CH3 domain variants or bispecific antibodies containing them

Abstract

The present invention relates to a heterodimer containing a CH3 domain variant pair or a bispecific antibody containing the same, and more specifically, to a heterodimer or a bispecific antibody to which a CH3 domain variant pair with amino acid substitutions at a specific position is applied.

Description

[Technical Field]

[0001] The present invention relates to a heterodimer containing a CH3 domain mutant pair or a bispecific antibody containing the same, and more specifically to a heterodimer or a bispecific antibody to which a CH3 domain mutant pair in which an amino acid at a specific position is substituted is applied. [Background technology]

[0002] In recent years, as the diverse causes and mechanisms of action for certain indications have become clearer, approaches to therapeutic drug development have shifted from single-target to multi-target approaches. Accordingly, for decades, various studies have been conducted in the development of antibody-based therapeutics to confer dual or multispecificity to monospecific antibodies, enabling them to specifically bind to two or more antigen proteins.

[0003] Bispecific antibodies are antibodies that have dual functions by simultaneously interacting with two different antigens. Effective strategies for generating bispecific antibodies are based on approaches that mutate CH3 amino acid residues to form a knob for half of the antibody and a hole for the other half. Such knob-into-hole (kiH) techniques have been reported to generate heterodimers of approximately 80% (Ridgway, Presta et al. 1996).

[0004] However, there is still a need for improvement / modification to enhance the bisspecificity of currently developed bispecific antibodies. [Overview of the Initiative] [Problems that the invention aims to solve]

[0005] Against this technological backdrop, the inventors of this application have discovered that, in addition to conventional knob-into-hole (kiH) technology, the bispecificity of antibodies can be improved through CH3 domain engineering by creating heterodimers containing CH3 domain variant pairs with specific amino acid substitutions, thereby completing the present invention.

[0006] The object of the present invention is to provide a heterodimer containing a CH3 domain mutant pair.

[0007] The object of the present invention is to provide a fusion protein containing the aforementioned heterodimer.

[0008] The object of the present invention is to provide a bispecific antibody containing a heterodimer including a CH3 domain mutant pair. [Means for solving the problem]

[0009] To achieve the above objective, the present invention provides a heterodimer comprising a first heavy chain CH3 domain and a second heavy chain CH3 domain, The first heavy chain CH3 domain or the second heavy chain CH3 domain contains one or more amino acid substitutions (according to the EU index) at positions selected from the group shown below: (1) The amino acid at position Q347, E356, or E357 is substituted with K or R. (2) The amino acid at position D399 is substituted with W, F, Y, or H. (3) The amino acid at position K360, K370, or K439 is substituted with Q, N, E, or D. (4) The amino acid at position K409 is substituted with A, V, T, or S. The present invention provides a heterodimer in which the first heavy-chain CH3 domain and the second heavy-chain CH3 domain bind to form a dimer.

[0010] The present invention also provides a fusion protein comprising the heterodimer.

[0011] The present invention relates to a bispecific antibody comprising a first arm that binds to a first antigen including VH1-CHa-Fc1 and VL1-CLb, and a second arm that binds to a second antigen including VH2-CH1-Fc2 and VL2-CL:

[0012] The aforementioned VH1 and VH2 are heavy chain variable regions that each contain the same or different antigen-binding regions.

[0013] The aforementioned VL1 and VL2 are light chain variable regions that each contain the same or different antigen-binding regions.

[0014] The CHa comprises i) an IgG heavy chain invariant region or IgD heavy chain invariant region CH1, and an IgG heavy chain invariant region CH2 or CH3.

[0015] The CLb comprises one or more selected from the group consisting of CL1 containing an IgG light chain invariant region λ or κ and IgG heavy chain invariant regions CH1, CH2, CH3, i)

[0016] CH1 is the IgG heavy chain invariant region CH1, and CL is the IgG light chain invariant region CL.

[0017] Fc1 of the first arm and Fc2 of the second arm combine to form a heavy chain invariant region dimer,

[0018] A bispecific antibody comprising a heterodimer containing a first heavy-chain CH3 domain and a second heavy-chain CH3 domain, where the CH3 of CHa, Fc1 of the first arm, or Fc2 of the second arm are located at a position selected from the group shown below, and each domain contains one or more amino acid substitutions (according to the EU index): (1) The amino acid at position Q347, E356, or E357 is substituted with K or R. (2) The amino acid at position D399 is substituted with W, F, Y, or H. (3) The amino acid at position K360, K370, or K439 is substituted with Q, N, E, or D. (4) The amino acid at position K409 is substituted with A, V, T, or S.

Brief Description of the Drawings

[0019] [Figure 1] It is a diagram showing the structure of a bispecific antibody containing a heterodimer containing a mutation according to the present invention. [Figure 2] It is a diagram showing the structure of the entire CH3 domain of a bispecific antibody and the target region of the mutation. [Figure 3] It is a schematic diagram of the region of the mutation candidate group. [Figure 4] It is a diagram showing the analysis result of the expression pattern through non-reducing SDS-PAGE of the filtered supernatant. [Figure 5] Figures 5a to 5f are diagrams showing the analysis results of non-reducing and reducing SDS-PAGE of the product obtained by primary purification of the bispecific antibody candidates. [Figure 6] It is a diagram showing the ELISA result of single antigen binding containing the ErbB2 and VEGF binding regions. [Figure 7] It is a diagram showing the ELISA result of dual antigen binding containing the ErbB2 and VEGF binding regions. [Figure 8] It is a diagram showing the result of quantifying the result of FACS analysis of the binding strength between ErbB2 expressed on the surface of JIMT-1 cells and the bispecific antibody.

Modes for Carrying Out the Invention

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the technical field to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the technical field.

[0021] In one aspect, the present invention relates to a heterodimer comprising a first heavy chain CH3 domain and a second heavy chain CH3 domain,

[0022] The first heavy chain CH3 domain or the second heavy chain CH3 domain contains one or more amino acid substitutions (according to the EU index) at a position selected from the group shown below: (1) The amino acid at position Q347, E356, or E357 is substituted with K or R. (2) The amino acid at position D399 is substituted with W, F, Y, or H. (3) The amino acid at position K360, K370, or K439 is substituted with Q, N, E, or D. (4) The amino acid at position K409 is substituted with A, V, T, or S. This invention relates to a heterodimer in which the first heavy-chain CH3 domain and the second heavy-chain CH3 domain bind to form a dimer.

[0023] The heterodimer is based on a first heavy chain CH3 domain and a second heavy chain CH3 domain, each of which can be considered a monomer, and "monomer" may mean half of the heterodimer. The heterodimer can be formed by assembling monomers. The heterodimer can also be formed by inducing amino acid mutations in each of the monomers and assembling the mutated monomers.

[0024] Specifically, the heterodimer may form a knob-into-hole (kiH) including mutations that cause steric hindrance, promoting heterodimer formation and suppressing homodimer formation. The heterodimer forms a mutated CH3 domain pair in each monomer, specifically in the first heavy chain CH3 domain and the second heavy chain CH3 domain, including mutations such as amino acid substitutions.

[0025] In relation to the knob-into-hole (kiH), a knob can be formed by a mutation in the first heavy chain CH3 domain, such as an amino acid substitution, to form a knob, and by a mutation in the second heavy chain CH3 domain, such as an amino acid substitution, to form a hole.

[0026] As a result, the first heavy chain CH3 domain or the second heavy chain CH3 domain contains one or more amino acid substitutions (according to the EU index) at a position selected from the group shown below. (1) The amino acid at position Q347, E356, or E357 is substituted with K or R. (2) The amino acid at position D399 is substituted with W, F, Y, or H. (3) The amino acid at position K360, K370, or K439 is substituted with Q, N, E, or D. (4) The amino acid at position K409 is substituted with A, V, T, or S. The first heavy-chain CH3 domain and the second heavy-chain CH3 domain bind to form a dimer.

