are more conducive to the formation of heterodimers and less conducive to the formation of homodimers, significantly increasing the proportion of heterodimers while greatly reducing the proportion of homodimers.

[0101] The embodiments of the invention described in this application will be described in detail below. However, those skilled in the art will understand that the following embodiments are only for illustrative purposes and should not be considered as limiting the scope of the invention. Unless otherwise specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0102] Implementation Plan

[0103] 1. A heterodimer molecule comprising a first polypeptide chain and a second polypeptide chain, the first polypeptide chain comprising a first CH3 domain of the antibody heavy chain constant region, and the second polypeptide chain comprising a second CH3 domain of the antibody heavy chain constant region, wherein, compared with the corresponding wild-type human antibody heavy chain constant region CH3 domain, the first CH3 domain and the second CH3 domain contain a mutation of an amino acid selected from the positions shown in (1) to (3) below:

[0104] (1) The Y349 and T366 of the first CH3 domain are mutated, and the 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) Mutations occur at T366 and K409 of the first CH3 domain, and mutations occur at T366, L368, Y407, and F405 of the second CH3 domain. Optionally, the first CH3 domain and / or the second CH3 domain also have mutations at one or two amino acid positions selected from K392, D399, Y349, S354, and E357; and

[0106] (3) Mutations occur at T366 and F405 of the first CH3 domain, and mutations occur at T366, L368, Y407 and K409 of the second CH3 domain, 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;

[0107] The positions of the amino acids mentioned above were determined based on the EU index of the KABAT number of the antibody Fc.

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

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

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

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

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

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

[0114] 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;

[0115] 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

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

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

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

[0119] 2b) A mutation occurs at Y349 in the first CH3 domain, and a mutation occurs at E357 in the second CH3 domain; and

[0120] 2c) Mutations occur at Y349 and S354 in the first CH3 domain and at E357 in the second CH3 domain.

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

[0123] 3b) A mutation occurs at Y349 in the first CH3 domain, and a mutation occurs at E357 in the second CH3 domain; and

[0124] 3c) Mutations occur at Y349 and S354D in the first CH3 domain, and at E357 in the second CH3 domain.

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

[0126] 6. The heterodimer molecule according to any one of embodiments 1-5, wherein the mutation in the first CH3 domain and / or the second CH3 domain comprises 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.

[0127] 7. The heterodimer molecule according to any one of embodiments 1-6, 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.

[0128] 8. A heterodimer molecule according to any one of embodiments 1-7, wherein 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.

[0129] 9. A heterodimer 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. A heterodimer molecule according to any one of embodiments 1-9, wherein the first CH3 domain comprises one or more mutations selected from the group consisting of Y349C, T366W, F405K, K409A, L368E, K392D, Y349D, S354D and / or D399S.

[0131] 11. A heterodimer molecule according to any one of embodiments 1-10, wherein the second CH3 domain comprises 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 heterodimer 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 heterodimer molecule according to any one of embodiments 1-12, wherein the first CH3 domain and the second CH3 domain contain mutations selected from the group consisting of:

[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 structural domain: Y349C+T366W+K409A, Second CH3 structural 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 structural domain: Y349C+T366W+K409A+K360E+Q347E, Second CH3 structural 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; and

[0151] 18) First CH3 domain: T366W+F405K+Y349D+S354D, Second CH3 domain: T366S+L368A+Y407V+K409A+E357A.

[0152] 14. A heterodimer molecule according to any one of embodiments 1-13, wherein the first CH3 domain and the second CH3 domain contain mutations selected from the group consisting of:

[0153] 2) First CH3 domain: Y349C+T366W+F405K, Second CH3 domain: D356C+T366S+L368A+Y407V;

[0154] 4) First CH3 structural domain: Y349C+T366W+K409A, Second CH3 structural domain: D356C+T366S+L368A+Y407V+F405K;

[0155] 9) First CH3 domain: Y349C+T366W+K409A+L368E, Second CH3 domain: D356C+T366S+L368A+Y407V+F405K;

[0156] 10) First CH3 domain: T366W+K409A+K392D, Second CH3 domain: T366S+L368A+Y407V+D399S+F405K;

[0157] 11) First CH3 domain: T366W+K409A, Second CH3 domain: T366S+L368G+Y407A+F405K;

[0158] 13) First CH3 domain: T366W+K409A+Y349D+S354D, Second CH3 domain: T366S+L368A+Y407V+F405K+E357A;

[0159] 15) First CH3 domain: T366W+F405K+D399S, Second CH3 domain: T366S+L368A+Y407V+K409A+K392D;

[0160] 16) First CH3 domain: T366W+F405K, second CH3 domain: T366S+L368G+Y407A+K409A; and

[0161] 18) First CH3 domain: T366W+F405K+Y349D+S354D, Second CH3 domain: T366S+L368A+Y407V+K409A+E357A.

[0162] 15. The heterodimer molecule according to any one of embodiments 1-14, wherein the first polypeptide chain and the second polypeptide chain further contain the CH2 domain of the antibody heavy chain constant region.

[0163] 16. The heterodimer molecule according to any one of embodiments 1-15, wherein the first polypeptide chain and the second polypeptide chain further contain a hinge region or a portion of the hinge region of the antibody heavy chain constant region, respectively.

