Fc region-based heterodimer molecule and use thereof

Abstract

The present invention relates to a heterodimeric Fc scaffold based on an immunoglobulin Fc region, a heteromultimeric protein comprising said heterodimeric Fc scaffold, in particular a monospecific or multispecific binding protein, as well as a composition comprising the same and use thereof.

Description

(12) International Application published under the Patent Cooperation Treaty Intellectual Property Organization > (19) arias DEHEs NOU NOMA YN JA — enn — (10) Ents (43) International Patent Classification: MU, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM, PA, C0O7K 16 / 00 (2006.01) A61P 37 / 02 (2006.01) PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, SC, SD, A61K 39 / 395 (2006.01) A6LP 35 / 00 (2006.01) SE, SG, SK, SL, ST, SV, SY, TH, TJ, TM, TN, TR, TT, TZ, CI2N 15 / 13 (2006.01) CO7K 16 / 46 (2006.01) UA, UG, US, UZ, VC, VN, WS, ZA, ZM, ZW. (21) International Application Number: PCT / CN2024 / 129416 (84) HERR AA indicates that each available region is required (22) International Application Date: 2024 42 11 A 1 A (01.11.2024) (4): ARIPO (BW, CV, GH, GM, KE, LR, LS, MW, MZ,, _ NA, RW, SC, SD, SL, ST, SZ, TZ, UG, ZM, ZW), Eurasian (25) Application Language: Chinese (AM, AZ, BY, KG, KZ, RU, TJ, TM), European (AL, AT, BE, (26) Publication Language: Chinese BG, CH, CY, CZ, DE, DK, EE, ES, FI, FR, GB, GR, HR,, HU, IE, IS, IT, LT, LU, LV, MC, ME, MK, MT, NL, NO, (30) RIAL: PL, PT, RO, RS, SE, SI, SK, SM, TR), OAPI (BF, BJ, CF, PCT / CN2023 / 129691 CG, CI,CM, GA, GN, GQ, GW, KM, ML, MR, NE, SN, 20234711 A3HA (03.11.2023) CN TD, TG). CD Applicant: PHRONTLINE BIOPHARMA (SUZHOU) CO.,LTD This international publication: os . [CNICN]; FH FBT 9 4 IRIN TH RH BC SB AR TE - PHRONTLINE BIOPHARMA International Search Report (Article 21(3) of the Treaty). —_ AWOILAG ULE Ki Bite 1 SKige - including the sequence listing portion of the specification (Details 5.2(a). = 1915 38 215124(CN), Shanghai PHRONTLINE BIOPHARMA CO., LTD) [CN / CN]; Room 301, No. 396, Lishizhen Road, Pudong New Area, Shanghai, China (CN). 3 (72) Inventors: Hu Siyi (Hg, SiyD; Tianyun Plaza, No. 111, Wusongjiang Avenue, Guobu Street, Wuzhong District, Suzhou, Jiangsu Province, China (CN), Chen Zhaoyuan (CHEN, Zhaoyuan); Tianyun Plaza, No. 111, Wusongjiang Avenue, Guobu Street, Wuzhong District, Suzhou, Jiangsu Province, China (CN). Huang Mao (GIUANG, Mao); Room 396, Lishizhen Road, Pudong New Area, Shanghai, China (CN). ms (74) Agent: Beijing Zhongzi Law Firm (7th Floor, New Era Building, No. 26 Pinganli West Street, Xicheng District, Beijing, 100034, China). = 81) As otherwise specified in the designated country, each of the following is required to provide national protection: AE, AG, AL, AM, AO, AT, AU, AZ, BA, BB, BG, =BH, BN, BR, BW, BY, BZ, CA, CH, CL, CN, CO, CR, CU, => CV, CZ, DE, DJ, DK, DM, DO, DZ, EC, EE, EG, ES, FI, = GB, GD, GE, GH, GM, GT, HN, HR, HU, ID, IL, IN, IQ, IR, IS, IT, JM, JO, JP, KE, KG, KH, KN, KP, KR, KW, KZ, = LA, LC, LK, LR, LS, LU, LY, MA, MD, MG, MK, MN, P x (54) Title: FC REGION-BASED HETERODIMER MOLECULE AND USE THEREOF SR (54) Invention Title: Fe-based heterodimer molecules and their uses = (57) Abstract: Provided are an immunoglobulin Fc region-based heterodimeric Fc stent, a heterologous multimeric protein containing a heterodimeric Fe stent, in particular a single or multispecific binding protein, and a composition comprising the same and a use. —} IV、 、 _ 、 (57) Abstract: Provided are immunoglobulin Fc region-based heterodimeric Fc stents, heterologous multimeric Fe stents containing said heterodimeric Fc stents and proteins, especially single or multispecific binding proteins, and compositions comprising the same and uses. WO 2025 / 092987 PCT / CN2024 / 129416 Fe region-based heterodimeric molecules and their uses Technical Field This invention relates to immunoglobulin Fe RH HORA Fe stents, heterologous multimeric proteins containing said heterodimeric Fe stents, especially single or multispecific binding proteins, and compositions comprising the same and uses. BackgroundBispecific antipodies are a rapidly developing class of novel antibody drugs in recent years. These antibodies can simultaneously bind to two different antigens or two different epitopes on the same antigen, thus offering potential additional clinical benefits for the treatment of complex diseases. Currently, bispecific antibodies exist in many different structural forms, including non-IgG-like forms based on the fusion of antibody fragments such as scFv, Fab, VH, and VL, and IgG-like forms based on the Fo domain. Compared to non-IgG-like forms, bispecific antibodies with structures similar to conventional IgG exhibit better physicochemical properties, stability, and pharmacokinetic characteristics, showing broad application prospects. The Fo region of the antibody is known to have dimerizing function, playing a crucial role in maintaining antibody function in vivo. Several heterodimerization Fc techniques have been reported to assist in the production of bispecific antibodies (Moore GL, Methods 2019, 154: 38-50). For example, Carter et al. invented the handle-into-hole model and successfully achieved the preparation of bispecific antibodies (Patent US7951917B1). In this method, specific small side-chain amino acids at the CH3 region interface of the first heavy chain are mutated into large side chains and amino acids (e.g., T366Y), and specific amino acids at the CH3 region interface of the second heavy chain are mutated into small side chains and amino acids (e.g., Y407T). This results in the mutated residues at the interface being spatially complementary, thereby