[0027] In other words, the present invention relates to a bispecific antibody comprising a first arm that binds to a first antigen comprising VH1-CHa-Fc1 and VL1-CLb, and a second arm that binds to a second antigen comprising VH2-CH1-Fc2 and VL2-CL:

[0028] The aforementioned VH1 and VH2 are heavy chain variable regions that each contain the same or different antigen-binding regions. The aforementioned VL1 and VL2 are light chain variable regions that each contain the same or different antigen-binding regions. The CHa comprises i) an IgG heavy chain invariant region or IgD heavy chain invariant region CH1, and an IgG heavy chain invariant region CH2 or CH3. The CLb comprises one or more selected from the group consisting of CL1 containing an IgG light chain invariant region λ or κ and IgG heavy chain invariant regions CH1, CH2, CH3, i) CH1 is the IgG heavy chain invariant region CH1, and CL is the IgG light chain invariant region CL. Fc1 of the first arm and Fc2 of the second arm combine to form a heavy chain invariant region dimer. The CH3 of CHa, Fc1 of the first arm, or Fc2 of the second arm, at a position selected from the group shown below, contains a heterodimer comprising a first heavy-chain CH3 domain and a second heavy-chain CH3 domain, each containing one or more amino acid substitutions (according to the EU index) at a position selected from the group shown below: (1) The amino acid at position Q347, E356, or E357 is substituted with K or R. (2) The amino acid at position D399 is substituted with W, F, Y, or H. (3) The amino acid at position K360, K370, or K439 is substituted with Q, N, E, or D. (4) The amino acid at position K409 is substituted with A, V, T, or S.

[0029] This invention provides a novel form of bispecific antibody, different from the bispecific antibody described in Korean Patent Publication No. 2022-0095163. By introducing different mutant pairs into the CH3 domains that overlap between Fc and Fab, mispairing is minimized, thereby improving the yield of heterologous dilutions compared to conventional bispecific antibodies.

[0030] The heterodimer is based on a first heavy chain CH3 domain and a second heavy chain CH3 domain, each of which can be considered a monomer, and "monomer" may mean half of the heterodimer. The heterodimer can be formed by assembling monomers. The heterodimer can also be formed by inducing amino acid mutations in each of the monomers and assembling the mutated monomers.

[0031] Specifically, a knob-into-hole (kiH) can be formed, including mutations that cause steric hindrance, promoting heterodimer formation and suppressing homodimer formation. The heterodimer forms a mutated CH3 domain pair in each monomer, specifically in the first heavy chain CH3 domain and the second heavy chain CH3 domain, including mutations such as amino acid substitutions.

[0032] In relation to the knob-into-hole (kiH), a knob-into-hole (kiH) can be formed by a mutation in the first heavy chain CH3 domain, such as an amino acid substitution, to form a knob, and by a mutation in the second heavy chain CH3 domain, such as an amino acid substitution, to form a hole.

[0033] As a result, the first heavy chain CH3 domain or the second heavy chain CH3 domain contains one or more amino acid substitutions (according to the EU index) at a position selected from the group shown below, (1) The amino acid at position Q347, E356, or E357 is substituted with K or R. (2) The amino acid at position D399 is substituted with W, F, Y, or H. (3) The amino acid at position K360, K370, or K439 is substituted with Q, N, E, or D. (4) The amino acid at position K409 is substituted with A, V, T, or S. The first heavy-chain CH3 domain and the second heavy-chain CH3 domain bind to form a dimer.

[0034] The antibody that binds to the heterodimer may be of any type, such as Fab, Fab', F(ab')2, vH, vL, Fv, scFv, scFv2, scFab, or dAb.

[0035] "Dual specificity" or "bispecificity" refers to the property of a binding protein that can specifically bind to two different targets and modulate their activity. For example, it can be produced by conjugating monoclonal antibodies or fragments thereof that specifically bind to each target, possessing two segmented antigen-binding arms (specificity for two targets) and being monovalent for each antigen to which it binds.

[0036] VH1 and VH2 are heavy chain variable regions, each containing the same or different antigen-binding regions. VL1 and VL2 are light chain variable regions, each containing the same or different antigen-binding regions.

[0037] A polypeptide is any polymer chain of amino acids. The terms "peptide" and "protein" can be used interchangeably with polypeptide, and these also refer to polymer chains of amino acids. Polypeptides include natural or synthetic proteins, protein fragments, and polypeptide analogs of protein sequences. Polypeptides may be monomers or polymers.

[0038] In relation to interactions between antibodies, polypeptides, proteins, or peptides, "specific binding" or "specific binding" means that the interaction occurs based on the presence of a specific structure on a chemical species (e.g., an antigen-determining factor or epitope). For example, antibodies generally recognize and bind to specific protein structures rather than the protein itself. If an antibody is specific to epitope "A", then in a reaction involving labeled "A" and the antibody, the presence of molecules containing epitope A (or free, unlabeled A) is expected to bind to the antibody and reduce the amount of labeled A.

[0039] An antibody refers to any immunoglobulin (Ig) molecule composed of four polypeptide chains, i.e., two heavy chains (H) and two light chains (L), or any functional fragment, mutant, variant, or derivative of such an Ig molecule that possesses the essential epitope-binding features. Specific examples of these variants, variants, or derivatives are discussed below, but are not limited to them.

[0040] A "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population; that is, an antibody that is identical to the individual antibodies in the population, excluding any possible spontaneous mutations that may exist in trace amounts. Monoclonal antibodies are highly specific and are induced against a single antigenic site. In contrast to conventional (polyclonal) antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen.

[0041] An "epitope" refers to a determinant site on a protein to which an antibody can specifically bind. Epitopes are typically composed of chemically active surface molecules, such as amino acids or sugar side chains, and generally possess not only specific three-dimensional structural features but also specific charge properties. Stereomorphic and non-stereomorphic epitopes are distinguished by the fact that their electron-binding properties disappear in the presence of a denaturing solvent, while the latter does not.

[0042] Humanized non-human (e.g., Murin) antibodies are chimeric antibodies containing minimal sequences derived from non-human immunoglobulins. In most cases, humanized antibodies are human immunoglobulins (recipient antibodies) in which residues from the recipient's hypervariable region are replaced with residues from the hypervariable region of a non-human species (donor antibody), such as mouse, rat, rabbit, or non-human primate, that possess the desired specificity, affinity, and capability.

[0043] A "human antibody" is a molecule derived from human immunoglobulin, meaning that the entire amino acid sequence constituting the antibody, including the complementarity-determining region and structural region, is composed of human immunoglobulin.

[0044] In a complete antibody, each heavy chain consists of a heavy chain variable region (represented as HCVR or VH) and a heavy chain invariant region. The heavy chain invariant region consists of three domains: CH1, CH2, and CH3. Each light chain consists of a light chain variable region and a light chain invariant region. The light chain invariant region consists of one domain, CL. The VH and VL regions are again divided into a hypervariable region called the complementarity-determining region (CDR), which contains a more conserved region called the skeletal region (FR). The "variable region" refers to the light and heavy chain portions of the antibody molecule, including the amino acid sequences of the complementarity-determining region (CDR; i.e., CDR1, CDR2, and CDR3) and the skeletal region (FR). VH refers to the variable region of the heavy chain. VL refers to the variable region of the light chain.

