CN113121697B - CH3 domain modification induced heterodimer and preparation method and application thereof - Google Patents

Abstract

The invention belongs to the field of antibody engineering, and provides a heterodimer, a preparation method and application thereof. The present invention combines the "pro-concave" model with electrostatic interactions by comprehensively considering various interactions between interfacial amino acids, such as ionic interactions, hydrophobic interactions, and steric interactions, to screen for preferred CH3 mutant sequences that are more prone to form heterodimers rather than homodimers, thus greatly increasing the yield of heterodimer molecules. Compared with references CN106883297A and US20150307628A1, the method for constructing the convex-concave model is simpler, has fewer point mutations and can improve the purity of the heterodimer to more than 95% at most under the condition of not introducing disulfide bonds.

Description

CH3 domain modification induced heterodimer and preparation method and application thereof

Technical Field

The invention belongs to the field of antibody engineering, and particularly relates to a hetero-dimer formed by modification and induction of a CH3 structural domain, and a preparation method and application thereof.

Background

Bispecific antibodies have a variety of modes of construction, in which IgG-type bispecific antibodies have similar structure, physicochemical properties, and Fc-segment functions as conventional antibodies. Typically, an IgG-type bispecific antibody consists of two heavy chains of different amino acid sequences (i.e., heavy chain hc_a of anti-antigen a and heavy chain hc_b of anti-antigen B) and two light chains of different amino acid sequences (i.e., light chain lc_a of anti-antigen a and light chain lc_b of anti-antigen B). When 4 polypeptide chains are combined, homodimers and heterodimers are formed between the two heavy chains, and mismatches are also formed between the light and heavy chains, thus yielding 8 different combinations, only one of which is the desired target antibody molecule. The efficiency of separating and purifying 8 molecules to obtain the target molecules is extremely low and very difficult.

There are various methods for constructing bispecific antibodies of the IgG type, one of which is important by engineering Fc to form heterodimers. As early as 90 s of the 20 th century, carter et al modified the Fc fragment of the antibody with a "knob-in-hole" model, which more successfully achieved the preparation of bispecific antibodies (Ridgway, presta et al 1996; carter 2001). Carter et al create a "bulge" in the CH3 domain of the first heavy chain of Fc by mutating an amino acid with a small side chain to an amino acid with a large side chain, and "recess" by mutating some amino acids in CH3 of the second heavy chain to amino acids with small side chains. The principle of the "male-female" model is that the "male-female" interactions support the formation of heterodimers, while the "male-male" model and the "female-female" model hinder the formation of homodimers. However, in their findings, the "concave-concave" model is still insufficient in its ability to hinder homodimer formation.

US2010286374A1 discloses a method for promoting heterodimer formation using electrostatic interactions. Specifically, the charged amino acids in the CH3 domains of two heavy chains are each mutated to an oppositely charged amino acid such that the CH3 domain of one heavy chain is generally positively charged and the CH3 domain of the other heavy chain is generally negatively charged, and electrostatic repulsion of the same charge inhibits homodimer formation. However, electrostatic interactions do not completely inhibit homodimer formation, and the introduction of excessive mutations instead causes a decrease in protein expression levels, reflecting the complexity of the relationship of interactions between interfacial amino acids in the case of multiple amino acid mutations.

Combining the "convex-concave" model with electrostatic action is an effective method of promoting heterodimer formation. CN106883297a discloses a method, specifically, introducing F405K mutation at one end of "concave" further on the basis of Carter et al's "concave-convex" model, enhancing electrostatic repulsion between "concave-concave" interface amino acids, and introducing K409A mutation at one end of "convex" at the same time, avoiding electrostatic repulsion between "convex-concave" interface amino acids, thereby inhibiting formation of "concave-concave" homodimer and maintaining formation of "convex-concave" heterodimer. Although the method disclosed in this patent can enhance the formation of heterodimers, one of these schemes can only enhance the proportion of heterodimers up to 93%.

US20150307628A1 discloses another method for promoting heterodimer formation using a "male-female" model and electrostatic interactions. Specifically, one scheme creates a "bulge" on the CH3 domain of the first heavy chain of Fc by a K409W point mutation, and a "recess" on CH3 of the second heavy chain by a F405T, D399V point mutation; the CH3 domain of the first heavy chain was negatively charged by a K360E point mutation, and the CH3 domain of the second heavy chain was positively charged by a Q347R point mutation. The scheme can increase the proportion of the heterodimer to 91.4+/-1.2% at the highest without introducing disulfide bonds.

Although both methods disclosed in CN106883297a and US20150307628A1 are superior to the simple "male-female" model in principle and further increase the proportion of heterodimers, new studies are still needed to increase the proportion of heterodimers to above 95%.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provides a hetero-dimer formed by modification and induction of a CH3 structural domain, and a preparation method and application thereof. The invention combines a 'convex-concave' model and electrostatic interaction by comprehensively considering intermolecular interactions such as ionic interaction, spatial interaction and hydrophobic interaction, and screens a preferable CH3 mutant sequence which is more prone to form a heterodimer rather than a homodimer, and can improve the proportion of the heterodimer molecules to more than 95% at most under the condition of not introducing disulfide bonds.