promoting the formation of Fo heterodimers. Besides the example mutations mentioned above, more widely used mutation combinations in this method include the handle T366W and hole T366S, and the L368A and Y407V mutation combinations, which can produce Fc HARKs in 80-90% of cases. However, the handle-hole model is less effective against homodimer (Chomodimer) impurities.The inhibitory effect of this method is still insufficient. Electrostatic reversal is also widely used to promote the formation of Fc heterodimers. This method mutates a specific amino acid in one heavy chain CHB to a positively charged aAAR (Lys or Arg), and a specific amino acid in another heavy chain CH3 to a negatively charged amino acid (Asp or Glu). Through electrostatic attraction between the mutated residues, the formation of Po heterodimers is promoted. For example, Kannam et al. generated a high proportion of heterodimers by introducing mutations D399K and E3S6K into the CH3 region of the first heavy chain and mutations K409D and K392D into the CH3 region of the second heavy chain (Patent US8592562B2). Igawa et al. also promoted Fo heterodimer formation by introducing mutations E356K and D399K into the CH3 region of the first heavy chain and mutations K439E and K409D into the CH3 region of the second heavy chain (Patent US10011858B2). Genmab invented the DuoBody technology. This technology involves introducing the F40SL mutation into the CH3 region of the heavy chain of one antibody and the CH3 K409R mutation into the heavy chain of another antibody. Controlled Fab arm exchange between the two antibodies is achieved by separately expressing and purifying them, followed by mixing them in the presence of a reducing agent (e.g., 2-MEA). This technique produces Fo HORA KB with a purity of 90-95% (patent US9150663B2). Fab exchange technology (SEED) is also used to promote Fo FF-HAA Ga (Muda et al. Proteins Eng. Des. Sel. 2011, 24: 447-454). Sequence differences between RANG SAF IgA and IgG in the CH3 domain lead to the generation of Fo heterodimers through complementary CH3 domain pairing, preventing homodimer formation. Zymeworks has reported a scaffold structure for improving the stability of Fo heterodimers.(Patent US20120149876A1). For example, a scaffold 1a design includes a heavy chain CH3 region with T366L K392M, T394W mutation and another heavy chain CH3 region with F40SA, Y407A mutation, achieving a Tm value of 74°C. Additionally, a scaffold 2a design includes a heavy chain CH3 region with L3S$1YY047A mutation and another heavy chain CH3 region with T366VK409F mutation, achieving a Tm value of 75.5°C. The proportion of Fo heterodimers reaches over 90%. Although various strategies for Fo heterodimer formation have been proposed, these strategies have shown certain shortcomings in application. Bispecific antibodies from Bio and BREAF0 exhibit homology mismatch and reduced thermal stability, thus affecting the drug-likeness of antibody drugs. For example, it has been found that most FeO HORA and HEA GG1 monoclonal antibodies formed using existing technologies have poor thermal stability. The Tm value of the CH3 domain of wild-type IgG1 monoclonal antibodies is generally greater than 80°C (Ionescu RM, J Pharm Sci. 2008, 97: 1414-1426). Mutations in the CH3 and HCl amino acids introduced through Fe heterodimerization technology can lead to a significant decrease in the melting temperature (Tm) of the CH3 domain. For example, CH3 HIRAM produced by handle-hole 154% Bl (T366W / T366S, L368A, Y407V) has a Tm value of 69.4°C (Atwell S, J. Mol. Biol. 1997, 270: 26-35). CH3 HIRAM produced by handle-hole 154% Bl (D399K, E356K / K409D, K392D) has a Tm value of 124°C at 68.8°C (Gunasekaran K, J. Biol. Chem. 2010, 285: 19637-19646). For structurally complex antibody drugs, lowerThe manufacturing and preservation of drugs. Although introducing non-natural disulfide bonds (such as Y349C and Y354C) into the handle-hole model can improve the efficiency of heterodimer formation and thermal stability, for some drugs, such as antibody-drug conjugates (ADCs), the introduction of additional disulfide bonds can adversely affect product quality. Furthermore, the proportion of Fo heterodimers generated by existing technologies is generally between 80-95%, with other impurities including Fe BAR, Fe homodimers, and high molecular weight polymers. These impurities need to be further removed by other methods such as ion exchange chromatography, especially the removal of homologous heavy chain mismatch impurities, which poses a challenge to downstream antibody purification processes. Moreover, the higher the impurity content, the lower the recovery rate of the purified product. Given the broad application prospects of IgG-like bispecific antibodies, there is still a need in this field to develop Fo heterodimerization technologies that address the chain mismatch and thermal stability issues of bispecific antibodies to improve their drugability. Summary of the Invention: The inventors have discovered that, in a scaffold structure based on a dimerized Fo region, by introducing a knock-in-hole (KIH) mutation and further introducing a specific CH3 domain interface mutation, antibodies encapsulated in this gold-plated Fo scaffold exhibit superior thermostability compared to corresponding antibodies encapsulated only with the gold-plated KIH mutation. Simultaneously, the advantages brought by the KIH mutation (especially the advantage of promoting heavy chain pairing in the target heterodimer) are maintained, and biological effects comparable to those of natural antibodies are preserved, including antigen-specific binding and FcRn LAR FeyR receptor