[0045] The "complementarity-determining regions" (CDRs; i.e., CDR1, CDR2, and CDR3) refer to the amino acid residues in the antibody variable region that are necessary for antigen binding. Each variable region typically has three CDR regions, identified as CDR1, CDR2, and CDR3. The "skeletal regions" (FRs) are variable region residues other than the CDR residues. Each variable region typically has four FRs, identified as FR1, FR2, FR3, and FR4. These are arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0046] A Fab fragment has a structure that contains variable regions of the light and heavy chains, an invariant region of the light chain, and a first invariant region (CH1) of the heavy chain, and has one antigen-binding site. A Fab' fragment differs from Fab in that it has a hinge region containing one or more cysteine ​​residues at the C-terminus of the heavy chain CH1 domain. F(ab')2 is produced when the cysteine ​​residues in the hinge region of Fab' form disulfide bonds. Fv is the smallest antibody fragment that contains only the heavy chain variable region and the light chain variable region. In a two-chain Fv, the heavy chain variable region and the light chain variable region are linked by non-covalent bonds, while in a single-chain Fv (scFv), the heavy chain variable region and the light chain variable region are generally linked by covalent bonds via a peptide linker or directly linked at the C-terminus, and can form a dimer-like structure similar to a two-chain Fv. Such antibody fragments can be obtained using proteolytic enzymes (for example, restrictive cleavage of the whole antibody with papain yields Fab, and cleavage with pepsin yields the F(ab')2 fragment), and can also be produced through genetic engineering techniques.

[0047] The present invention comprises an "antigen-binding region," which is one or more antibody fragments having specific binding ability to an antigen, and can specifically bind to other antigens, thereby being bispecific, bispecific, or multiplespecific. In the present invention, the antigen-binding region is contained in the heavy chain variable region of VH1 or VH2 and the light chain variable region of VL1 or VL2, each containing the same or different antigen-binding regions.

[0048] According to the present invention, the IgG-like structure does not deviate significantly. Structures that significantly impair the IgG structure are likely to greatly increase the instability of the protein. Furthermore, we attempted to significantly reduce the frequency of light chain mispairing by introducing an immunoglobulin structure-invariant region into the light chain mispairing region. By introducing a conventionally known heavy chain constant region domain into one of the two Fab arms, we induced a mechanism to prevent light chain mispairing.

[0049] The aforementioned bispecific antibody has two target-binding Fab portions, with no structural deformation in the left arm (second arm), and the right arm (first arm) further incorporating an Ig-invariant domain (CH3). In addition to IgG1 CH1, various types of CH1 can be introduced in place of IgG1 CH1, and the heavy chain heterodimer configuration can incorporate a knob-in-hole structure.

[0050] The CHa may include i) an IgG heavy chain invariant region or an IgD heavy chain invariant region CH1, and an IgG heavy chain invariant region CH2 or CH3.

[0051] For example, CHa may include, in the first arm, CH3 derived from IgG1, IgG2, IgG3, IgG4, IgD, or IgM, in the order from the N-terminus to the C-terminus, and CH3 derived from IgG1, IgG2, IgG3, or IgG4.

[0052] The CLb comprises one or more selected from the group consisting of i) CL1 containing an IgG light chain invariant region λ or κ and IgG heavy chain invariant regions CH1, CH2, and CH3.

[0053] The CLb may include one or more selected from the group consisting of CL1 containing a light chain invariant region λ or κ derived from IgG1, IgG2, IgG3, IgG4, IgD, or IgM, and heavy chain invariant regions CH1, CH2, CH3 derived from IgG1, IgG2, IgG3, IgG4. For example, the CLb may include, in the first arm, CL1 containing a light chain invariant region λ or κ derived from IgG1, IgG2, IgG3, IgG4, IgD, or IgM, in the order from the N-terminus to the C-terminus, and heavy chain invariant region CH3 derived from IgG1, IgG2, IgG3, IgG4. For example, the CLb may include, in the first arm, CH3 derived from IgG1, IgG2, IgG3, IgG4, and CL1 containing a light chain invariant region λ or κ derived from IgG1, IgG2, IgG3, IgG4, IgD, or IgM, in the order from the N-terminus to the C-terminus.

[0054] CH1 is the IgG heavy chain invariant region CH1, and CL is the IgG light chain invariant region CL. Each of CH1 and CL may be derived from IgG1, IgG2, IgG3, IgG4, or IgD. CH1 and CL may have a structure like a Fab fragment through non-covalent mutual bonding.

[0055] The term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain that can be generated by the complete papain degradation of an antibody. The Fc region can be a specific sequence Fc region or a mutant Fc region. The Fc region of an immunoglobulin generally contains two invariant domains, the CH2 domain and the CH3 domain, and optionally a CH4 domain.

[0056] In one embodiment, CHa and CLb may each contain CH3. Specifically, CHa and CLb may each contain CH3 derived from IgG1, IgG2, IgG3, or IgG4. The CHa and CLb may form a dimer through covalent or non-covalent mutual bonding.

[0057] In some cases, the CH3 domain and the CH1 domain may be linked by a linker. The linker may be a peptide linker and may contain about 5–25 aa residues, or more specifically, about 5–10 aa residues. For example, it may contain, but is not limited to, hydrophilic amino acids such as glycine and / or serine.

[0058] Specifically, the linker may be, for example, a glycine linker (G, Gly)p (p is 1-10), a GS linker (G n S) m (n and m may each be 1 to 10). Specifically, the linker may contain GGGGS or (GGGGS)2, or 5 to 10aa of glycine in (G, Gly)p where p is 5 to 10.

[0059] The first and second arms may be connected via a hinge. The first and second arms may be connected via a hinge formed by including one or more arrays selected from the group shown below: DKTHTCPPCP, EPKSSDKTHTCPPCP, and ESKYGPPCPPCP.

[0060] The Fc1 of the first arm and the Fc2 of the second arm each contain CH2 and CH3 monomers of the heavy chain invariant region. The monomers represent one domain of a dimer formed through two invariant domains CH2-CH3 having identical amino acid sequences in the heavy chain invariant region Fc. The Fc1 of the first arm and the Fc2 of the second arm combine to form a heavy chain invariant region dimer.

[0061] The aforementioned dimer may include a homodimer formed by the binding of immutable CH3 domains having the same amino acid sequence, or a heterodimer formed by the binding of immutable CH3 domains with different amino acid sequences.

[0062] "Amino acid substitution" or "substitution" can mean the replacement of an amino acid at a specific position within the amino acid sequence of the CH3 domain. In particular, in some embodiments, the substitution involves an amino acid that does not occur naturally at the specific position or an amino acid sequence different from the naturally occurring amino acid sequence.

[0063] The CH3 domain is derived from an immunoglobulin (Ig) domain, which is a region of an immunoglobulin having a tertiary structure, and may be derived from an immunoglobulin selected from the group consisting of IgG, IgM, IgA, IgD, and IgE, but specifically from IgG. The IgG may be human IgG.

[0064] In relation to human IgG, the "CH" domains are as follows: "CH1" corresponds to positions 118-220 according to the EU index, "CH2" corresponds to positions 237-340 according to the EU index, and "CH3" corresponds to positions 341-447 according to the EU index.

[0065] According to the EU index, the sequence of the first domain among the CH3 domains at positions 341-447 is as follows: GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(Sequence ID 1).

[0066] According to the EU index, the sequence of the second domain among the CH3 domains at positions 341-447 is as follows: GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(Sequence ID 2).

[0067] The following explanation is based on the CH3 domain at IgG positions 341-447, according to the EU index.

[0068] Specifically, the amino acids at positions Q347, E356, or E357 of the first heavy chain CH3 domain may be substituted with K or R, or the amino acid at position D399 may be substituted with W, F, Y, or H.