The heterodimer of the present invention refers to an antibody molecule or fragment containing two heavy chains with different amino acid sequences, including but not limited to bispecific antibodies, monovalent antibodies, fc fusion proteins, and the like.

In a first aspect the present invention provides a heterodimer comprising a first polypeptide chain and a second polypeptide chain, said first and second polypeptide chains comprising an antibody heavy chain constant region CH3 domain, designated ch3_a domain and ch3_b domain, respectively, said ch3_a and ch3_b domains comprising mutations in amino acids at positions compared to the wild-type human antibody heavy chain constant region CH3 domain: the ch3_a domain is mutated at K409, F405, and the ch3_a domain and ch3_b domain also have mutations at one or more amino acid positions selected from Q347, Y349, S354, E356, E357, K360, S364, L368, K370, K392, D399 and K439, respectively.

The positions of the amino acids described above are determined according to the EU index of the KABAT numbering.

Preferably, the ch3_a domain and ch3_b domain have one or more of the following mutations selected from:

1a) S364, E357 of the CH3_B domain is mutated, and K370 of the CH3_A domain is mutated;

1b) D399 of the ch3_b domain was mutated and K392 of the ch3_a domain was mutated;

1c) E356, E357 of the CH3_B domain is mutated and K439 of the CH3_A domain is mutated;

1d) S364 of the CH3_B domain is mutated and L368 of the CH3_A domain is mutated;

1e) Q347 of the CH3_B domain was mutated and K360 of the CH3_A domain was mutated.

Further preferred, the ch3_a domain and ch3_b domain also have the following mutations:

1f) S354 of the CH3_B domain is mutated and Y349 of the CH3_A domain is mutated; or 1 g) Y349 of the CH3_B domain is mutated and S354 of the CH3_A domain is mutated.

Preferably, the mutation is selected from one or several of the following mutations: Q347R, Y349C, S354C, E K, E357K, E357S, K E, S364R, S364K, L368D, K370D, K392D, D399K, K439E, F405E and K409F.

Q347R means that glutamine Gln347 is replaced with arginine (R). Y349C means that tyrosine Tyr349 is replaced with cysteine (C). S354C refers to the substitution of serine Ser354 for cysteine (C). E356K refers to the substitution of glutamic acid Glu356 with lysine (K). E357K refers to the substitution of glutamic acid Glu357 with lysine (K). E357S refers to the substitution of Glu357 with serine (S). K360E means that lysine Lys360 is replaced with lysine (K). S364R refers to the replacement of serine Ser364 with arginine (R). S364K refers to the substitution of serine Ser364 with lysine (K). L368D means that leucine Leu368 is replaced with aspartic acid (D). K370D means that lysine Lys370 is replaced with aspartic acid (D). K392D means that lysine Lys392 is replaced by aspartic acid (D). D399K means that aspartic acid Asp399 is replaced by lysine (K). K439E means that lysine Lys439 was replaced with glutamic acid (E). F405E means that phenylalanine Phe405 is replaced with glutamic acid (E). K409F means that lysine Lys409 is replaced with phenylalanine (F).

Preferably, the ch3_a domain and ch3_b domain of the heterodimer contain mutations selected from the group consisting of:

(a1) Ch3_a domain: f405e+k409f+k370d, ch3_b domain: s364r+e357S;

(a2) Ch3_a domain: f405e+k409f+k370d+s354C, ch3_b domain: s364r+e357s+y349C;

(a3) Ch3_a domain: f405e+k409f+k370d+y349C, ch3_b domain: s364R+E357S+S354C

(b1) Ch3_a domain: f405e+k409f+k392D, ch3_b domain: D399K;

(b2) Ch3_a domain: f405e+k409 f+k407d+s354C, ch3_b domain: d399k+y349C;

(b3) Ch3_a domain: f405e+k409f+k392d+y349C, ch3_b domain: d399k+s354C;

(c1) Ch3_a domain: f405e+k409f+k439D, ch3_b domain: e356k+e357K;

(c2) Ch3_a domain: f405e+k409f+k439d+s354C, ch3_b domain: e356k+e357k+y349C;

(c3) Ch3_a domain: f405e+k409f+k439d+y349C, ch3_b domain: e356k+e357k+s354C;

(d1) Ch3_a domain: f405e+k409f+l368D, ch3_b domain: S364R;

(d2) Ch3_a domain: f405e+k409 f+l19d+s354C, ch3_b domain: s364r+y349C;

(d3) Ch3_a domain: f405e+k409 f+l19d+y349C, ch3_b domain: s364r+s354C;

(e1) Ch3_a domain: f405e+k409f+l368D, ch3_b domain: S364K;