binding. Based on this discovery, the inventors propose a Fo heterodimerization mutation combination that can reduce the chain mismatch ratio and increase the stability of the target antibody product. Applying the mutation combination of this invention can effectively improve the bispecificity of the antibody.Drug-likeness and stability of heterologous antibodies. In a first aspect, the present invention provides a CH3 heterodimer containing a tungsten-modified combination of the present invention and a heterodimer Fc scaffold. In a second aspect, the present invention provides the use of the heterodimer Fc scaffold and the CH3 heterodimer of the present invention as components for constructing heteropolymeric proteins, particularly single / multi-specific binding proteins, and corresponding heteropolymeric proteins comprising said Fe scaffold or CH3 heterodimer. In a third aspect, the present invention particularly provides binding proteins comprising the heterodimer Fc scaffold according to the present invention. In a fourth aspect, the present invention provides isolated polynucleotides encoding the heterodimer Fe scaffold, CH3 HORA, FRA RAE AR binding proteins according to the present invention; vectors (particularly expression vectors) comprising the isolated polynucleotides of the present invention; and host cells comprising the isolated polynucleotides of the present invention or vectors (particularly expression vectors). This invention also provides uses and methods for producing the heterodimeric Fo scaffold, CH3 heterodimer, heteropolymeric protein, and binding molecules of this invention using the polynucleotide, carrier, or host cells of this invention. 2 WO 2025 / 092987 PCT / CN2024 / 129416 In a fifth aspect, this invention provides compositions comprising the heteropolymeric protein or binding protein of this invention, and conjugates comprising a therapeutic agent conjugated with the heteropolymeric protein or binding protein of this invention. In some embodiments, the compositions are pharmaceutical compositions comprising OHRERAEA of this invention (especially the multispecific binding protein of this invention) and a pharmaceutically acceptable carrier. In a sixth aspect, this invention also provides heteropolymeric proteins, binding proteins, compositions, and slowing agents according to this invention.The invention provides methods of use and applications of the heteropolymer protein, binding protein, composition, or sustained-release compound according to the invention. In some embodiments, the invention provides the use of the heteropolymer protein, binding protein, composition, or sustained-release compound according to the invention as a medicament. In some embodiments, the invention provides the use of the heteropolymer protein, binding protein, composition, or sustained-release compound according to the invention for treating a disease in an individual in need. In a specific embodiment, the disease is cancer. The invention also provides the use of the heteropolymer protein, binding protein, composition, or sustained-release compound according to the invention in the preparation of a medicament for treating a disease in an individual in need, and a method of treating an individual disease using the heteropolymer protein, binding protein, composition, or sustained-release compound according to the invention, wherein the method comprises administering a therapeutically effective amount of the heteropolymer protein, binding protein, composition, or sustained-release compound according to the invention to the individual. In some embodiments, the heteropolymer protein, binding protein, composition, or sustained-release compound is formulated into a pharmaceutically acceptable form. In some embodiments, the disease is cancer. In some embodiments, the individual is preferably a mammal, particularly a human. Figure 1 shows a schematic diagram of the local crystal structure of the CH3-CH3 region of the Fe dimer. Figure 1A shows the hydrophobic interaction of the CH3-CH3 interface in wild-type IgG1 (PDB: 1HZH). Figure 1B shows the hydrophobic interaction of the CH3-CH3 interface in the handle-hole model (PDB: 4NQS). The left side of the figure represents the handle (knob) chain, and the right side represents the hole (hole) chain. Figure 1C shows the 3D structure of the CH3-CH3 region based on computer simulation of the M2 mutation combination. The L3S1Y mutation is associated with the handle T366W and E357 (CRRA).Figure 1D shows the CH3-CH3 region 3D 24744.10-F K409D and hole D399R mutations of the M3 mutant combination forming a new hydrogen bond network (indicated by dashed lines) with multiple amino acids at the CH3-CH3 interface, based on computer simulation. Figure 2 shows the non-reducing SDS-PAGE electrophoresis of the Ska VHH-Fe / Fe mutant combination. In Figure 2A, the transfection ratio of VHH-Fc and Fec plasmids is 1:1. In Figure 2B, the transfection ratio of VHH-Fe and Fe Fe plasmids is 0.6:1. For each mutant combination shown, the heterodimer (AB) and other impurities (LISA ROR AFH HBA) contained in the purified protein and protein A products exhibit different migration distances due to differences in molecular weight. Figure 3 shows the Tm value of the VHH-Fc / Fc mutant combination determined by DSC method. The Tm values ​​of the samples were analyzed using a microcalorimetric differential scanning calorimeter (Microcal PEAQ-DSC). Each mutant combination indicated one to three Tm values ​​based on changes in Cp values, with the highest Tm value reflecting the stability of the CH3 domain. Figure 4 shows the non-reducing SDS-PAGE electrophoresis of the VHH-Fc / Fab-Fc mutant