[0069] In one embodiment, the first heavy chain CH3 domain may include one or more selected from the group consisting of Q347K, Q347R, E357K, E357R, E356K, E356R, D399W, D399F, D399Y, and D399H. Specifically, the first heavy chain CH3 domain may include one or more selected from the group consisting of Q347K, E357K, E356K, D399W, D399F, D399Y, or D399H.

[0070] Furthermore, the amino acids at positions K360, K370, or K439 of the second heavy chain CH3 domain may be substituted with Q, N, E, or D, or the amino acid at position K409 may be substituted with A, V, T, or S.

[0071] In one embodiment, the second heavy-chain CH3 domain may include one or more selected from the group consisting of K360Q, K360N, K360E, K360D, K370E, K370D, K370N, K370Q, K439E, K439D, K439N, K439Q, K409A, K409V, K409T, and K409S. Specifically, the second heavy-chain CH3 domain may include one or more selected from the group consisting of K360Q, K360N, K360E, K360D, K370E, K439E, K439D, K409A, K409T, and K409S.

[0072] In one embodiment, this may include one or more, two or more, three or more, or all four of the substitutions shown below: The first heavy chain CH3 domain contains Q347K or Q347R, and the second heavy chain CH3 domain contains K360Q, K360N, K360E or K360D, The first heavy-chain CH3 domain includes E357K or E357R, and the second heavy-chain CH3 domain includes K370Q, K370N, K370E, or K370D, The first heavy chain CH3 domain includes E356K or E356R, and the second heavy chain CH3 domain includes K439Q, K439N, K439E, or K439D, The first heavy-chain CH3 domain includes D399W, D399F, D399Y, or D399H, and the second heavy-chain CH3 domain includes K409A, K409T, K409V, or K409S.

[0073] In one embodiment, this may include one or more, two or more, three or more, or all four of the substitutions shown below: The first heavy-chain CH3 domain contains Q347K, and the second heavy-chain CH3 domain contains K360Q, K360N, K360E, or K360D. The first heavy chain CH3 domain contains E357K, and the second heavy chain CH3 domain contains K370E, The first heavy-chain CH3 domain includes E356K, and the second heavy-chain CH3 domain includes K439E or K439Q, The first heavy-chain CH3 domain includes D399W, D399F, D399Y, or D399H, and the second heavy-chain CH3 domain includes K409A, K409T, or K409S.

[0074] In a specific embodiment of the present invention, the first heavy chain CH3 domain may include one or more selected from the group consisting of Q347K, E357K, E356K, D399W, D399F, D399Y, and D399H, and the second heavy chain CH3 domain may include one or more selected from the group consisting of K360Q, K360N, K360E, K360D, K370E, K439E, K439D, K409A, K409T, and K409S.

[0075] A specific embodiment of the present invention may include one or more combinations of substitutions selected from the following group. The first heavy chain CH3 domain includes Q347K, the second heavy chain CH3 domain includes K360Q, the first heavy chain CH3 domain includes E357K, and the second heavy chain CH3 domain includes K370E. The first heavy chain CH3 domain includes Q347K, the second heavy chain CH3 domain includes K360Q, and the first heavy chain CH3 domain includes E356K and the second heavy chain CH3 domain includes K439E. The first heavy chain CH3 domain includes E357K, the second heavy chain CH3 domain includes K370E, and the first heavy chain CH3 domain includes E356K and the second heavy chain CH3 domain includes K439E. The first heavy chain CH3 domain includes Q347K, the second heavy chain CH3 domain includes K360Q, and the first heavy chain CH3 domain includes D399W and the second heavy chain CH3 domain includes K409A. The first heavy chain CH3 domain includes E357K, the second heavy chain CH3 domain includes K370E, and the first heavy chain CH3 domain includes D399W and the second heavy chain CH3 domain includes K409A. It includes E356K from the first heavy chain CH3 domain, K439E from the second heavy chain CH3 domain, and D399W from the first heavy chain CH3 domain and K409A from the second heavy chain CH3 domain, The first heavy chain CH3 domain includes Q347K, the second heavy chain CH3 domain includes K360Q, the first heavy chain CH3 domain includes E357K, the second heavy chain CH3 domain includes K370E, and the first heavy chain CH3 domain includes E356K and the second heavy chain CH3 domain includes K439E, The first heavy chain CH3 domain includes E357K, the second heavy chain CH3 domain includes K370E, the first heavy chain CH3 domain includes D399W, the second heavy chain CH3 domain includes K409A, and the first heavy chain CH3 domain includes E356K and the second heavy chain CH3 domain includes K439E. The first heavy chain CH3 domain includes Q347K, the second heavy chain CH3 domain includes K360Q, the first heavy chain CH3 domain includes D399W, the second heavy chain CH3 domain includes K409A, the first heavy chain CH3 domain includes E356K, and the second heavy chain CH3 domain includes K439E. The first heavy chain CH3 domain includes Q347K, the second heavy chain CH3 domain includes K360Q, the first heavy chain CH3 domain includes E357K, the second heavy chain CH3 domain includes K370E, the first heavy chain CH3 domain includes D399W, the second heavy chain CH3 domain includes K409A, the first heavy chain CH3 domain includes E356K, and the second heavy chain CH3 domain includes K439E. Depending on the circumstances, it may further include one or more selected from the group consisting of Y349C, S354C, T366S, T366W, L368A and Y407V among the first heavy chain CH3 domains or the second heavy chain CH3 domain.

[0076] In one embodiment, one of the first heavy chain CH3 domain or the second heavy chain CH3 domain further comprises one or more selected from the group consisting of Y349C, S354C, 366S, T366W, L368A and Y407V, and the other of the first heavy chain CH3 domain or the second heavy chain CH3 domain may further comprise one or more selected from the group consisting of S354C, Y349C, T366W, T366S, L368A and Y407V.

[0077] Specifically, this may include residue mutations according to the present invention. JPEG2026517992000002.jpg170115JPEG2026517992000003.jpg181115JPEG2026517992000004.jpg182115JPEG202 6517992000005.jpg181115JPEG2026517992000006.jpg180115JPEG2026517992000007.jpg180115JPEG2026517992 000008.jpg181115JPEG2026517992000009.jpg180115JPEG2026517992000010.jpg181115JPEG2026517992000011. jpg180115JPEG2026517992000012.jpg181115JPEG2026517992000013.jpg180115JPEG2026517992000014.jpg95115

[0078] In other aspects, the present invention relates to a fusion protein comprising the heterodimer.

[0079] The heterodimer may bind to one end of a polypeptide or protein, such as peptides, cytokines like IL-2, IL-10, IL-12, GCSF, and GM-CSF, chemokines like RANTES, CXCL9, CXCL10, and CXCL12, hormones, immune barrier proteins like CTLA-4, TNFR1, TNFRII, TNFSF, and TNFRSF, or blood factors.

[0080] The heterodimer may be bound to the N-terminus or C-terminus of a polypeptide or protein. Optionally, the heterodimer may be linked to the polypeptide or protein by a linker. The linker may be a peptide linker and may contain about 5–25 aa residues, or more specifically, about 5–10 aa residues. For example, it may contain, but is not limited to, hydrophilic amino acids such as glycine and / or serine.

[0081] In the bispecific antibody format according to the present invention, the heavy and light chains of the first arm that bind to the first antigen are configured as follows: The heavy chain of the first arm is configured in the order of VH-CH3a-Linker-CH1a-Hinge-CH2-CH3b. The CH1a domain may be the IgG1CH1 domain. The CH3a domain may contain a hole mutation and a mutation for the formation of a disulfide bond with the CH3c of the light chain. The CH3b domain may contain a mutation for the formation of a disulfide bond. An elbow sequence AS may optionally be added between the VH region and the CH3a region.