[0164] 17. The heterodimer molecule according to any one of embodiments 1-16, wherein the CH3 domain of the heavy chain constant region of the wild-type human antibody is selected from the CH3 domain of the heavy chain constant region of human IgG, the CH3 domain of the heavy chain constant region of human IgA, the CH3 domain of the heavy chain constant region of human IgD, the CH3 domain of the heavy chain constant region of human IgE, and the CH3 domain of the heavy chain constant region of human IgM.

[0165] 18. The heterodimer molecule according to any one of embodiments 1-17, wherein the CH3 domain of the wild-type human antibody heavy chain constant region is the CH3 domain of the human IgG1 heavy chain constant region.

[0166] 19. The heterodimer molecule according to any one of embodiments 1-18, wherein the first and / or second polypeptide chain further comprises a molecular binding region selected from an antigen-binding region, a receptor-binding region, and an enzyme-binding region.

[0167] 20. The heterodimer molecule according to embodiment 19, wherein the antigen-binding region contains an antibody variable region.

[0168] 21. The heterodimer molecule according to any one of the embodiments 1-20 is a bispecific antibody, a bispecific fusion protein, or an antibody-fusion protein chimera.

[0169] 22. A composition comprising a heterodimeric molecule according to any one of embodiments 1-21, and optionally a pharmaceutically acceptable carrier or excipient.

[0170] 23. A nucleic acid molecule encoding a first polypeptide chain and / or a second polypeptide chain of a heterodimer molecule according to any one of embodiments 1-21.

[0171] 24. A carrier containing nucleic acid molecules as described in implementation scheme 23.

[0172] 25. A host cell containing the vector as described in embodiment 24.

[0173] 26. Use of the heterodimer 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 bispecific antibodies, bispecific fusion proteins, or antibody-fusion protein chimeras.

[0174] 27. A method for preparing a heterodimer molecule, comprising the step of expressing the heterodimer molecule using a host cell according to embodiment 25.

[0175] 28. The method according to embodiment 27, wherein the host cell simultaneously contains a vector encoding a first polypeptide chain and a second polypeptide chain in the heterodimer molecule, and the method includes using the host cell to express, recover, and obtain the heterodimer molecule.

[0176] 29. The method according to embodiment 28, wherein the host cells include a first group of cells and a second group of cells, each of the first group of cells and the second group of cells respectively containing a vector encoding a first polypeptide chain and a second polypeptide chain of the heterodimer molecule, the method comprising: 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 a first polypeptide chain homodimer and a second polypeptide chain homodimer, and then mixing the first polypeptide chain homodimer and the second polypeptide chain homodimer under suitable conditions to prepare the heterodimer molecule.

[0177] Example 1: Obtaining the first round of candidate mutation sequences

[0178] 1. Fc structure modeling and acquisition of interfacial amino acids

[0179] Forty-eight human IgG1 antibody crystal structures containing Fc regions were obtained from the protein database (PDB, www.pdb.org). Using a structural similarity search algorithm (reference: Yuzhen Ye and Adam Godzik. FATCAT: a web server for flexible structure comparison and structure similaritysearching. Nucleic Acids Res., 2004, 32(Web Server issue): W582-585.), it was determined that the Fc segments of these 48 antibodies originated from 1DN2 (PDB number).

[0180] The protein contact amino acid recognition software CMA (http: / / ligin.weizmann.ac.il / cma / ) was used to screen and identify amino acid contacts between CH3-CH3 in the antibody (PDB number: 1DN2) based on the distance of amino acid interactions. According to the amino acid contact rules, interfacial amino acids refer to some amino acids whose distance between a side chain heavy atom and any amino acid heavy atom of another chain is less than a threshold. In this embodiment, the threshold was chosen to be 4.5 Å, but 5.5 Å can also be chosen (e.g., B. Erman, I. Bahar and RL Jernigan. Equilibrium states of rigid bodies with multiple interaction sites. Application to protein helices. J. Chem. Phys. 1997, 107: 2046-2059.). The conservation of amino acid contact interfaces in human and mouse IgG subtypes can be obtained through sequence multiple alignment. Table 1 shows the 34 interfacial amino acids of antibody 1DN2 that passed the amino acid contact screening (i.e., amino acid distance less than 4.5 Å), where chain A and chain B represent the first and second chains of antibody 1DN2, respectively. The following amino acid positions are named according to the EU index of the KABAT number of the antibody Fc.

[0181] Table 1. Amino acid list of the CH3-CH3 interface of antibody 1DN2

[0182]

[0183] 2. Mutating amino acids to alter ion interactions.

[0184] Based on the results in Table 1, amino acid pairs containing charged amino groups were selected, and one amino group on one of the chains was mutated (a non-charged amino acid became a charged amino acid, or a charged amino acid became a non-charged amino acid, or the charge property of the charged amino acid was changed) to make the ionic interaction between Fc chain A and Fc chain B unbalanced, thereby reducing the probability of homodimer formation and / or increasing the probability of heterodimer formation.