(e2) Ch3_a domain: f405e+k409 f+l19d+s354C, ch3_b domain: s364k+y349C;

(e3) Ch3_a domain: f405e+k409 f+l19d+y349C, ch3_b domain: s364k+s354C;

(f1) Ch3_a domain: f405e+k409f+k360E, ch3_b domain: Q347R;

(f2) Ch3_a domain: f405e+k409 f+k120e+s354C, ch3_b domain: q347R+Y349C;

(f3) Ch3_a domain: f405e+k409f+k360e+y349C, ch3_b domain: q347R+S354C;

(g1) Ch3_a domain: f405e+k409f+k370d+k360E, ch3_b domain: s364r+e357s+q347R;

(g2) Ch3_a domain: f405e+k409 f+k370d+k350e+s354C, ch3_b domain: s364r+e357s+q347r+y349C;

(g3) Ch3_a domain: f405e+k409 f+k370d+k350e+y349C, ch3_b domain: s364r+e357s+q347r+s354C.

In embodiments of the invention, the antibody constant regions are derived from IgG (e.g., igG1, igG2, igG3, igG 4), igA (e.g., igA1, igA 2), igD, igE, or IgM.

In a second aspect, the present invention provides a composition comprising: (1) The heterodimer of any one of claims 1-5, and (2) a pharmaceutically acceptable carrier and/or diluent and/or excipient.

In a third aspect of the invention there is provided a polynucleotide comprising: a nucleotide molecule a encoding a first polypeptide chain of the heterodimer of any one of claims 1-5, and a nucleotide molecule B encoding a second polypeptide chain of the heterodimer of any one of claims 1-5;

a fourth aspect of the present invention provides a carrier assembly comprising: a recombinant vector A containing the nucleotide molecule A, and a recombinant vector B containing the nucleotide molecule B.

The expression vectors used in the recombinant vector A and the recombinant vector B are conventional expression vectors in the art, and refer to expression vectors comprising appropriate regulatory sequences, such as promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and/or sequences, and other appropriate sequences. The expression vector may be a virus or plasmid, such as a suitable phage or phagemid, see, e.g., sambrook et al Molecular Cloning for further technical details: a Laboratory Manual, second edition, cold Spring Harbor Laboratory Press,1989. A number of known techniques and protocols for nucleic acid manipulation are described in Current Protocols in Molecular Biology, second edition, ausubel et al. The expression vector of the present invention is preferably pDR1, pcDNA3.1 (+), pcDNA3.1/ZEO (+), pDHFR, pTT5, pDHFF, pGM-CSF or pCHO 1.0, more preferably pTT5.

In a fifth aspect the invention provides a recombinant host cell comprising said vector combination.

The original host cell of the recombinant host cell of the present invention may be various host cells conventional in the art, as long as it can stably self-replicate the recombinant vector described above, and the nucleotide carried therein can be expressed efficiently. Wherein the primary host cell may be a prokaryotic or eukaryotic expression cell, preferably comprising: COS, CHO (chinese hamster ovary ), NS0, sf9, sf21, DH5 a, BL21 (DE 3) or TG1, more preferably e.coli TG1, BL21 (DE 3) cells (expressing single chain antibodies or Fab antibodies) or CHO-K1 cells (expressing full length IgG antibodies). The expression vector is transformed into a host cell, so that the preferred recombinant host cell of the invention can be obtained. Wherein the conversion process is conventional in the art, preferably chemical, heat shock or electrotransformation.

Preferably, the primary host cell is a eukaryotic cell, and more preferably a CHO cell or 293E cell.

In a sixth aspect the invention provides the use of a heterodimer according to the first aspect of the invention, a composition according to the second aspect of the invention, a polynucleotide according to the third aspect of the invention, a vector combination according to the fourth aspect of the invention, or a recombinant host cell according to the fifth aspect of the invention in the preparation of bispecific antibodies, bispecific fusion proteins and antibody-fusion protein chimeras.

In a seventh aspect, the invention provides a method of preparing a heterodimer according to the first aspect of the invention, using a recombinant host cell according to the fifth aspect of the invention to express the heterodimer.

In the invention, the recombinant host cell contains a recombinant vector A encoding a first polypeptide chain in the heterodimer and a recombinant vector B encoding a second polypeptide chain in the heterodimer, and the heterodimer molecule is obtained by utilizing the expression of the recombinant host cell and recovery.

Wherein the heterodimer can be purified from the recombinant host cell using standard experimental means. For example, when the heterodimeric protein comprises an antibody Fc fragment, protein a may be used for purification. Purification methods include, but are not limited to, chromatographic techniques such as size exclusion, ion exchange, affinity chromatography, and ultrafiltration, or suitable combinations of the above.

In the present invention, the transfection ratio of recombinant vector A and recombinant vector B in the recombinant host cell is 1:3 to 3:1, e.g., 1:2 to 2:1, e.g., 1:1.5 to 1.5:1, e.g., about 1:1.