[0082] Considering mutations with biologically equivalent activity, the substitutions according to the present invention are interpreted to include substitutions to sequences that exhibit substantial identity with those of the described sequences.

[0083] Substantial identity means sequences that, when aligned to maximize correspondence between the sequence of the present invention and any other sequence, and analyzed using algorithms commonly used in the industry, exhibit at least 61% homology, more preferably 70% homology, more preferably 80% homology, and most preferably 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher homology. Alignment methods for sequence comparison are publicly known in the industry. The NCBI Basic Local Alignment Search Tool (BLAST) is accessible through NBCI and other sources and can be used in conjunction with sequence analysis programs such as blastp, blasm, blastx, tblastn, and tblastx on the internet. BLAST is accessible at www.ncbi.nlm.nih.gov / blast / . Methods for comparing sequence homology using this program can be found at www.ncbi.nlm.nih.gov / blast / blast_help.html. [Examples]

[0084] The present invention will be described in more detail below with reference to examples. These examples are provided solely to illustrate the present invention more concretely, and it will be obvious to those ordinary skill in the art that the scope of the present invention is not limited by these examples. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

[0085] Example 1. Design and Engineering of S-DUAL 2G antibody Introduction of CH3 domain amino acid residue variants The bispecific antibody format that simultaneously binds to the first and second antigens is composed of the following: The bispecific antibody format is formed by assembling a total of four polypeptides, namely two heavy chains (H) and two light chains (L). The heavy and light chains of the first arm that binds to the first antigen are composed of the following: The heavy chain has the structure VH-CH3D-CH1a-Hinge-CH2-CH3B. Here, CH3B and CH3D contain sequences of the IgG1 CH3 domain, and the CH1a region may contain sequences of the IgG1 CH1 domain. Of the domains that make up the heavy chain, the IgG1 CH3B and CH3D domains contain mutations of either a knob (T366W) or a hole (T366S / L368A / Y407V). The IgG1 CH3B and CH3D mains may include mutations that generate cysteine ​​to form a disulfide bond between the domains, i.e., knobs (S354C / T366W), knobs (Y349C / T366W), or holes (S354C / T366S / L368A / Y407V), or holes (Y349C / T366S / L368A / Y407V). The IgG1 CH3 mutant candidate group was derived by screening amino acid residues that primarily act on the CH3A-CH3B domain binding constituting the heavy chain invariant site in Bioluminate® (Schrodinger, LLC)'s "protein interaction analysis" panel, and then applying substituted mutant amino acids that do not conflict with the side chain structure of adjacent amino acid residues. Based on the aforementioned candidate group, four amino acid residue mutation pairs were selected through structural modeling that exhibited high interaction energy values ​​between heterologous diluents (CH3A-CH3B) but low interaction energy values ​​between homologous diluents (CH3A-CH3A, CH3B-CH3B). Subsequently, these amino acid residue mutations were introduced into bispecific antibodies by combining them single, double, or triple in the CH3 domain pairs of the heavy chain invariant region.

[0086] The light chain of the first arm that binds to the first antigen consists of VL-CH3C-CLb. Here, CH3C represents the IgG1 CH3 domain, but may include a knob (T366W) or a hole (T366S / L368A / Y407V) mutation. The IgG1 CH3 domain may also include mutations that produce cysteine ​​for disulfide bonding, i.e., a knob (S354C / T366W), a knob (Y349C / T366W), a hole (S354C / T366S / L368A / Y407V), or a hole (Y349C / T366S / L368A / Y407V). The heavy and light chains of the second arm that binds to the second antigen are as follows: The heavy chain consists of VH-CH1-Hinge-CH2-CH3A. The IgG1 CH3A domain contains mutations for a knob (T366W) or a hole (T366S / L368A / Y407V). The IgG1 CH3A domain may also contain mutations to generate cysteine ​​for disulfide bonding, i.e., a knob (S354C / T366W), a knob (Y349C / T366W), a hole (S354C / T366S / L368A / Y407V), or a hole (Y349C / T366S / L368A / Y407V). The light chain of the second arm is composed of VL-CLb. Based on the above bispecific antibody format, we attempted to introduce additional IgG1 CH3 mutations to improve the yield of heterologous diluent formation. The IgG1CH3 mutant candidate group consisted of a single combination of four amino acid residue mutations, introduced into the CH3B domain in the VH-CH3D-CH1a-Hinge-CH2-CH3B heavy chain of the first arm that binds to the first antigen, and into the CH3C domain in the CH3D domain and VL-CH3C-CLb light chain.

[0087] [Table 1] CH3 mutant candidate group confirmed through in-silico simulation

[0088] 1.2 Design by anti-VEGF x anti-HER2 bisspecific antibody The first antigen was determined to be the HER2 protein, and the amino acid sequences of the heavy chain variable region and the light chain variable region of trastuzumab that bind to HER2 were used in the VH or VL bispecific antibody format. The second antigen was determined to be the VEGF-A target, and the amino acid sequences of the heavy chain variable region and the light chain variable region of bevacizumab were used in the VH or VL bispecific antibody format. The amino acid sequence information was confirmed via https: / / go.frugbank.com / .

[0089] [Table 2] JPEG2026517992000017.jpg228153Fc, Information on a bispecific antibody in which amino acid residue mutations were applied to the FabCH3 domain pair.

[0090] The specific composition of each candidate is shown in Table 3. [Table 3] JPEG2026517992000019.jpg245153JPEG2026517992000020.jpg246153JPEG2026517992000021.jpg248153JPEG2026517992000022.jpg247153JPEG2026517992000023.jpg246153JPEG2026517992000024.jpg247153JPEG2026517992000025.jpg245153JPEG2026517992000026.jpg246153JPEG2026517992000027.jpg248153JPEG2026517992000028.jpg245153JPEG2026517992000029.jpg248153JPEG2026517992000030.jpg245153JPEG2026517992000031.jpg247153JPEG2026517992000032.jpg247153JPEG2026517992000033.jpg245153JPEG2026517992000034.jpg246153JPEG2026517992000035.jpg247153JPEG2026517992000036.jpg246153JPEG2026517992000037.jpg246153JPEG2026517992000038.jpg248153JPEG2026517992000039.jpg245153JPEG2026517992000040.jpg244153JPEG2026517992000041.jpg247153JPEG2026517992000042.jpg247153JPEG2026517992000043.jpg249153JPEG2026517992000044.jpg248153JPEG2026517992000045.jpg247153JPEG2026517992000046.jpg248153JPEG2026517992000047.jpg247153JPEG2026517992000048.jpg245153JPEG2026517992000049.jpg247153JPEG2026517992000050.jpg247153JPEG2026517992000051.jpg246153JPEG2026517992000052.jpg246153JPEG2026517992000053.jpg204153 Heavy and light chain sequences of each candidate.

[0091] Example 2. Production of bispecific antibodies containing CH3 domain variants Vector plasmids containing the coding genes for the heavy and light chain regions (Q-SBL) of the first arm and vector plasmids containing the coding genes for the heavy and light chain regions of the second arm were constructed. To express the second arm, the heavy and light chain coding genes were inserted into a single vector plasmid. The promoter used was CMV, and a WPRE (woodchuck hepatitis virus post-transcriptional regulatory element) was inserted after the coding gene to increase the expression level during transient expression.