[0185] As an example, consider a mutation in chain A's Phe405 residue, resulting in Phe405Lys (also written as F405K), while chain B remains unchanged. Since the contacting amino acid residues on chain B surrounding the 405th amino acid residue contain two Lys residues, both positively charged, the positive charge from the F405K mutation on both chains will introduce a strong repulsive force when chains A pair up. However, when chains A pair up with chain B, only one chain (A) experiences the repulsive force introduced by the F405K mutation, while the other chain (B) retains Phe405 and does not exhibit this repulsive force. In this case, the mutual repulsion between the two chains AA is significant, much greater than that between AB or BB, thus effectively reducing the formation of AA homodimers.

[0186] If, while introducing the F405K mutation into chain A, the contact amino acid residue Lys409 on chain B corresponding to the F405K mutation residue in chain A is mutated to K409E or K409A, then when chains A pair up, the positive charge introduced by the F405K mutation on both chains A will still introduce a strong repulsive force. However, when chains A pair up with chains B, the F405K mutation on chain A interacts with the K409E or K409A mutation on chain B without repulsion, and may even generate attraction (K409E). When chains B pair up, neither repulsion nor attraction is introduced. In this case, the mutual repulsion between the two chains AA is very significant, while the repulsion between AB is reduced or attraction is introduced. Therefore, this effectively reduces the formation of AA homodimers while simultaneously promoting the formation of AB heterodimers.

[0187] Similarly, the mutation combinations obtained in this embodiment are shown in the table below:

[0188] Table 2: List of Heterodimer Mutation Combinations

[0189]

[0190] Example 2: Preparation and investigation of ScFv-Fc / Fc heterodimers

[0191] 1. Construct recombinant vectors expressing the Fc fragment of mutant human IgG1 and the ScFv-Fc fusion protein.

[0192] Based on the constant region amino acid sequence (P01857) of human immunoglobulin gamma1 (IgG1) in the Uniprot protein database, the amino acid sequence of the human IgG1-Fc region was obtained (SEQ ID NO: 1). By reverse transcription PCR, the nucleic acid fragment encoding human IgG1-Fc (SEQ ID NO: 2, named Fc gene) was obtained from total RNA of human PBMCs. The coding sequence of mouse kappaIII signal peptide was added to its 5' end by overlap PCR (as shown in SEQ ID NO: 3). Then, it was subcloned into the vector pcDNA4 (Invitrogen, Cat V86220) to obtain a recombinant expression vector for expressing human IgG1-Fc (Fc) protein in mammalian cells.

[0193] The ScFv-Fc fusion protein encoding gene (as shown in SEQ ID NO: 5, where ScFv refers to a single-chain antibody against HER2) was artificially synthesized. The sequence of the ScFv-Fc fusion protein encoded by this gene is shown in SEQ ID: 4. It was then subcloned into the mammalian cell expression vector pcDNA4 (Invitrogen, Cat V86220) to obtain a recombinant expression vector for mammalian cell expression of the ScFv-Fc fusion protein.

[0194] According to Table 2 of Example 1, combined mutations were performed on the encoding genes of scFV-Fc and Fc using overlap PCR. The mutation targeting the A strand was located on the scFV-Fc fusion protein, and the mutation targeting the B strand was located on the Fc protein. The mutated genes were subcloned into pcDNA4 (Invitrogen, Cat V86220), ultimately yielding recombinant expression vectors for expressing the mutated scFV-Fc fusion protein and the mutated Fc protein in mammalian cells.

[0195] 2. Transiently express ScFv-Fc / Fc heterodimers and detect the effect of different mutant combinations on heterodimer content.

[0196] The expression vectors for the four mutation combinations from step 1, the KH combination (as the reference group), and one 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 co-transfection with its corresponding A-chain (referring to the scFV-Fc fusion protein chain) and B-chain (referring to the Fc protein chain) recombinant expression vectors at a 1:1 ratio. After 5 to 6 days of culture, the transient expression culture supernatant was collected, and the transient transfection products of the four mutation combinations, the KH mutation combination, and the wild-type negative control group were obtained by Protein A affinity chromatography. These transient transfection products contained different proportions of homodimeric protein (ScFv-Fc / ScFv-Fc, Fc / Fc) and heterodimeric protein (ScFv-Fc / Fc). Because these three proteins (ScFv-Fc / ScFv-Fc, Fc / Fc, and ScFv-Fc / Fc) have different molecular weights, the composition of homodimeric proteins (ScFv-Fc / ScFv-Fc, Fc / Fc) and heterodimeric proteins (ScFv-Fc / Fc) in each product group can be detected by SDS-PAGE electrophoresis under non-reducing conditions. Simultaneously, the proportions of homodimeric proteins (ScFv-Fc / ScFv-Fc, Fc / Fc) and heterodimeric proteins (ScFv-Fc / Fc) are analyzed using BioRad's ImageLab professional image analysis software. The electrophoresis results are as follows: Figure 1 As shown in Table 3.

[0197] Table 3. Ratio of homodimers and heterodimers in transient conversion products for each mutant combination

[0198]

[0199] Compared to the wild-type negative control, the proportion of heterodimers (ScFv-Fc / Fc) in all four candidate mutant combinations and the KH combination increased significantly. Furthermore, the introduction of new mutations based on KH also altered the proportion of heterodimers, with some showing a significant increase (e.g., combinations 2 and 4) and others a moderate increase (e.g., combinations 1 and 3). It is noteworthy that because these new mutant combinations involve adjustments to both the steric and ionic interactions of the side chain groups at the contact surfaces, their impact on heterodimer content cannot be simply considered as a superposition of these two interactions. For example, while both combinations introduce the F405K mutation, increasing the repulsion between homodimers, the effect on heterodimer increase is far greater in mutant combination 2 than in mutant combination 1 (the heterodimer content in mutant combination 2 is approximately 70%, while in combination 1 it is approximately 58%). Furthermore, for the mutation introduced at the K409 site, the increase in heterodimer content (77%) brought about by the non-charged mutation in mutation combination 4 is far better than the opposite charge mutation (57%) in mutation combination 3; and theoretically, if we simply consider the superposition of the two interactions, the effects of the two mutations should be similar.