In the present invention, both the first polypeptide chain and the second polypeptide chain comprise the CH3 domain of an Fc fragment of an antibody, and the two polypeptide chains interact via the CH3 domain or the CH 3-containing Fc fragment to form a dimer, in particular a heterodimer. The two polypeptide chains may be in different combinations, for example, the first polypeptide chain is an antibody, the second polypeptide chain is a fusion protein, or both polypeptide chains are fusion proteins, or both polypeptide chains are antibodies, targeting different antigens or epitopes. When the fusion protein comprises the Fc segment of an antibody and the extracellular domain of a cell adhesion molecule, it is also known as an immunoadhesin. The cell adhesion molecule is primarily a molecule that recognizes a specific ligand cell surface receptor, including, for example, cadherins, selectins, immunoglobulin superfamily, integrins, and hyaluronan.

In the present invention, the CH3 is derived from an antibody Fc fragment, preferably from a human antibody Fc fragment. In general, the CH3 domain of a human antibody Fc fragment is derived from a wild-type human antibody Fc fragment. Wild-type human antibody Fc refers to an amino acid sequence present in the human population, although the Fc fragment may vary slightly among individuals. Human antibody Fc fragments in the present invention also include alterations to individual amino acids of the wild-type human antibody Fc sequence, including, for example, certain amino acids mutated at glycosylation sites, or other nonsense mutations. For the CH3 and CH2 domains, it is possible to contain other mutations than those mentioned in the present invention which do not affect the function of the antibody, in particular the Fc fragment.

In the present invention, when the first polypeptide chain and/or the second polypeptide chain contains a hinge region, the hinge region is connected between two polypeptide segments as a flexible segment to ensure the function of each polypeptide segment; the length of the hinge region can be selected by one skilled in the art as desired, for example, the full length sequence or a portion thereof can be selected.

In the present invention, the numbering of the amino acid positions in the Fc or CH2, CH3 domains or hinge regions thereof is determined according to the position of the EU numbering index of Kabat. Those skilled in the art know that even if the amino acid sequence is changed due to an insertion or deletion of an amino acid or other mutation in the above region, the position number of each amino acid corresponding to the standard sequence, which is determined according to the EU numbering index of Kabat, remains unchanged. The EU index is described in Kabat et al, sequences of Proteins of Immunological Interest, 5 th edition Public Health Service, national Institutes of Health, bethesda, MD. (1991).

The invention has the beneficial effects that:

the present invention combines the "pro-concave" model with electrostatic interactions by comprehensively considering various interactions between interfacial amino acids, such as ionic interactions, hydrophobic interactions, and steric interactions, to screen for preferred CH3 mutant sequences that are more prone to form heterodimers rather than homodimers, thus greatly increasing the yield of heterodimer molecules. Compared with references CN106883297A and US20150307628A1, the method for constructing the convex-concave model is simpler, has fewer point mutations and can improve the purity of the heterodimer to more than 95% at most under the condition of not introducing disulfide bonds.

Drawings

FIG. 1 is a schematic diagram of the crystal structure of the CH3 domain. The figure shows that K409 of CH3_A is located in the cavity enclosed by F405, D399 and K370 of CH3_B and forms an ionic interaction with the side chain of D399.

FIG. 2 is a schematic representation of heterologous and homologous antibodies. From left to right are ch3_a/ch3_b heterodimer, ch3_a/ch3_a homodimer, and ch3_b/ch3_b homodimer, respectively. Square modules represent electrostatic interactions and circular modules represent spatial interactions. There is a repulsive interaction between the different shaped modules. There is mainly electrostatic-hydrophobic repulsion between ch3_a/ch3_a homodimers, and there is electrostatic repulsion and electrostatic-hydrophobic repulsion between ch3_b/ch3_b homodimers.

FIG. 3 is an electrophoretic analysis of transiently expressed scFv-Fc/Fc heterodimers. 4-12% SDS-PAGE protein gel electrophoresis. Lanes are from left to right: protein molecular weight standard, combination 1, combination 2, combination 3 and combination 4. The homodimers and heterodimers contained in each set of products had different migration distances in gel electrophoresis due to molecular weight differences. The positions of the different homodimeric or heterodimeric proteins are indicated in the figure.

FIG. 4 is an electrophoretic analysis of the effect of different transfection ratios on scFv-Fc/Fc heterodimers. 4-12% SDS-PAGE protein gel electrophoresis. Lanes are from left to right: the cotransfection ratio is ch3_a recombinant vector: ch3_b recombinant vector=1:1, the cotransfection ratio is ch3_a recombinant vector: ch3_b recombinant vector=1.5:1 and the protein molecular weight standard. The homodimers and heterodimers contained in each set of products had different migration distances in gel electrophoresis due to molecular weight differences. The positions of the different homodimeric or heterodimeric proteins are indicated in the figure.