[0092] ExpiCHO-S animal cells (Thermo Fisher, A29127) were co-transfected with vector plasmids containing the coding genes for the first arm and the second arm. The ratio of the vector plasmids used for expressing the first arm to the vector plasmids used for expressing the second arm was 1:1 (Q-SBL). For phenotypic infection, the ExpiFectamine CHO Transfection Kit (Thermo Fisher, A29130) was used according to the manufacturer's manual. Thirteen days after phenotypic infection, the supernatant was obtained by centrifugation (10000 rpm, 15 mins) to obtain bispecific antibodies, and residual cell debris was removed from the supernatant using 0.22 μm filter paper.

[0093] Example 3. Expression of a bispecific antibody containing a CH3 domain variant. The expression yield of bispecific antibodies containing CH domain variants was measured using an Octet instrument (Table 4). A calibration curve was determined using a Protein A biosensor (Sartorius 18-5010) and an IgG1 type standard, and the concentration of bispecific antibodies in the CHO cell culture supernatant (HCCF) was determined through this calibration curve.

[0094] [Table 4] Antibody concentration in culture supernatant quantified by octet analysis

[0095] Example 4. Purification of bispecific antibodies containing CH3 domain mutants For research and diverse analyses of bispecific antibodies to which the candidate CH3 domain mutations of the present invention are applied, the culture medium obtained by expressing the candidates was purified using a MabSelect Sure (Cytiva) column, a Protein A affinity chromatography technique, to produce primary bispecific antibodies. The bispecific antibody material obtained in the primary purification step was identified by non-reducing SDS-PAGE using a 3-8% Tris-acetate gel and Tris-acetate buffer. Furthermore, to confirm the presence of other fragments unrelated to the bispecific antibody, reduced SDS-PAGE was performed using a 4-12% Bis-Tris gel and Bis-Tris buffer.

[0096] Specifically, a MabSelect Sure column was equilibrated with a 50 mM Tris-HCl (pH 7.0) buffer solution (equilibrium buffer solution), and then a bispecific antibody culture medium containing mutants, filtered through 0.22 μm filter paper, was loaded onto the column. Proteins that did not bind to the column were washed with the aforementioned equilibrium buffer solution, and impurities nonspecifically bound to the column were removed sequentially using an equilibrium buffer solution containing 0.5 M Sodium Chloride and a 20 mM Bis-Tris (pH 5.8) buffer solution. Subsequently, the bispecific antibodies that specifically bound to the column were eluted by running a 0.2 M Glycine-HCl (pH 3.2) buffer solution over them. The eluted samples were neutralized to pH 5.0 using a 1.0 M Tris solution and then filtered through 0.22 μm filter paper.

[0097] The next purification step was performed using a Capto SP (Cytiva) column, a type of cation exchange chromatography, and the details are as follows: After stabilizing the Capto SP column with a 50 mM Bis-Tris (pH 6.0) buffer solution, a bispecific antibody sample titrated at pH 6.0 was applied, and impurities that did not bind to the column were washed with the same buffer solution. The bispecific antibody bound to the column was eluted using sodium chloride between 0.1 M and 1.0 M.

[0098] The final purification step involved hydrophobic interaction chromatography to remove high molecular weight (HMW) and low molecular weight (LMW) impurities from the bispecific antibody sample that had undergone secondary purification. In this step, a butyl-based sepharose column was used, and the sample was prepared by replacing the purified bispecific antibody, which had been purified by ion exchange chromatography, with a high-concentration salt buffer solution to achieve a salt concentration of 1.0 M to 1.5 M. After equilibrating the column with a 50 mM Bis-Tris (pH 6.0) buffer solution having the same salt concentration as the sample, the prepared sample was loaded. The bound bispecific antibody was eluted using a gradient elution method over 30 CV with 50 mM Bis-Tris (pH 6.0) without salt. The final purified bispecific antibody was concentrated to a concentration of 1-2 mg / mL using a 50 kDa molecular-weight cut-off ultrafiltration tube, and then replaced with a buffer solution suitable for the analytical conditions.

[0099] Example 5. Purity analysis by sice exclusion high-pressure chromatography (SEC-HPLC). To confirm the purity of the agglutination and heterodimer of the bispecific antibody candidate, sieve exclusion high-pressure chromatography was performed. Approximately 50 μg of the purified product that passed through protein A resin was passed through a TSKGel G3000WXL (Tosoh) column at a flow rate of 0.5 mL / min using a Water Alliance 2695 HPLC and 2489 UV / Vis detector system with a mobile phase of 250 mM Potassium phosphate, 200 mM Potassium Chloride, pH 7.2, and UV absorbance was measured at 280 nm. System compatibility was confirmed using Bio-Rad's Gel filtration standard (151-1901), and after analysis, the data was integrated using the manufacturer's Empower 3.0 software to determine purity as a percentage area ratio. The SEC-HPLC analysis results are shown in Table 5.

[0100] [Table 5] Purity analysis of bispecific antibody candidate products purified using rPA with SEC-HPLC.

[0101] Example 6. CE-SDS analysis For purity analysis of bispecific antibody candidates under non-reducible or reducing conditions, PA800Plus (Sciex) and the IgG Purity and Heterogeneity Assay Kit (Sciex) were used for sodium dodecyl sulfate capillary electrophoresis (CE-SDS). 50 μL of protein at a maximum concentration of 2 mg / mL was used for analysis of purified samples using Protein A resin. For analysis, each sample was mixed with 5 μL of 250 mM iodoacetamide or 14.2 M 2-mercaptoethanol, 2 μL of an internal standard, and 45 μL of sodium dodecyl sulfate sample buffer solution, and then heated at 70°C for 10 minutes (CE-SDS method 1). Alternatively, 20 μL of protein at a maximum concentration of 3.5 mg / mL was used for analysis of purified samples using Protein A resin. For analysis, each sample was mixed with 5 μL of 250 mM iodoacetamide or 14.2 M 2-mercaptoethanol, 2 μL of an internal standard, and 75 μL of sodium dodecyl sulfate sample buffer solution, and then heated at 70°C for 10 minutes (CE-SDS method 2).

[0102] After the analysis is complete, the data will be provided by the manufacturer in 32Karat. TM Analyzed with software version 10.3.

[0103] Table 6 shows the results of analysis on CE-SDS under non-reducing conditions after purification using Protein A resin. After purifying the bispecific antibody candidate group using Protein A resin, the purity of heterologous dimers was confirmed by sodium dodecyl sulfate capillary electrophoresis (CE-SDS).

[0104] [Table 6] Purity of bispecific antibodies under non-reducing conditions

[0105] Example 7. Single Antigen Binding ELISA Since the biantibodies constituting this invention have multiple target types, ELISA experiments for both single-antigen binding and biantigen binding were performed. The specific implementation process for single-antigen binding ELISA is as follows: Depending on the target to be confirmed, rhErbB2-his tag and rhVEGF165 recombinant protein (R&D Systems) were coated onto a 96-well immunoassay ELISA plate using 1x PBS pH 7.4. At this time, the coating concentrations were prepared at 1 μg / mL for rhErbB2-his tag and 0.5 μg / mL for rhVEGF, with 100 μl processed per well. After coating with rhErbB2-his tag and rhVEGF 165 at 37°C for 1 hour, the plates were washed five times with 0.05% PBS-T. To prevent nonspecific binding, 200 μl per well was treated with 2% BSA to perform a blocking process, incubated at 37°C for 30 minutes, and then washed five times with 0.05% PBS-T. The bispecific antibodies specified in this invention were sequentially diluted with 2% BSA (20-0.0001 nM), and then 100 μl of each antibody was added to each well of a plate coated with recombinant protein. After incubation at 37°C for 1 hour, the plates were washed five times with 0.05% PBS-T. Next, the HRP-conjugated secondary antibody was diluted using PBS containing 2% BSA (anti-hIgG FC-HRP 1:10,000), and 100 μl was added per well. The plates were incubated at 37°C for 30 minutes and washed five times with 0.05% PBS-T. 100 μl of TMB (Bio-Rad), which reacts with the HRP in the secondary antibody to develop color, was added per well. After development at room temperature for 5 minutes, 100 μl of 1 M H2SO4 was added per well to complete the color development, and the absorbance was measured at a wavelength of 450 nm using a SpectraMax ABS Plus (Molecular Devices) instrument. As a result, EC for single antigen binding in the CH3 engineering progression group, including ErbB2 and the VEGF binding region, respectively. 50Table 7 shows a comparison of the values. Figure 6 is a graph of the 4-parameter fitting analysis of the binding affinity of the candidate group ErbB2 and VEGF to single antigens.