[0200] To further investigate the effect of the co-transformation ratio of the A and B chains of the recombinant expression vector on the homodimer and heterodimer ratios, the two optimal mutant combinations (2 and 4), as well as the co-transformation expression vector used in the KH combination, were transfected into suspension-cultured 293H cells (ATCC CRL-1573) using PEI at ratios of 4:1 and 1:4, respectively. After 5-6 days of culture, the cell supernatant was collected. The transient transformation products were obtained by Protein A affinity chromatography. The composition of homodimer protein (ScFv-Fc / ScFv-Fc, Fc / Fc) and heterodimer protein (ScFv-Fc / Fc) was detected by SDS-PAGE electrophoresis under non-reducing conditions. Specific results are shown in Table 4. The results show that the co-transformation ratio of the recombinant expression vector has a significant impact on the homodimer and heterodimer ratios in the products. Regardless of the co-transformation ratio (4:1 or 1:4), the content of heterodimers in the products was significantly reduced. These results indicate that while these three combinations significantly increase the proportion of heterodimers and decrease the proportion of homodimers in the product when the expression of A and B chains is relatively balanced, when the expression of A and B chains in the product is unbalanced, resulting in an excess of either A or B chain, the excess product will have an increased proportion of homodimers and a relatively lower content of heterodimers. Specifically, in the KH combination, an excess of either chain resulted in a significant decrease in heterodimer content; in mutant combination 2, an excess of B chain (Fc) had a greater impact; and in mutant combination 4, an excess of A chain (ScFv-Fc) had a greater impact. However, even with an excess of either B or A chain in mutant combinations 2 or 4, the proportion of heterodimers formed was still significantly higher than in the control KH combination. Further analysis of the results reveals that while the interaction between the A and B chains was significantly enhanced in these three mutation combinations, the weakening of the interactions between A chains or between B chains was insufficient. This resulted in the imbalance between homodimer and heterodimer formation being disrupted when one component was overexpressed, leading to the production of more homodimers. Specifically, the new mutation combinations 2 and 4 showed a significant improvement in preventing homodimer formation compared to the KH combination.

[0201] Table 4. Effect of different co-transformation ratios on the ratio of homodimers and heterodimers

[0202]

[0203] Example 3: Obtaining the second round of candidate mutation sequences

[0204] 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 disclosed three-dimensional crystal structure of wild-type Fc, further interface amino acid mutations are introduced to further reduce the mutual attraction between A-chain and B-chain, and inhibit the formation of homodimeric proteins.

[0205] Based on the results in Table 1, we further selected paired amino acids containing charged amino acids in the contact amino acids near the mutation sites in mutation combinations 2 and 4, and mutated one amino acid 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). This further increased the imbalance of ionic interactions between chain A and chain B, and reduced the probability of homodimer formation or simultaneously increased the probability of heterodimer formation.

[0206] Mutating the contacting amino acid pair Lys360 in chain A and Gln347 in chain B alters the ionic interactions between them. On one chain (e.g., chain A), both amino acid residues are mutated to negatively charged residues, such as by introducing mutations K360E and Q347E; while on the other chain (e.g., chain B), uncharged amino acid residues are mutated to positively charged residues, such as by introducing the mutation Q347R. In this case, when chain A interacts with chain A, the negative charges at positions 360 and 347 repel each other; when chain B interacts with chain B, the positive charges at these two sites repel each other; only when chain A interacts with chain B will their respective positive and negative charges attract each other. This mutation is expected to increase the repulsion between chains AA and BB, while increasing the attraction between chains AB.

[0207] We also examined the amino acid residue Leu368. This residue is surrounded by two charged amino acid residues, Glu357 and Lys409. Considering that we introduced the K409A mutation in the previous mutation combination 4, in this case, on the same Fc strand where the K409A mutation was already introduced (designated as strand A here according to Example 2), we further mutated Leu368 to a negatively charged amino acid residue (e.g., 368E). Now, when strand A pairs with strand A, the negative charge of L368E on both strands interacts with the negative charge on E357, introducing repulsion; while when strand A pairs with strand B, the negative charge of L368E on strand A repels the negative charge on E357 on strand B, but simultaneously attracts K409 on strand B. Overall, not much repulsion or attraction is introduced. This mutation is expected to increase the mutual repulsion between the two strands AA, but does not affect the interaction between strands AB or BB.

[0208] Based on the preferred Fc mutation combinations (mutation combination 2 and mutation combination 4) mentioned in Examples 1 and 2, and with the addition of newly introduced mutation combinations, the resulting mutation combinations are shown in Table 5:

[0209] Table 5: List of Heterodimer Mutation Combinations - 2

[0210]

[0211] Example 4: Preparation and investigation of a new round of ScFv-Fc / Fc heterodimer mutant combinations

[0212] 1. Construct recombinant vectors expressing the Fc fragment of mutant human IgG1 and the ScFv-Fc fusion protein.