Detailed Description

The following examples are further illustrative of the invention and should not be construed as limiting the invention. Examples do not include detailed descriptions of conventional methods, such as those used to construct vectors and plasmids, methods of inserting genes encoding proteins into such vectors and plasmids, or methods of introducing plasmids into host cells such methods are well known to those of ordinary skill in the art and are described in numerous publications, including Sambrook, j., fritsch, e.f. and maniis, t. (1989) Molecular Cloning: A Laboratory Manual,2 ^nd edition,Cold spring Harbor Laboratory Press.

1. Experimental materials:

293E cells from NRC biotechnology Research Institute.

2. Experimental reagent:

PBS: purchased from the company division of bioengineering (Shanghai), cat No. B548117.

Citric acid: purchased from national pharmaceutical group chemical company, inc.

Prime star HS DNA polymerase: purchased from Takara corporation under the product number R010A.

Endotoxin-free plasmid large extraction kit: available from tengen company under the designation DP117.

3. Experimental instrument:

HiTrap MabSelectSuRe column: purchased from GE company.

AKTA-FPLC rapid protein liquid chromatography system: purchased from GE company.

C1000 Touch Thermal Cycler PCR instrument: purchased from Bio-Rad corporation.

Chemidoc MP gel imager: purchased from Bio-Rad corporation.

Centrifuge: purchased from Eppendorf corporation.

G1600AX capillary electrophoresis apparatus: purchased from agilent.

MicroCal PEAQ-DSC micro thermal differential scanning calorimeter: purchased from malvern corporation.

Example 1 design, construction, expression and purification of first round mutation candidate combinations

1. Design of first round CH3 Domain amino acid modifications

Most antibodies that have been marketed belong to the IgG1 or IgG4 subclass, and the Fc-segment amino acid sequences of IgG1 and IgG4 are highly conserved. In the present invention, igG1 is preferred as a template for designing amino acid modifications of the CH3 domain, and these amino acid modifications are equally applicable to the IgG4 subtype without specific description.

The CH3 domain forms a homodimer, the CH3 domain crystal structure of the Fc segment of IgG1 antibody (pdbcode: 4 BSW) is shown in FIG. 1, K409 of CH 3-A is located within the cavity enclosed by F405, D399 and K370 of CH 3-B, and forms an ionic interaction with the side chain of D399. The above sites are highly conserved on IgG4, and differ from IgG1 only in that the amino acid at position 370 of the heavy chain of IgG4, EU numbering, is Arg. US20150307628A1 mentions that mutating K409 of ch3_a to Trp (or K409W) creates a "convexity", requiring the simultaneous introduction of F405T and D399V point mutations at ch3_b creating a "concavity", which would otherwise cause steric and hydrophobic-electrostatic repulsion. Surprisingly, it was found in the present invention that mutation of K409 of CH 3-A to Phe (or called K409F) does not cause steric hindrance rejection with amino acid F405 of CH 3-B and hydrophobic-electrostatic rejection with amino acids K370 and D399 of CH 3-B. Hydrophobic-electrostatic repulsion also does not occur when amino acid K370 on ch3_b is mutated to Arg (or called K409R). Conversely, mutating D399 on CH 3B to an amino acid that is nonpolar and has smaller side chains, such as Ala (or D399A), creates a "pocket" that is rather detrimental to heterodimer formation (see FIG. 3 below). Thus, the present invention has found a more convenient way of constructing a "male-female" model than the control patent US20150307628 A1.

To inhibit the formation of ch3_a homodimers, further introducing an F405E or F405D point mutation on ch3_a; to inhibit the formation of homodimers by ch3_b, the s364r+e357S point mutation was introduced on ch3_b, while the K370D or K370E point mutation was introduced on ch3_a, as shown in fig. 2. As described above, the heterodimer mutant combinations in table 1 were obtained.

TABLE 1 heterodimer mutation combination list-1

2. Construction of Fc fragment of human IgG1 with mutation and recombinant vector of scFv-Fc fusion protein

The gene encoding scFv-Fc fusion protein (scFv-Fc fusion protein sequence is shown in SEQ ID: 1) is obtained through artificial synthesis, wherein scFv refers to a single-chain antibody against CD 3. Then subcloning into mammalian cell expression vector pTT5 to obtain recombinant expression vector for mammalian cell expression of scFv-Fc fusion protein. The Fc fragment (Fc fusion protein sequence shown in SEQ ID NO: 2) of the gene is subcloned into a mammalian cell expression vector pTT5 to obtain a recombinant expression vector for expressing the Fc fusion protein by mammalian cells. According to table 1 of example 1, the scFv-Fc and Fc-encoding genes were subjected to combinatorial mutation using an overlap PCR method, wherein the mutation for the ch3_a chain was located on the Fc fusion protein and the mutation for the ch3_b chain was located on the scFv-Fc fusion protein. Subcloning the mutated gene into pTT5 to obtain recombinant expression vectors for expressing mutated scFv-Fc fusion proteins and mutated Fc proteins, respectively, in mammalian cells.