[0106] [Table 7] EC for single antigen-binding ELISA of bispecific antibodies containing ErbB2 and VEGF binding regions 50 value

[0107] Example 8. Dual Antigen Binding ELISA The specific implementation process for the dual antigen-binding ELISA is as follows: Depending on the target to be confirmed, recombinant rhVEGF 165 protein (R&D Systems) was coated onto a 96-well immunosorbent ELISA plate using 1x PBS pH 7.4. The coating concentration was 100 μl per 1 μg / mL well. The rhVEGF 165 was coated at 4°C for 18 hours, and then washed five times with 0.05% PBS-T. To prevent nonspecific binding, the plates were blocked with 200 μl per well of 2% BSA, incubated at 37°C for 2 hours, and then washed five times with 0.05% PBS-T. A bispecific antibody, continuously diluted in a ratio of 1 / 3 (100~0.0016 nM) using 2% BSA based on a maximum concentration of 100 nM, was mixed in a 1:1 ratio with rhErbB2-his (R&D Systems) diluted to 1 μg / mL, and incubated at 37°C for 2 hours. After washing the microplate five times with 0.05% PBS-T, 100 μl each of the previously mixed antibody and rhErbB2-his sample was added to each well and incubated at 37°C for 2 hours, followed by five washes with 0.05% PBS-T. Subsequently, the HRP-conjugated anti-secondary antibody was diluted using PBS containing 2% BSA (anti-his tag-HRP 1:10,000), 100 μl was added per well, incubated at 37°C for 1 hour, and washed five times with 0.05% PBS-T. 100 μl of TMB (Bio-Rad) was added to each well, and after developing color at room temperature for 5 minutes, 100 μl of 1 M H2SO4 was added to each well to terminate the color development. Absorbance was measured at a wavelength of 450 nm using a SpectraMax ABS Plus (Molecular Devices) instrument. As a result, the EC reaction against the double antigen binding of the CH3 engineering progression group containing ErbB2 and the VEGF binding region was determined. 50 Table 8 shows a comparison of the values. Figure 7 is a graph of the 4-parameter fitting performed on the ErbB2 and VEGF biantigen binding affinity analysis of the candidate group.

[0108] [Table 8] EC for bispecific antibody containing ErbB2 and VEGF binding region in biantigen-binding ELISA 50 value

[0109] Example 9. Analysis of fluorescently activated cells (Fluorecscence-Activated Cell Sorter, FACS) To confirm whether antigen-binding ability at the cellular level is maintained when the CH3 domain mutant according to the present invention is introduced into a bispecific antibody, fluorescence-activated cell analysis was performed. The specific implementation process of fluorescence-activated cell analysis is as follows: JIMT-1 cells expressing HER2, the target to be examined, were subcultured in DMEM medium containing 1% penicillin-Streptomycin (Gibco, 15140-122) and 10% fetal bovine serum (Gibco, 16140071) at 37°C in a 5% CO2 incubator, and the cell density was kept below 80% using 75-T Flasks. JIMT-1 cells were placed in 96-well plates in a 2x10⁶ format. 5After preparing cells at the specified cell / well density, the bispecific antibody specified in this invention was prepared to 100 nM using FACS staining buffer (Invitrogen, 00-4222-26), and 100 μL was added per well. The cells were then reacted at 4°C for 1 hour. The supernatant was removed by centrifugation (1,000 rpm, 5 minutes), and the cells reacted with the antibody were washed with 100 μL of FACS buffer three times to prevent nonspecific binding. The FITC-conjugated secondary antibody was diluted using FACS buffer (Abcam, ab239228, 1:40), and 100 μL was added per well. The cells were then reacted at 4°C for 30 minutes, and the same washing process was repeated three times. Cells reacted with the antibody were analyzed using a fluorescent cell classifier (Satorius, iQue®3) to detect the fluorescence emission signals of exite cells in the 498 nm region and emite cells in the 475-650 nm region through a FITC filter. The results are shown in the figure, and the quantified results using Mean Fluorescence Intensity (MFI) are shown in Table 9 and Figure 8.

[0110] [Table 9] The table shows the quantitative comparison of the binding strength between ErbB2 expressed on the surface of JIMT-1 cells and biantibodies, measured by MFI values.

[0111] Example 10. Analysis of the coupling dynamics of FcγRIIa, FcγRIIIa, and C1q. The Fc region (hinge-CH2) of IgG1 interacts with the Fcγ receptor (FcγR) and complement protein (C1q) to induce immune effector function. To confirm whether the binding ability to FcγRIIa, FcγRIIIa, and C1q is maintained when the CH3 domain variant according to the present invention is introduced into the IgG1 skeleton, binding dynamics analysis was performed using the Octet® HTX system. The specific implementation process of the binding dynamics analysis is as follows.

[0112] FcγRIIa: After aligning the baseline with the analysis buffer (1x PBS containing 0.25% Sodium azide + 0.5% BSA + 0.1% Tween-20) for 60 seconds on the FAB2G Biosensor, 100 nM of the bispecific antibody was immobilized for 300 seconds. After aligning the baseline again for 60 seconds, serially diluted FcγRIIa (4000, 2000, 1000, 500, 250, 125, 62.5, 0 nM) was allowed to bind and dissociate for 60 seconds each. K D values were calculated using Data Analysis 12.0 software.

[0113]

Table 10

[0114] FcγRIIIa: After aligning the baseline with 1x kinetics buffer for 60 seconds on the FAB2G Biosensor, 100 nM of the bispecific antibody was immobilized for 300 seconds. After aligning the baseline again for 60 seconds, serially diluted FcγRIIIa (2000, 1000, 500, 250, 125, 62.5, 31.25, 0 nM) was allowed to bind and dissociate for 60 seconds each.

[0115]

Table 11

[0116] C1q: The baseline was aligned to the FAB2G Biosensor with analytical buffer (1x PBS containing 200mM NaCl + 0.3% BSA + 0.05% Tween-20) for 60 seconds. A 100nM bispecific antibody was then fixed for 300 seconds, the baseline was aligned again for 60 seconds, and then sequentially diluted C1q (200, 100, 50, 25, 12.5, 6.25, 3.125, 0nM) were bound for 30 seconds and dissociated for 60 seconds.

[0117] [Table 12] C1q-bound K of bispecific antibodies D value

[0118] Example 11. Analysis of FcRn coupling dynamics The Fc region (CH2-CH3) of IgG1 interacts with FcRn and plays a crucial role in improving the antibody's half-life by mediating antibody recirculation. To confirm whether the binding ability to FcRn is maintained when the CH3 domain variant according to the present invention is introduced into a bispecific antibody, binding dynamics analysis was performed using the Octet® HTX system. The specific implementation process of the binding dynamics analysis is as follows: After aligning the baseline to the FAB2G Biosensor with 1x kinetics buffer for 60 seconds, the bispecific antibody diluted to 100 nM was fixed for 300 seconds. After aligning the baseline with pH 6.0 analytical buffer for 180 seconds, sequentially diluted FcRn (1600, 800, 400, 200, 100, 50, 25, 0 nM) were bound for 30 seconds and dissociated for 60 seconds. Data Analysis 12.0 software was used. D The value was calculated.