[0213] Using the wild-type scFV-Fc and Fc protein expression recombinant vector constructed in Example 2 as a template, and according to Table 5 of Example 3, the encoding genes of scFV-Fc and Fc were subjected to combined mutations using overlap PCR. The mutation targeting the A strand was located on the scFV-Fc fusion protein, and the mutation targeting the B strand was located on the Fc protein. The mutated genes were subcloned into pcDNA4 (Invitrogen, Cat V86220), ultimately obtaining a recombinant expression vector for expressing the newly mutated scFV-Fc fusion protein and the mutated Fc protein in mammalian cells.

[0214] 2. Transiently express ScFv-Fc / Fc heterodimers and detect the effect of different mutant combinations on heterodimer content.

[0215] Following the method described in Example 2-2, five new mutant combinations (5 to 9) and the first-round preferred mutant combinations (2 and 4) were transiently expressed using 293H cells (ATCC CRL-1573). The recombinant expression vectors for the A and B chains were co-transformed at a ratio of 1:1. After culturing for 5 to 6 days, the transient expression culture supernatant was collected, and the transient transduction products of the five new mutant combinations and the two first-round preferred combinations were obtained by Protein A affinity chromatography. These transient transduction products all contained different proportions of homodimeric proteins (ScFv-Fc / ScFv-Fc, Fc / Fc) and heterodimeric proteins (ScFv-Fc / Fc). Because these three proteins (ScFv-Fc / ScFv-Fc, Fc / Fc, and ScFv-Fc / Fc) have different molecular weights, the composition of homodimeric proteins (ScFv-Fc / ScFv-Fc, Fc / Fc) and heterodimeric proteins (ScFv-Fc / Fc) in each product group can be detected by SDS-PAGE electrophoresis under non-reducing conditions. Simultaneously, the proportions of homodimeric proteins (ScFv-Fc / ScFv-Fc, Fc / Fc) and heterodimeric proteins (ScFv-Fc / Fc) are analyzed using BioRad's ImageLab professional image analysis software. The electrophoresis results are as follows: Figure 2 As shown in Table 6.

[0216] Table 6. Ratio of homodimers to heterodimers in the transient products of each mutant combination -2

[0217]

[0218] Compared to the preferred combinations from the first round of mutations, some newly introduced mutations slightly increased the proportion of heterodimer formation, such as combination 5 compared to combination 2; several combinations showed little change, such as combination 6 compared to combination 2, and combinations 7 and 8 compared to combination 4; however, the introduction of a new mutation in combination 9 significantly reduced the proportion of heterodimer formation. This is presumably because the negative charge of L368E newly introduced in chain A repulses the negative charge of E357 on chain B more than it attracts K409 on chain B, making the heterodimer unstable. Overall, while some of the newly introduced mutations provided some assistance in heterodimer formation, they did not result in a significant increase.

[0219] To further investigate the effects of the newly introduced mutations on A-chain-A and B-chain-B homodimers, we transiently expressed either the A-chain or B-chain protein separately and examined the homodimer formation trend by comparing the expression levels of homodimer proteins under the same transient transfection conditions. The recombinant expression vector was transfected into suspension-cultured 293H cells (ATCCCRL-1573) using PEI. After 5-6 days of culture, the cell supernatant was collected. The transient transfection products were obtained using Protein A affinity chromatography, and their expression levels were detected using OD280. The results are shown in Table 7. From the expression levels, the partial mutations introduced into chain A in combination 4 (combinations 8 and 9) reduced the homodimer formation trend; the partial mutations introduced into chain B in combination 2 (combination 5) reduced the homodimer formation trend; the other new mutations had little effect on homodimer formation. The results also show that, compared to combination 4 and its derived mutant combinations (7, 8, 9), combination 2 and its mutant derivatives (5, 6) exhibit a lower tendency for chain A to form homodimers; while the latter shows a lower tendency for chain B to form homodimers. This result is consistent with the results obtained in Example 2, further demonstrating the feasibility of using this method to preliminarily examine homodimer formation trends. Furthermore, in terms of expression levels, all chains B are significantly lower than chains A. We also performed individual transient expression tests on wild-type chains A and B, finding that without any mutations, the expression level of wild-type B homodimers was lower than that of wild-type chains A (approximately half that of wild-type A). This suggests that the N-terminus of the Fc sequence in chain A, fused with the ScFv sequence, contributes to its increased expression level; however, the difference in expression levels between chains A and B does not directly reflect the difference in homodimer formation trends between chains A and B.

[0220] Table 7. Comparison of homodimer expression levels in each mutant combination with transient transduction of chain A or chain B alone.

[0221]

[0222] Example 5: Obtaining the third round of candidate mutation sequences

[0223] Based on the crystal structure of mutant combination 4, and with structural modeling, new candidate sequences for amino acid mutations at the contact interface will be identified in order to further inhibit the formation of homodimeric proteins or promote the formation of heterodimeric proteins based on the original mutant combinations (such as mutant combinations 2 or 4).