3. Transiently expressing scFv-Fc/Fc heterodimers and detecting the effect of different combinations of mutations on heterodimer content

The corresponding expression vectors of the 4 mutation combinations in the step 1 are transfected into 293E cells in suspension culture by PEI, each group of mutation combinations comprises the corresponding recombinant expression vectors of A chain (referring to scFV-Fc fusion protein chain) and B chain (referring to Fc protein chain) which are transfected together, and the cotransformation ratio of the recombinant expression vectors of the A chain and the B chain is 1:1. After 5-6 days of culture, collecting the transient expression culture supernatant, and obtaining the initially purified 4 groups of mutation combinations by Protein A affinity chromatography. These transient products all contain different proportions of homodimeric proteins (scFv-Fc/scFv-Fc, fc/Fc) and heterodimeric proteins (scFv-Fc/Fc). Due to the difference in molecular weight between the three proteins (scFv-Fc/scFv-Fc, fc/Fc, and scFv-Fc/Fc), the composition of the homodimeric protein (scFv-Fc/scFv-Fc, fc/Fc) and the heterodimeric protein (scFv-Fc/Fc) in each group of products can be detected by SDS-PAGE under non-reducing conditions, and the results of the electrophoresis are shown in FIG. 3. The homodimers of combination 1 and combination 2 were significantly more than those of combination 3 and combination 4, indicating that the D399A mutation on ch3_b was not detrimental to heterodimer formation. The homodimer of combination 4 was less than that of combination 3, indicating that the F405E mutation was superior to F405D.

Example 2 design, construction, expression and purification of second round mutant candidate combinations

In example 1, the formation of homodimers (scFv-Fc/scFv-Fc) was inhibited by electrostatic repulsion by introducing positively charged amino acids on the ch3_b chain. This example aims to further explore methods of reducing the inter-attraction between ch3_b chains and inhibiting homodimeric protein formation by introducing a combination of other charged amino acid mutations. Based on the existing K409F and F405E point mutation combination of the CH3_A domain, the following charged amino acid mutation combination is further introduced into the CH3_A domain and the CH3_B domain:

ch3_a domain: k392D, ch3_b domain: D399K;

ch3_a domain: k439D, ch3_b domain: e356k+e357K;

ch3_a domain: L368D, ch3_b domain: S364R;

ch3_a domain: L368D, ch3_b domain: S364K;

ch3_a domain: k360E, ch3_b domain: Q347R;

ch3_a domain: k370d+k360E, ch3_b domain: s364r+e357s+q347R.

Thus, combinations 5 to 10 shown in Table 2 were obtained. The scFv-Fc and Fc encoding genes were subjected to combinatorial mutation using an overlap PCR method, wherein the mutation for the CH 3-A chain was located on the Fc fusion protein and the mutation for the CH 3-B chain was located on the scFv-Fc protein. Subcloning the mutated gene into pTT5 to obtain recombinant expression vectors for expressing mutated scFv-Fc fusion proteins and mutated Fc proteins, respectively, in mammalian cells. Combination 4 was used as a control group and combinations 5-10 were used as test groups, and the expression purification method was as shown in step 3 of example 1. The purified protein fractions were analyzed by non-reducing capillary gel electrophoresis and the percentage of peak area of each product fraction was calculated. As shown in Table 3, combinations 5 to 10 each gave a better heterodimer purity, wherein combination 7 was comparable to the heterodimer obtained in combination 4, and combination 10 was obtained by adding a new charged amino acid mutation to combination 4.

TABLE 2 heterodimer mutation combination List-2

TABLE 3 influence of combinations of differently charged amino acids on the ratio of homodimers and heterodimers

Example 3 Effect of transfection proportion on scFv-Fc/Fc heterodimer formation

To further examine the effect of co-transformation ratio of recombinant vectors for the a-and B-chains on the ratio of homodimers to heterodimers, the co-transformed expression vectors used for the preferred mutant combination 4 were transfected with ch3_a recombinant vector: ch3_b recombinant vector=1:1 and ch3_a recombinant vector: ch3_b recombinant vector=1.5:1, respectively, with PEI into 293E cells in suspension culture, and after 5-6 days of culture, the cell supernatants were collected. The respective transient products were obtained by Protein A affinity chromatography. The composition of homodimeric proteins (scFv-Fc/scFv-Fc, fc/Fc) and heterodimeric proteins (scFv-Fc/Fc) was detected by SDS-PAGE electrophoresis under non-reducing conditions. The specific results are shown in FIG. 4. From the results, it can be seen that: the cotransformation ratio of the recombinant expression vector has a relatively obvious effect on the ratio of homodimers to heterodimers in the product. Ch3_b forms homodimers relatively more readily than ch3_a, and the homodimeric protein scFv-Fc/scFv-Fc is significantly more when the co-transfection ratio is ch3_a recombinant vector ch3_b recombinant vector = 1:1; when the transfection ratio of ch3_a recombinant vector, e.g. the cotransfection ratio is ch3_a recombinant vector: ch3_b recombinant vector=1.5:1, the homodimeric protein scFv-Fc/scFv-Fc is significantly reduced.