[0119] [Table 13] FcRn-conjugated K of bispecific antibodies D value [Industrial applicability]

[0120] According to the present invention, it is possible to provide a heterodimer containing a CH3 domain in which the heterodimer formation yield is enhanced.

[0121] Furthermore, according to the present invention, by introducing an asymmetric amino acid mutation into the CH3 domain of the heavy chain invariant region, the formation of allogeneic diluents that occur during the production of bispecific antibodies can be minimized, and the yield of heterogeneous diluent formation can be improved.

[0122] Furthermore, bispecific antibodies produced using the CH3 domain variant of the heavy chain invariant region according to the present invention maintain the target antigen binding ability and the intrinsic function of the heavy chain invariant region (Fc: Hinge-CH2-CH3) of conventional wild-type antibodies, namely the binding ability to the Fcγ receptor (FcγR, Fcγ Receptor), complement protein (C1q, Complement component 1q), and FcRn (Neonental Fc receptor), thereby maintaining antibody-dependent immunoeffector function and blood half-life.

[0123] Furthermore, the present invention provides a bispecific antibody format that improves upon the nonspecific binding of heavy and light chains and homodimer formation in conventional bispecific antibody formats that simultaneously bind a first and second antigen. By introducing asymmetric amino acid mutations into the CH3 domain of the heavy chain invariant region, and further introducing other asymmetric amino acid mutations into the CH3 domain of the antibody variable region applied to the company's bispecific antibody format, it is possible to minimize the formation of allogeneic diluents that occur during bispecific antibody production and improve the yield of heterogeneous diluent formation.

[0124] Although specific aspects of the present invention have been described in detail above, it will be obvious to those with ordinary skill in the art that this particular technology is merely a preferred embodiment and does not limit the scope of the invention. Therefore, the substantial scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A heterodimer comprising a first heavy chain CH3 domain and a second heavy chain CH3 domain, The first heavy chain CH3 domain or the second heavy chain CH3 domain contains one or more amino acid substitutions (according to the EU index) at a position selected from the group shown below: (1) The amino acid at position Q347, E356, or E357 is substituted with K or R. (2) The amino acid at position D399 is substituted with W, F, Y, or H. (3) The amino acid at position K360, K370 or K439 is substituted with Q, N, E or D, and (4) The amino acid at position K409 is substituted with A, V, T or S. A heterodimer characterized in that the first heavy chain CH3 domain and the second heavy chain CH3 domain bind to form a dimer.

2. The heterodimer according to claim 1, characterized in that the amino acid at position Q347, E356, or E357 of the first heavy chain CH3 domain is substituted with K or R, or the amino acid at position D399 is substituted with W, F, Y, or H.

3. The heterodimer according to claim 1, characterized in that the amino acid at position K360, K370, or K439 of the second heavy chain CH3 domain is substituted with Q, N, E, or D, or the amino acid at position K409 is substituted with A, V, T, or S.

4. The heterodimer according to claim 1, characterized in that the first heavy chain CH3 domain contains Q347K or Q347R, and the second heavy chain CH3 domain contains K360Q, K360N, K360E or K360D.

5. The heterodimer according to claim 1, characterized in that the first heavy chain CH3 domain includes E357K or E357R, and the second heavy chain CH3 domain includes K370Q, K370N, K370E, or K370D.

6. The heterodimer according to claim 1, characterized in that the first heavy chain CH3 domain includes E356K or E356R, and the second heavy chain CH3 domain includes K439Q, K439N, K439E, or K439D.

7. The heterodimer according to claim 1, characterized in that the first heavy chain CH3 domain includes D399W, D399F, D399Y, or D399H, and the second heavy chain CH3 domain includes K409A, K409T, K409V, or K409S.

8. The heterodimer according to claim 1, further comprising one or more selected from the group consisting of Y349C, S354C, T366S, T366W, L368A and Y407V among the first heavy chain CH3 domain or the second heavy chain CH3 domain.

9. A fusion protein characterized by comprising a heterodimer according to any one of claims 1 to 8.

10. VH1-CHa-Fc １ and a first arm that binds to the first antigen including VL1-CLb, and VH2-CH1-Fc ２ A bispecific antibody comprising a second arm that binds to a second antigen including VL2-CL, The VH1 and VH2 are heavy chain variable regions that each contain the same or different antigen-binding regions. The VL1 and VL2 are light chain variable regions each containing the same or different antigen-binding regions. The CHa comprises an IgG heavy chain invariant region or an IgG heavy chain invariant region CH1, and an IgG heavy chain invariant region CH2 or CH3. The CLb comprises one or more selected from the group consisting of CL1 containing an IgG light chain invariant region λ or κ and IgG heavy chain invariant regions CH1, CH2, CH3. CH1 is the IgG heavy chain invariant region CH1, and CL is the IgG light chain invariant region CL. Fc of the first arm １ and the Fc of the second arm ２ They bind to form a heavy chain invariant region dimer, CH3 of CHa, Fc of the first arm １ or the Fc of the second arm ２ A bispecific antibody comprising a heterodimer containing a primary heavy-chain CH3 domain and a secondary heavy-chain CH3 domain, each containing one or more amino acid substitutions (according to the EU index) at a position selected from the group shown below: (1) The amino acid at position Q347, E356, or E357 is substituted with K or R. (2) The amino acid at position D399 is substituted with W, F, Y, or H. (3) The amino acid at position K360, K370 or K439 is substituted with Q, N, E or D, and (4) The amino acid at position K409 is substituted with A, V, T, or S.

11. The bispecific antibody according to claim 10, characterized in that the amino acid at position Q347, E356, or E357 of the first heavy chain CH3 domain is substituted with K or R, or the amino acid at position D399 is substituted with W, F, Y, or H.

12. The bispecific antibody according to claim 10, characterized in that the amino acid at position K360, K370, or K439 of the second heavy chain CH3 domain is substituted with Q, N, E, or D, or the amino acid at position K409 is substituted with A, V, T, or S.

13. The bispecific antibody according to claim 10, characterized in that it contains Q347K or Q347R among the first heavy chain CH3 domains and K360Q, K360N, K360E or K360D among the second heavy chain CH3 domains.

14. The bispecific antibody according to claim 10, characterized in that the first heavy chain CH3 domain contains E357K or E357R, and the second heavy chain CH3 main contains K370Q, K370N, K370E, or K370D.

15. The bispecific antibody according to claim 10, characterized in that it contains E356K or E356R among the first heavy chain CH3 domains and K439Q, K439N, K439E or K439D among the second heavy chain CH3 domains.

16. The bispecific antibody according to claim 10, characterized in that it contains D399W, D399F, D399Y, or D399H among the first heavy chain CH3 domains, and K409A, K409T, K409V, or K409S among the second heavy chain CH3 domains.

17. The bispecific antibody according to claim 10, further comprising one or more selected from the group consisting of Y349C, S354C, T366S, T366W, L368A and Y407V among the first heavy chain CH3 domain or the second heavy chain CH3 domain.

18. The bispecific antibody according to claim 10, characterized in that the CH3 and CH1 of CHa or the CH3 and CL1 of CLb are linked by a linker.

19. The bispecific antibody according to claim 18, characterized in that the linker contains 5 to 10 aa residues.

20. The bispecific antibody according to claim 18, characterized in that the first arm and the second arm are connected by a hinge.

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