[0224] Crystal structure analysis of mutant combinatorial heterodimer protein

[0225] Mutant combination 4 was selected, transiently expressed in 293H cells (ATCC CRL-1573), and the heterodimeric protein of mutant combination 4 was purified and subjected to crystal structure determination. Here, we used molecular cloning to insert a His-tag sequence at the C-terminus of chain B of mutant combination 4, so that after Protein A affinity chromatography, a relatively pure chain A-chain B heterodimeric protein could be obtained for crystallization using the IMAC method.

[0226] The crystal structure analysis process is as follows:

[0227] Heterodimeric Fc crystals were formed under the following conditions: 2 μL of crystallization buffer (15% PEG3350, 1M LiCl, 0.1M 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 standing droplet at 22 °C. Crystals grew after approximately 3 days. The crystals were then placed in a solution containing 17% PEG3350, 1M LiCl, 0.1M MES, pH 6.0, and 20% glycerol; followed by rapid wetting and freezing in liquid nitrogen. X-ray diffraction data were collected using an SSRF BL17U. Molecular substitution structure was determined using the structure of wild-type Fc (PDB accession number: 3AVE) as a framework.

[0228] Crystal structure analysis shows that the overall structure of the mutated Fc heterodimer is similar to that of the wild-type Fc, but it is altered at the CH3 interface due to the interaction of different side chain groups. The specific crystal structure of the CH3 interface is shown in [reference needed]. Figure 3 .

[0229] 2. Obtaining new candidate combinations of mutations

[0230] Based on the results of the crystal structure of mutation combination 4, new candidate mutations were further screened.

[0231] First, through the three-dimensional crystal structure analysis, we discovered 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; instead, they become a pair of free thiol groups. Based on this result, we will cancel this pair of mutations in the third round of mutagenesis, restoring it to the wild-type amino acid sequence before the mutation.

[0232] Second, through three-dimensional structural modeling and comparison, further mutations were introduced near the mutated amino acid residue pair F405K-K409A to alter ionic and hydrogen bonds. Assuming K409A is located on chain A and F405K is located on chain B, a K392D mutation was introduced on chain A and a D399S mutation was introduced on chain B. For example... Figure 4As shown, for the interaction between chain A and chain B, the newly introduced mutation pair adds an ionic bond of K392D-F405K and a hydrogen bond of K392D-D399S, which is expected to effectively increase the tendency for heterodimer formation. In the A-chain-A interaction, electrostatic repulsion between K329D and D399 is introduced, inhibiting the formation of homodimers in chain A. In the B-chain-B interaction, the original ionic bond between K409 and D399 disappears due to the introduction of the D399S mutation, reducing the tendency for homodimer formation in chain B.

[0233] Third, comparing the crystal structure of mutant combination 4 with that of the wild-type Fc protein revealed an outward shift of chain A (i.e., away from chain B). This is presumably due to steric hindrance caused by the enlarged side chain group in the T366W mutation in chain A. Based on this, we further mutated the amino acid residues on chain B that were in contact with the T366W residues 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 Y407A and L386G mutations, leaving sufficient space for the T366W mutation. This could potentially further stabilize the heterodimer structure.

[0234] Fourth, mutations were introduced on other contact amino acid pairs surrounding the F405K-K409A mutant amino acid pair to alter the interfacial electrostatic interactions. Here, we examine the Y349 and E357 contact amino acid pair. Chain A introduced the mutation Y349D, and chain B introduced E357A. The electrostatic repulsion introduced between Y349D and E357A between A and B inhibits the formation of AA homodimers; no new forces were introduced between A and B, or between B and C. Building on this, chain A was further mutated with the S354D mutation, strengthening the electrostatic repulsion between it and E357A, further inhibiting the formation of AA homodimers.

[0235] First, based on mutation combination 4, the above mutations are introduced, and the resulting mutation combinations are shown in Table 8:

[0236] Table 8: List of Heterodimer Mutation Combinations - 3

[0237]

[0238] Subsequently, based on mutation combination 2, the above mutations were introduced while referring to mutation combination 4, and the resulting mutation combinations are shown in Table 9:

[0239] Table 9: List of Heterodimer Mutation Combinations - 4

[0240]

[0241] Example 6: Preparation and investigation of the third round of ScFv-Fc / VhH-Fc heterodimer mutant combinations

[0242] 1. Construct recombinant vectors expressing the Fc fragment of mutant human IgG1 and the ScFv-Fc fusion protein.

[0243] Considering that the expression level of the pure Fc fragment is lower than that of scFv-Fc, in order to better control the expression ratio of the two strands, we fused a variable region sequence of a camel single-domain antibody (labeled VhH) to the N-terminus of the original B strand (pure Fc strand). The gene encoding the VhH-Fc fusion protein, as shown in SEQ ID NO: 36, was artificially synthesized. The sequence of the VhH-Fc fusion protein encoded by this gene is shown in SEQ ID: 35. It was then subcloned into the mammalian cell expression vector pcDNA4 (Invitrogen, Cat V86220) to obtain a recombinant expression vector for expressing the VhH-Fc fusion protein in mammalian cells.