combinations. For each mutant combination shown, heterodimers (ABC) and other impurities (homodimers, haptens, free monomers) contained in the purified protein A product exhibited different migration distances due to differences in molecular weight. Figure 5 shows the non-reducing SDS-PAGE electrophoresis of the Fab-Fc mutant. For each mutant shown, the heavy chain to light chain plasmid transfection ratio was 1:1.3. For each mutant shown, haptens, disulfide-linked homodimers, and other impurities (free light chain and free heavy chain) contained in the purified protein A product exhibited different migration distances due to differences in molecular weight. Figure 6 shows the HIC-HPLC detection.In vitro assembly effect of Fab-Fc mutant combination. The half-antibody purified by protein A affinity shows two main peaks, representing either monomers or homodimers. The bispecific antibody generated by in vitro assembly shows a heterodimer main peak, with other smaller peaks representing half-antibody monomers or homodimer impurities. Figure 7 shows the binding activity of the bispecific antibody to the antigen as measured by ELISA. Figure 8 shows the binding affinity constants of the bispecific antibody to FcRn and FoFoyRI as measured by Fortebio. Detailed Description of the Invention Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. Furthermore, the materials, methods, and examples described herein are illustrative only and are not intended to be limiting. Other features, objects, and advantages of the invention will be apparent from this specification and the accompanying drawings and from the appended claims. The term "about" when used in conjunction with a numerical value means to encompass a range of numerical values ​​having a lower limit of 58% less than the specified numerical value and an upper limit of 59% greater than the specified numerical value. In this document, the terms "comprising" or "including" mean including the stated element, integer, or step, but do not exclude any other element, integer, or step. For example, when referring to the Fc RAS CH domain, it encompasses the Fc region consisting of or primarily consisting of the CH3 domain, and also the Fe region that also contains other constant domains such as CH2. In this document, RIE Fo RRA, a scaffold protein dimer consisting of or primarily consisting of paired and dimerized immunoglobulin and white Fc regions.The polymeric structure serves as a scaffold, allowing for the attachment or ligation of other functional molecules at its N-terminus or C-terminus, including, but not limited to, binding domains, biologically active peptides or fragments thereof, and toxins. In some respects, this scaffold can be particularly used to construct multispecific (e.g., bispecific) antibodies or single- or multispecific binding proteins with domains that differ from the first peptide in at least one amino acid residue. In some aspects, the first and second polypeptides of the heteropolymeric protein include polymerizing domains (e.g., the first and second CHB domains of the CH3 heterodimer according to the invention; the first and second Fo regions of the RARE AR A ROR MA Fo scaffold), wherein the polymerizing domains facilitate the interaction of the first and second polypeptides at the interface and the formation of a "heterodimer". In some aspects, the heteropolymeric protein may include a "heterodimer" formed by the first and second polypeptides, or other polypeptides may be present in addition to the first and second polypeptides to form a higher-order tertiary structure. Examples of heteropolymeric proteins include, but are not limited to, monospecific or multispecific binding proteins, such as bispecific antibodies, bispecific immunoadhesins, or antibody / immunoadhesin complexes. As used herein, the terms "binding protein" and "binding molecule" are used interchangeably and, in their broadest sense, refer to a protein molecule that specifically binds to at least one target. An example of a binding protein is an antibody and an immunoadhesin. "ETP, RIK Fo scaffold-binding protein" refers to a protein containing a Fo scaffold and at least one target bound to it.A binding protein, or a binding protein consisting of or primarily of a Fo scaffold and at least one target-binding domain connected thereto. In some embodiments, the binding protein comprises one (and only one) Fc scaffold. In other embodiments, the binding protein may comprise more than one Fc scaffold. In this document, the term "single-specific" means that the binding molecule is capable of specifically binding to one and only one specific target (e.g., one epitope). The term "multi-specific" means that the binding molecule is capable of specifically binding to at least two different targets (e.g., at least two different epitopes). Typically, a multi-specific binding molecule comprises at least two target-binding domains that are specific to different targets. In some embodiments, the different targets may be polypeptides or proteins or portions thereof located on the same cell, different cells, the same antigen, or different antigens, such as receptors, ligands, or antigenic epitopes. In some implementations, the multispecific binding molecule is bispecific, capable of simultaneously binding to two different targets, such as two different epitopes, for example, two different epitopes expressed on two different cells, or two different epitopes on two different antigens, or two different epitopes on the same antigen. Hereinafter, the term “valence” indicates the presence of a specific number of target-binding domains in the binding molecule. Therefore, the term “monovalent” associated with the binding molecule indicates the presence of one (and no more than one) target-specific binding domain in the binding molecule. Accordingly, the term “multivalent” indicates the presence of multiple target-specific binding domains in the binding molecule. For example, a “tetravalent” binding molecule refers to…The binding molecule contains a total of four target-specific binding domains, regardless of whether the targets targeted by these binding domains are the same. Hereinafter, the term "target-binding domain" refers to a portion of a polypeptide or protein that provides interaction with a target. Exemplary binding domains include an antigen-binding domain of an antibody, a ligand, a receptor-binding domain of a ligand, and a ligand-binding domain of a receptor. In some aspects of the invention, a preferred target-binding domain is the antigen-binding domain of an antibody. The antigen-binding domain of an antibody typically contains amino acid residues from the complementarity-determining region (CDR) of the antibody. Natural immunoglobulin molecules typically have two antigen-binding domains, and Fab molecules typically have a single antigen-binding domain. Hereinafter, the term "antigen-binding domain" refers to an antibody or fragment thereof that specifically binds to an antigen epitope. In some specific embodiments, the antigen-binding domain comprises an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL), or, when the antibody is a heavy chain antibody, comprises a single heavy chain variable region (VHHD). In some embodiments, the antigen-binding domain may also comprise an antibody constant region. Available heavy chain constant regions include any one of the following five types: o, β, s, γ, or he. Available light chain constant regions comprise any one of the following two types: K and λ. Thus, in some aspects, a specific antigen-binding domain may be selected from, including, but not limited to: antibody fragments comprising an antibody heavy chain variable region (VHD) and an antibody light chain variable region (VD), such as Fv, scFv, Fab, scFab, crossFab, and antibody fragments comprising a heavy chain variable region of a heavy chain antibody, such as VHH. In this document, the term "antigen determination and determination" is used in conjunction with..."Epitope" is used interchangeably and refers to a site on a macromolecule, such as a polypeptide or protein, that interacts with an antigen-binding domain to form a complex. In some aspects, an epitope can be, for example, a linear epitope consisting of consecutive amino acid segments, or a conformational configuration consisting of discontinuous amino acid residues located in different regions. According to the invention, useful antigens can be, for example, on the surface of tumor cells, virus-infected cells, other diseased cells, immune cells, or present in free macromolecules in body fluids and / or the extracellular matrix (ECM). In this document, the term "antigen" refers to a macromolecule that can elicit an immune response in mammals, thereby producing specific antibodies against it. In some aspects, useful antigens according to the invention are polypeptides or proteins, including, but not limited to, naturally occurring proteins and their variants, fragments, or derivatives derived from vertebrates. The vertebrates include mammals, such as primates, for example, humans. In this document, the term "immunoglobulin" refers to a protein having a structure that naturally presents antibodies. For example, IgG immunoglobulins are heterotetrameric glycoproteins of approximately 150,000 Daltons, composed of two light chains and two heavy chains linked by disulfide bonds. From the N-terminus to the C-terminus, each immunoglobulin heavy chain has a heavy chain variable region (VH), also called a heavy chain variable domain, followed by three heavy chain constant domains (CH1, CH2, and CH3), also called heavy chain constant regions. Similarly, from the N-terminus to the C-terminus, each immunoglobulin light chain has a light chain variable region (VL), also called a light chain variable domain, followed by a light chain constant domain (CL), also called a light chain constant region. In IgG antibody molecules, typically...The VH-CH1 region of the heavy chain pairs with the VL-CL region of the light chain to form a Fab fragment that specifically binds to the antigen. Therefore, an IgG immunoglobulin is essentially composed of a Fab molecule and two dimerized Fc regions. The heavy chain of an immunoglobulin can be classified into one of three types based on the type of its constant domain, called α(IgA), β(IgD), γ(IgE), γ(IgG), γ(IgM), and some of these categories can be further subdivided into subtypes, such as γ1(IgG1), γ2(IgG2), γ3(IgG3), γ4(IgG4), γ3(IgA2), γ4(IgA2), and γ5(IgA2). The light chain of an immunoglobulin can also be classified into one of two types based on the amino acid sequence of its constant domain, called K and γ6. In this document, the term "antibody" refers to a polypeptide containing one or more domains that bind to epitopes on antigens of interest, wherein the binding domain has a variable region sequence derived from or sequence-identical to immunoglobulins. The term encompasses a wide range of antibody structures, including but not limited to, single-chain antibodies, single / multispecific antibodies, mismatched antibodies, humanized antibodies, human sequence antibodies, full-length antibodies, and antibody fragments, as well as antigen-binding polymers and proteins assembled from them, provided they exhibit the desired antigen-binding activity. Typically, antibodies form antigen-binding