Example 4 Effect of disulfide bonding on scFv-Fc/Fc heterodimer formation

It is mentioned in US7695936 b2 and US20150307628A1 that disulfide bonds may promote the formation of heterodimers and increase the thermal stability of the molecules. In this example, cysteine mutations were introduced on the basis of mutation combination 4, resulting in the mutation combinations shown in Table 4. Genes encoding antibody heavy chain HC_A (SEQ ID NO: 3), antibody heavy chain HC_B (SEQ ID NO: 4) and antibody common light chain LC (SEQ ID NO: 5) were synthesized artificially and then subcloned into mammalian cell expression vector pTT5. The combined mutation of the HC_A and HC_B encoding genes was performed using an overlap PCR method, wherein the mutation for the CH 3-A chain was located on the HC_A chain and the mutation for the CH 3-B chain was located on the HC_B chain. Subcloning the mutated genes into pTT5 to finally obtain recombinant expression vectors for expressing the mutations in mammalian cells, respectively. Expression purification method as shown in step 3 of example 1, the co-transformation ratio of the recombinant expression vector was adjusted to HC_A chain: hc_b chain: common light chain lc=1.5:1:2. The purified protein fractions were analyzed by LC-MS and the percentage of each product fraction was calculated. Tm values of the samples were measured using a MicroCal PEAQ-DSC using a micro thermal differential scanning calorimeter. As a result, as shown in table 5, combination 11 was used as a control group, and combinations 12 and 13 were introduced with disulfide bonds based on combination 11, and the obtained heterodimers were found to have a proportion of 99.56% and 96.89%, respectively, similar to combination 11; the Tm values of Fc for combination 12 and combination 13 were 69.21 ℃ and 70.23 ℃, respectively, which were increased by about 3 ℃ over combination 11. This example shows that, although the control group (combination 11) already had a heterodimer proportion of greater than 95%, the introduction of disulfide bonds still further promotes heterodimer formation and increases the thermal stability of the antibody molecule. Similarly, the introduction of disulfide bonds should also increase the heterodimer ratio and thermal stability of combinations 5-10.

TABLE 4 cysteine mutant combinations

TABLE 5 Effect of disulfide bond on the proportion and thermal stability of heterodimers

The above description of the specific embodiments of the present invention has been given by way of example only, and the present invention is not limited to the above described specific embodiments. Any equivalent modifications and substitutions for the present invention will occur to those skilled in the art, and are also within the scope of the present invention. Accordingly, equivalent changes and modifications are intended to be included within the scope of the present invention without departing from the spirit and scope thereof.

Sequence listing

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Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

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Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr

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Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe

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Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr

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Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

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Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly

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Thr Thr Leu Thr Val Ser Ser Val Glu Gly Gly Ser Gly Gly Ser Gly

115 120 125

Gly Ser Gly Gly Ser Gly Gly Val Asp Asp Ile Gln Leu Thr Gln Ser

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Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys

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Arg Ala Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser

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Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser

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Gly Val Pro Tyr Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser

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Leu Thr Ile Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys

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Gln Gln Trp Ser Ser Asn Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu

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

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

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

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

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

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

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

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

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

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Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

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

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

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

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

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

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

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

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

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Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr

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Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

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Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

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Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

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

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Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

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Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

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

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

275 280 285

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

290 295 300

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

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

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

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Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

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

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Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu

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Gly Val Ile Trp Ser Gly Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr

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Ser Arg Leu Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln Val Phe Phe

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Arg Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp Gly Gln Gly

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

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

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

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

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Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

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

210 215 220

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

225 230 235 240

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

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

260 265 270

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

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

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

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

325 330 335

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

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Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

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

370 375 380

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

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

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

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Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

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Lys

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Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gln Ser Ile Gly Thr Asn

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Ile His Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile

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Lys Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly

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Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Ser

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Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Asn Asn Asn Trp Pro Thr

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Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

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Phe Asn Arg Gly Glu Cys

210

Claims (13)

1. A heterodimer comprising a first polypeptide chain and a second polypeptide chain, said first polypeptide chain and said second polypeptide chain comprising an antibody heavy chain constant region CH3 domain, designated ch3_a domain and ch3_b domain, respectively, said antibody heavy chain constant region being derived from IgG, which is IgG1, igG2, igG3 or IgG4;

the ch3_a and ch3_b domains contain mutations in the amino acids at the following positions compared to the wild-type human antibody heavy chain constant region CH3 domain:

the k409, F405 of the ch3_a domain is mutated, and the ch3_a domain and the ch3_b domain also have mutations at one or more amino acid positions selected from Q347, Y349, S354, E356, E357, K360, S364, L368, K370, K392, D399 and K439, respectively;

the ch3_a domain and ch3_b domain are mutated selected from the group consisting of:

(a1) Ch3_a domain: f405e+k409f+k370d, ch3_b domain: s364r+e357S;

(a2) Ch3_a domain: f405e+k409f+k370d+s354C, ch3_b domain: s364r+e357s+y349C;

(a3) Ch3_a domain: f405e+k409f+k370d+y349C, ch3_b domain: s364r+e357 s+s354C;

(b1) Ch3_a domain: f405e+k409f+k392D, ch3_b domain: D399K;

(b2) Ch3_a domain: f405e+k409 f+k407d+s354C, ch3_b domain: d399k+y349C;

(b3) Ch3_a domain: f405e+k409f+k392d+y349C, ch3_b domain: d399k+s354C;

(c1) Ch3_a domain: f405e+k409f+k439D, ch3_b domain: e356k+e357K;

(c2) Ch3_a domain: f405e+k409f+k439d+s354C, ch3_b domain: e356k+e357k+y349C;

(c3) Ch3_a domain: f405e+k409f+k439d+y349C, ch3_b domain: e356k+e357k+s354C;

(d1) Ch3_a domain: f405e+k409f+l368D, ch3_b domain: S364R;

(d2) Ch3_a domain: f405e+k409 f+l19d+s354C, ch3_b domain: s364r+y349C;

(d3) Ch3_a domain: f405e+k409 f+l19d+y349C, ch3_b domain: s364r+s354C;

(e1) Ch3_a domain: f405e+k409f+l368D, ch3_b domain: S364K;

(e2) Ch3_a domain: f405e+k409 f+l19d+s354C, ch3_b domain: s364k+y349C;

(e3) Ch3_a domain: f405e+k409 f+l19d+y349C, ch3_b domain: s364k+s354C;

(f1) Ch3_a domain: f405e+k409f+k360E, ch3_b domain: Q347R;

(f2) Ch3_a domain: f405e+k409 f+k120e+s354C, ch3_b domain: q347R+Y349C;

(f3) Ch3_a domain: f405e+k409f+k360e+y349C, ch3_b domain: q347R+S354C;

(g1) Ch3_a domain: f405e+k409f+k370d+k360E, ch3_b domain: s364r+e357s+q347R;

(g2) Ch3_a domain: f405e+k409 f+k370d+k350e+s354C, ch3_b domain: s364r+e357s+q347r+y349C;

(g3) Ch3_a domain: f405e+k409 f+k370d+k350e+y349C, ch3_b domain: s364r+e357s+q347 r+s354C;

the positions of the amino acids described above are determined according to the EU index of the KABAT numbering.

2. A composition characterized in that it comprises: (1) The heterodimer of claim 1, and (2) a pharmaceutically acceptable carrier and/or diluent and/or excipient.

3. A polynucleotide, vector combination, or recombinant host cell, wherein the polynucleotide comprises: a nucleotide molecule a encoding a first polypeptide chain of the heterodimer of claim 1, and a nucleotide molecule B encoding a second polypeptide chain of the heterodimer of claim 1;

the carrier combination comprises: a recombinant vector A containing the nucleotide molecule A and a recombinant vector B containing the nucleotide molecule B;

the recombinant host cell contains the vector combination.

4. The polynucleotide, vector combination, or recombinant host cell of claim 3, wherein the original host cell of the recombinant host cell is a eukaryotic cell.

5. The polynucleotide, vector combination, or recombinant host cell of claim 3 or 4, wherein the original host cell of the recombinant host cell is a CHO cell or a 293E cell.

6. Use of the heterodimer of claim 1, the composition of claim 2, the polynucleotide of any one of claims 3-5, the vector combination, or the recombinant host cell in the preparation of an antibody-fusion protein chimera.

7. Use of the heterodimer of claim 1, the composition of claim 2, the polynucleotide of any one of claims 3-5, the vector combination, or the recombinant host cell in the preparation of a bispecific fusion protein.

8. Use of the heterodimer of claim 1, the composition of claim 2, the polynucleotide of any one of claims 3-5, the vector combination, or the recombinant host cell in the preparation of a bispecific antibody.

9. A method of preparing the heterodimer of claim 1, wherein the heterodimer is expressed using the recombinant host cell of any one of claims 3-5.

10. The method of claim 9, wherein the transfection ratio of recombinant vector a to recombinant vector B in the recombinant host cell is 1:3 to 3:1.

11. The method of claim 10, wherein the transfection ratio of recombinant vector a to recombinant vector B in the recombinant host cell is 1:2 to 2:1.

12. The method of claim 10, wherein the transfection ratio of recombinant vector a to recombinant vector B in the recombinant host cell is 1:1.5 to 1.5:1.

13. The method of claim 10, wherein the transfection ratio of recombinant vector a and recombinant vector B in the recombinant host cell is 1:1.

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