[0244] Using the wild-type scFV-Fc protein expression recombinant vector constructed in Example 2 and the above-mentioned VhH-Fc fusion protein recombinant expression vector as templates, according to Table 8 of Example 5, the encoding genes of scFV-Fc and VhH-Fc (SEQ ID NO: 5 and SEQ ID NO: 36) were subjected to combined mutations using overlapping PCR. The mutation targeting the A strand was located on the scFV-Fc fusion protein, and the mutation targeting the B strand was located on the VhH-Fc protein. The mutated genes were subcloned into pcDNA4 (Invitrogen, Cat V86220), ultimately obtaining recombinant expression vectors for expressing the third-round mutated scFV-Fc fusion protein and the mutated VhH-Fc protein (SEQ ID NO: 4 to SEQ ID NO: 35) in mammalian cells.

[0245] 2. Transiently express ScFv-Fc / VhH-Fc heterodimers and detect the effect of different mutant combinations on heterodimer content.

[0246] Following the method described in Examples 2-2, the 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-transformation ratios of the A and B chains in the recombinant expression vectors were 4:1, 1:1, and 1:4. After culturing for 5 to 6 days, the transient expression culture supernatant was collected, and the four preliminarily purified new mutant combinations, as well as the transient transformation product of mutant combination 4, were obtained by Protein A affinity chromatography. These transient transformation products all contained different proportions of homodimeric proteins (ScFv-Fc / ScFv-Fc, VhH-Fc / VhH-Fc) and heterodimeric 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) differ, the composition of homodimeric proteins (ScFv-Fc / ScFv-Fc, VhH-Fc / VhH-Fc) and heterodimeric 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 homodimeric proteins (ScFv-Fc / ScFv-Fc, VhH-Fc / VhH-Fc) and heterodimeric proteins (ScFv-Fc / VhH-Fc) was analyzed using BioRad's ImageLab professional image analysis software. The electrophoresis results are shown in Table 10.

[0247] Table 10. Ratio of homodimers and heterodimers in transient products of each mutant combination -3

[0248]

[0249] To further investigate the effects of the newly introduced mutations on A-chain-A and B-chain-B homodimers, we transiently expressed either the A-chain or B-chain protein separately. By comparing the expression levels of homodimer proteins under the same transient transfection conditions, we examined the trend of homodimer formation. The recombinant expression vector was transfected into suspension-cultured 293H cells (ATCCCRL-1573) using PEI. After 5-6 days of culture, the cell supernatant was collected. The transient transfection products were obtained using Protein A affinity chromatography, and their expression levels were detected using OD280. The results are shown in Table 11.

[0250] Table 11. Comparison of expression levels of chain A or chain B homodimers in each mutant combination -2

[0251]

[0252] In summary, the results show that while the third round of mutations in mutant combination 4 did not significantly inhibit the formation of homodimers in chain A, it significantly inhibited the formation of homodimers in chain B and effectively promoted the formation of heterodimers. When the expression of the two chains was close to equilibrium (1:1), the heterodimer content in several new mutant combinations reached over 80%, a significant improvement compared to mutant combination 4. In mutant combination 11, the new mutation targeting chain B essentially completely inhibited the formation of homodimers in chain B. Even at a transient conversion ratio of 1:4 (A:B), no homodimers of chain B were observed, and the heterodimer content reached 89%.

[0253] Based on the results of combinations 10 to 13, we further selected mutant combinations 15, 16, and 18 and examined their effect on promoting heterodimer formation through transient expression.

[0254] Following the method described in Examples 2-2, the three mutant combinations (15, 16, 18) in Table 9, as well as mutant combination 2, were transiently expressed using 293H cells (ATCC CRL-1573). The co-transformation ratios of the recombinant expression vectors for the A and B chains were 4:1, 1:1, and 1:4. After culturing for 5 to 6 days, the transient expression culture supernatant was collected, and the three preliminarily purified new mutant combinations and the transient transformation products of mutant combination 2 were obtained by Protein A affinity chromatography. These transient transformation products all contained different proportions of homodimeric proteins (ScFv-Fc / ScFv-Fc, VhH-Fc / VhH-Fc) and heterodimeric proteins (ScFv-Fc / VhH-Fc). Because the molecular weights of these three proteins (ScFv-Fc / ScFv-Fc, VhH-Fc / VhH-Fc, and ScFv-Fc / VhH-Fc) differ, the composition of homodimeric proteins (ScFv-Fc / ScFv-Fc, VhH-Fc / VhH-Fc) and heterodimeric proteins (ScFv-Fc / VhH-Fc) in each group of products can be detected by SDS-PAGE electrophoresis under non-reducing conditions. Simultaneously, the proportions of homodimeric proteins (ScFv-Fc / ScFv-Fc, VhH-Fc / VhH-Fc) and heterodimeric proteins (ScFv-Fc / VhH-Fc) were analyzed using BioRad's ImageLab professional image analysis software. The electrophoresis results are shown in Table 12. It can be seen that... After introducing a third round of mutations into mutant combination 2, it also showed a more significant effect in inhibiting homodimer formation of chain B and effectively promoted heterodimer formation. When the expression of the two chains was close to equilibrium (1:1), the heterodimer content in several new mutant combinations reached over 80%, which was significantly higher than that of mutant combination 4. Among them, mutant combinations 16 and 18, with appropriate changes in the ratio of transient transduction vectors (excessive chain B plasmid or equilibrium of the two plasmids), still achieved a heterodimer content of over 80%.