domains on the surface of a VH-VL dimer via three complementarity-determining regions (HCDR1-3) in their heavy chain variable region (VH) and three complementarity-determining regions (LLCDR1-3) in their light chain variable region (VDL). The six CDRs confer specific binding of the antibody to the antigen. However, in the case of heavy chain antibodies, such as those derived from camelid heavy chain antibodies, the antibody can communicate through three complementary molecules within its single VH domain (also referred to as the VHH domain in this paper).The determining regions (CDR1-3) confer the specific binding of the antibody to the antigen. The VHH domain, like the variable regions of the heavy and light chains of a conventional IgG antibody, contains four conserved framework regions (FR) and three complementary determining regions (CDR), arranged in the order of FR1-CDR1-FR2-CDR2-FR3-CD3-FR4. In this text, "complementary determining region" or "CDR region" or "<CDR" or "hypervariable region" refers to the region in the variable region of the antibody that is highly variable in sequence and forms structurally defined loops (<hypervariable loops>] and / or contains antigen-contact residues (:antigen contact points). CDRs are mainly responsible for binding to the antigen epitope. In the VHH domain and the VHVVL domain of the antibody of the present invention, the CDRs are sequentially numbered starting from the N-terminus and are commonly referred to as CDR1, CDR2, and CDR3. One can use the well-known methods in the art to determine the CDR sequences in a given VHH domain and a given VHVAVL domain. A person skilled in the art can easily determine the CDR sequence ranges of any given amino acid sequence of the antibody variable region on http: / / www.abysis.org / abysis / , including the region ranges and their combined ranges of CDRs defined by the Kabat, AbM, Chothia, Contact, and IMGT methods. Unless otherwise stated, and otherwise in the present invention, the term "CDR" or "CDR sequence" encompasses the CDR sequences determined in any of the above ways and their combinations, but preferably the CDR sequences defined according to the Kabat method. In this text, the term "antibody fragment" refers to the non-integral part of an antibody that contains an antigen-binding domain. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab’, Fab’-SH, F(ab’)2, diabody, linear antibody, single-chain antibody (e.g., scFv,scFab and single-domain antibodies (sdAb). The amino acid sequence of VH or VL in the antibody fragment can be modified by substitution, deletion, addition, and / or insertion, as long as the antigen binding energy is maintained. Furthermore, the variable regions of the antibody fragment can be conjugated and humanized. In this paper, "Fv domain" refers to the smallest antibody fragment including both the complete antigen recognition and binding sites. "Fw is a dimer formed by a VH and a VL bond strongly bound together by non-covalent bonds (VH-VL = RA). In this paper, the term "scFv domain" refers to a single-chain polypeptide in which two variable regions (usually a VH and a VL) are linked via a linker to form an antigen-binding domain necessary for antigen binding. In this paper, the term "Fab BHAA, AMP RIL eG" refers to an antigen-binding domain in antibodies formed by the pairing of the heavy chain variable region VH and the heavy chain constant region CHI (VH-CHI1) with the complementary light chain variable region VL and the light chain constant region CL (VL-CL). In this paper, the term "crossFab" or "crossFab domain" refers to a Fab domain in which CHI and CL are exchanged, i.e., an antigen-binding domain formed by the pairing of VH-CL and VL-CHI1. In this paper, the term "ScFab" refers to a Fab domain linked as a single polypeptide chain by an artificial linker." In this paper, the term "VHH" or "VHH domain?" is used to refer to the variable heavy chain domain derived from heavy chain antibodies lacking a light chain. Therefore, VHHs differ from the conventional VHs of four-chain immunoglobulins in that they do not require pairing with a light chain variable domain to form an antigen-binding domain. Such VHH molecules can be derived from antibodies produced in camelid species (e.g., camels, alpacas, dromedaries, llamas, and guanacos). Other species besides camelids also...Antibodies against heavy chains that are naturally lacking the light chain can be generated; such VHHs are also within the scope of this invention. In some cases, for the therapeutic application of VHHs, it is desirable to reduce their immunogenicity. Therefore, preferably, the VHH domain used in the binding protein of this invention comprises a humanized sequence. Hereinafter, “immunoglobulin constant domain” means a constant domain derived from or obtained from or derived from the immunoglobulin heavy chain (e.g., human IgG1 heavy chain) or light chain, including heavy chain constant domains CH1, CH2, CH3, and optionally CH4; and light chain constant domain CL. This term includes both native and variant sequence constant domains. Hereinafter, the “type” or “subtype” of an immunoglobulin constant domain refers to the type or subtype determined based on its amino acid sequence. Heavy chain constant domains can be classified into five different types based on their sequence: IgA, IgD, IgE, IgG, and IgM, or further into subtypes (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. Therefore, in this document, reference to the IgG1 Fe region or CH3 domain means that the Foe region or CH3 domain, based on its amino acid sequence, can be classified as IgG type, and can be further classified as IgG1 