[0255] Table 12. Ratio of homodimers and heterodimers in the transient products of each mutant combination -4

[0256]

[0257] Example 7: Evaluation of other indicators of heterodimers

[0258] 1. Accelerated stability testing of heterodimers

[0259] We selected heterodimers from mutant combinations 4, 11, and 16 for accelerated stability testing over a 31-day period at 45°C using PBS as the buffer. Non-reducing CE-SDS was measured on days 0, 8, 18, and 31, and compared with the corresponding wild-type Fc protein. The 31-day accelerated stability SDS-PAGE results showed that the decrease in the main peak content of the three mutant samples and the wild-type control sample did not exceed 2% until day 31. This indicates that the heterodimers possess the same thermal stability as the wild type.

[0260] Although 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 disclosed, and these changes are all within the scope of protection 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.

Claims

1. A heterodimeric molecule comprising a first polypeptide chain and a second polypeptide chain, the first polypeptide chain comprising a first CH3 domain of the antibody heavy chain constant region, and the second polypeptide chain comprising a second CH3 domain of the antibody heavy chain constant region, wherein the first CH3 domain and the second CH3 domain contain a mutation of an amino acid selected from the group consisting of the following positions as indicated, compared to the corresponding wild-type human antibody heavy chain constant region CH3 domain: (I) Mutations occur at T366 and K409 of the first CH3 domain, and mutations occur at T366, L368, Y407 and F405 of the second CH3 domain, and optionally the first CH3 domain and / or the second CH3 domain also have mutations at one or two amino acid positions selected from K392, D399, Y349, S354 and E357; (II) Mutations occur at T366 and F405 of the first CH3 domain, and mutations occur at T366, L368, Y407, and K409 of the second CH3 domain, and optionally, the first CH3 domain and / or the second CH3 domain also have mutations at one or two amino acid positions selected from K392, D399, Y349, S354, and E357; and (III) Mutations occur at Y349 and T366 of the first CH3 domain, and mutations occur at D356, T366, L368, and Y407 of the second CH3 domain; furthermore, mutations occur at 1 to 3 amino acid positions selected from F405, K409, K360, Q347, and L368 in the first CH3 domain and / or the second CH3 domain; and The positions of the amino acids mentioned above were determined based on the EU index of the KABAT number of the antibody Fc.

2. The heterodimer molecule according to claim 1, wherein the first CH3 domain and / or the second CH3 domain optionally further comprises one of the mutations selected from: 2a) A mutation occurs at K392 in the first CH3 domain and a mutation occurs at D399 in the second CH3 domain; 2b) A mutation occurs at Y349 in the first CH3 domain, and a mutation occurs at E357 in the second CH3 domain; and 2c) Mutations occur at Y349 and S354 in the first CH3 domain and at E357 in the second CH3 domain.

3. The heterodimer molecule according to claim 1, wherein each mutation is independently selected from uncharged amino acid mutation to charged amino acid, charged amino acid mutation to uncharged amino acid, or charged amino acid mutation to amino acid with opposite charge.

4. The heterodimer molecule according to claim 1, wherein the mutation in the first CH3 domain and / or the second CH3 domain comprises one or more mutations selected from the following: Y349C, Y349D, T366W, T366S, L368A, L368E, L368G, F405K, Y407V, Y407A, K409E, K409A, K392D, D399S, E357A, and S354D.

5. The heterodimer molecule according to claim 1, 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 T366, L368, Y407, F405, D399, E357, K409 and / or K392.

6. The heterodimer molecule according to claim 1, 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 T366S, L368A, Y407V, F405K, D399S, L368G, Y407A, E357A, K409A and / or K392D.

7. The heterodimer molecule according to claim 1, wherein the first CH3 domain and the second CH3 domain contain a mutation of an amino acid selected from the positions shown in (1) to (4) below: (1) First CH3 domain: T366+K409+K392, second CH3 domain: T366+L368+Y407+D399+F405; (2) First CH3 domain: T366+K409, second CH3 domain: T366+L368+Y407+F405; (3) First CH3 domain: T366+K409+Y349, Second CH3 domain: T366+L368+Y407+F405+E357; and (4) First CH3 domain: T366+K409+Y349+S354, Second CH3 domain: T366+L368+Y407+F405+E357; in, In the first CH3 domain, the mutation at T366 is T366W, the mutation at K409 is K409A, the mutation at K392 is K392D, the mutation at Y349 is Y349D, and the mutation at S354 is S354D; in the second CH3 domain, the mutation at T366 is T366S, the mutation at L368 is L368A, and the mutation at Y407 is Y407V, or, the mutation at L368 is L368G, and the mutation at Y407 is Y407A, the mutation at D399 is D399S, the mutation at F405 is F405K, and the mutation at E357 is E357A.

8. The heterodimer molecule according to claim 1, wherein the CH3 domain of the heavy chain constant region of the wild-type human antibody is selected from the CH3 domain of the heavy chain constant region of human IgG, the CH3 domain of the heavy chain constant region of human IgA, the CH3 domain of the heavy chain constant region of human IgD, the CH3 domain of the heavy chain constant region of human IgE, and the CH3 domain of the heavy chain constant region of human IgM.

9. The heterodimer molecule according to claim 1, wherein it is a bispecific antibody, a bispecific fusion protein, or an antibody-fusion protein chimera.

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