subtype. Similarly, light chain constant domains can be classified into KK and CL light chain domains based on their sequence. Those skilled in the art can readily determine the type or subtype to which the constant domain belongs by comparing the amino acid sequence of the constant domain with the sequences of the corresponding constant domains of different types or subtypes of natural immunoglobulins. In this document, when the heterodimeric Fo scaffold or heteromultimeric protein molecule of the present invention contains multiple immunoglobulin heavy chain constant domains, it should be understood that it can...The selection is made independently of each other based on the intended function or use of the molecule. As an example, for the heteropolymer of the present invention containing a CHI1 domain and CH2 and CH3 domains, the three constant domains can all be of the IgG1 subtype, such as human IgG1, or only the CH3 domain or the CH2 and CH3 domains can be of the IgG1 subtype, such as human IgG1. In this document, “IgG conformation” means that the antibody conformation is the same as that of IgG immunoglobulin, essentially consisting of two Fab domains linked to the N-terminus of a dimerized Fe domain via an immunoglobulin and sclerochain region (or, where appropriate, via a flexible linker peptide). Thus, in a typical case, an IgG-like antibody consists of two heavy chains and two light chains, where each heavy chain has VH, CH1, CH2, and CH3 domains at its AN-SiC terminal; and each light chain has VL and CL domains from its N to C terminals. In this paper, "IgG-like conformation" refers to an antibody that retains the same stereotyped structural features as IgG immunoglobulin, but with one Fab domain missing, and / or at least one Fab domain replaced by a binding domain of a different conformation (e.g., scFv, VHH, ligand, or receptor ligand-binding domain). Such IgG-like conformation antibodies can be described by indicating the type of binding domain attached to the Fo-dimer scaffold. For example, VHH-Fc / Fc is a monovalent IgG-like conformation antibody composed of VHH-EFc 4 Jk Fe Fe peptides; VHH-Fc / Fab-Fc antibody is a bivalent IgG-like conformation antibody composed of VHH-Fc peptides and Fab-Fc peptides. In this paper, the term "immunoadhesin" refers to an antibody formed by the desired binding of a molecule to an immunoadhesin.An antibody-like protein molecule is formed by the fusion of a specific non-immunoglobulin-binding domain (e.g., a binding domain derived from a cell surface receptor or ligand, or the ligand itself) with an immunoglobulin constant domain (e.g., the Fo region or CH3 domain). The immunoglobulin constant domain can be derived from any immunoglobulin, such as IgG, especially IgG1, IgG2, IgG3, or IgG4. In this document, the term "FcMBCAFo region" is used to define an immunoglobulin and albumin heavy chain constant region portion containing the CH3 domain or a fragment thereof. In some cases, the immunoglobulin and albumin portion may also contain one or more other immunoglobulin constant domains or fragments thereof, including the CH4 or CH2 domains. In this article, when referring to amino acids in the Fc region and constant domains, the EU numbering system (also known as the EU index) described in Kabat et al., Sequences of Proteins of Immunological Interes, 1991, Public Health Service, National Institutes of Health, Bethesda, MD, 1991, is used. When referring to the Fc region of human IgG, the EU numbers for the amino acid residues can also be found in the IMGT Scientific chart (https: / / www.imgtore / IMGTScientificCharVNumbering / Hu_IGHGnberphtoml), which is hereby incorporated for reference. According to this numbering, in the human IgG1 immunoglobulin heavy chain, amino acids 118-215 are CH2 domains, amino acids 216-230 are CH2 domains, and amino acids 231-340 are CH3 domains. In some cases, the CH3 domain C Kae BER (Lys447) may not exist. Those skilled in the art can readily understand this.Sequence alignment with the published constant regions of natural immunoglobulins was used to determine the constant domains contained in the heterodimer Fc region and binding protein according to the present invention, as well as their types / subtypes and species origins. In some embodiments, the first and second Fe regions of the heterodimer Fc scaffold according to the present invention comprise or consist of CH3 domains. In other embodiments, the Fo region also comprises a CH2 domain. In still other embodiments, the Fo region also comprises a homogeneous region or a partially sharp-chain region, for example, including the lower sharp-chain portion of the tungsten sequence “CPPCP”. In this document, as those skilled in the art will understand, the CHB heterodimer of the present invention is a specific form of the heterodimer Fc scaffold according to the present invention, in which the first and second Fe regions of the Fo scaffold consist of or are substantially composed of CH3 domains. Therefore, unless explicitly stated otherwise, the description of the heterodimer Fc scaffold of the present invention is also equivalently applicable to the CH3 heterodimer according to the present invention. In this paper, the term "Fc CEH BOA Fe region" encompasses both native and variant Fc regions. The term "native sequence Fo region" encompasses the naturally occurring Fc region sequences of various immunoglobulins, such as the Fc region sequences of various Ig subtypes and their allotypes (Gestur Vidarsson et al., IgG subclasses and allotypes: from structure to effector functions, 20 October 2014, doi: 10.3389 / fimmu.2014.00520.). "RAAF Fe region" refers to the Fo region polypeptide relative to the native sequence FeO RSMAS MH. The modification can be the addition, deletion, or substitution of amino acid residues. Substitutions can include naturally occurring and variant amino acid residues.And non-naturally occurring amino acids. The purpose of modification can be to alter the physicochemical properties of the Fe region, such as thermal stability and heterodimerization tendency, and / or the binding of the Fc region to its receptor and its effector function. In this paper, “Fo scaffold” is also referred to as “Fec scaffold dimer” or “dimer Fo RR”. ALP, RIE RO RG’ Fe scaffold 7 WO 2025 / 092987 PCT / CN2024 / 129416 means that the first and second Fc regions constituting the Fo scaffold are sequence-identical. Accordingly, a “heterodimer” Fec scaffold means that the first and second Fo scaffolds constituting the Fc scaffold are RAPALA EY-PRRRARE FH. In this paper, the term “CH3 dimer” refers to a pair of paired and dimerized CH3 domains. The term "CH3 MARA, AP, FOCOR" refers to a dimer in which the first and second CD3 domains are identical in sequence. Correspondingly, "CH3 heterodimer" refers to a dimer in which the first and second CD3 domains differ in sequence by at least one amino acid residue. In this paper, when a Fo-based heteropolymeric protein (e.g., a mono / multi-specific binding protein) encapsulates only one Fc scaffold, such a multimeric protein can be considered, based on the two Fo regions of the scaffold, as one Fo member of the scaffold and a linked polypeptide (e.g., a target-binding domain, which can be 0, 1, or more), constituting one protein unit (i.e., monomer) of the dimer; while the other Fe member of the Fe scaffold and a linked polypeptide (e.g., a target-binding domain, which can be 0, 1, or more), constitute the other protein unit (i.e., monomer) of the dimer. In this case, it should be understood that the dimer...Each monomer can be a single or multiple polypeptide chains. When two monomers are identical in sequence structure, i.e., copies of each other, the dimer is referred to herein as a homodimer. For example, native IgG immunoglobulin is a typical example of this homodimer. When two monomers are different, for example, differing in the Fo sequence and / or the binding domain sequence linked thereto, the dimer is referred to herein as a heterodimer. For example, bispecific IgG antibodies or IgG-like antibodies are typical examples of such heterodimers. Mono / multispecific antibodies based on heterodimer Fe scaffolds are another typical example of such heterodimers. In this document, the expression "purity" associated with the heteropolymeric protein according to the invention (e.g., the binding protein according to the invention) refers to the ratio of the heteropolymeric protein to the total protein in the purified product after expression and purification from host cells. The purity of the target heteropolymeric protein can be determined by measuring the ratio of the target heteropolymeric protein product to various chain mismatch products in the purified product using SEC-HPLC. Preferably, the purity is greater than 90%. In this document, the expression "thermal stability" related to the heteropolymeric protein according to the invention (e.g., the binding protein according to the invention) refers to, as determined by DSC, the protein exhibiting a CH3 Tm value greater than 705°C; or, when the protein is an antibody in the IgG conformation, exhibiting a CH3 Tm value similar to that of natural human IgG1 immunoglobulin (e.g., +25°C, preferably +15°C). For the heteropolymeric protein of the invention based on a heterodimeric Fe scaffold, preferably, the Tm value is obtained without introducing non-natural diacid bonds into the heterodimeric Fo scaffold.The mutations were determined. In this paper, when referring to a single mutation, it is described by the position of the amino acid residue at which the mutation occurred and the amino acid residues before and after the mutation, denoted as [original and amino acid residues] mutated residue position [mutated amino acid residues]. For example, the substitution of threonine at position 366 of the Fc region with chromoic acid is denoted as T366W; the substitution of uric acid at position 409 of the Fe region with chromoic acid is denoted as Y407V. When referring to multiple mutation combinations, mutation combinations located on the same polypeptide chain are connected by the symbol ", for example, the mutation combination of L351Y and D399R occurring in hole #14 can be represented as "L351Y-D399R"; for mutation combinations located on different polypeptide chains, the symbol "1" is used to separate them, for example, the combination of L351Y and D399R mutations occurring on the hole chain and the region 409D mutation occurring on the knob chain can be represented as "hole L351Y-D399R / knob K409D". In this document, the terms “flexible linker peptide” or “peptide linker” are used interchangeably and refer to a short amino acid sequence consisting of amino acids, such as glycosyl (G) and / or serine (S) and / or threonine residues (T) used alone or in combination, or homogeneous regions of immunoglobulins or their modified forms. In this document, the term “modifier” refers to a modified or derivative of a binding protein formed by covalently linking or modifying other molecules (e.g., therapeutic or diagnostic molecules) to a target protein (e.g., a heteropolymeric protein or binding protein according to the invention). Examples of such other molecules include, but are not limited to, proteins / peptides / peptides, markers, drugs, and cytotoxic agents, such as: radioisotopes; chemotherapeutic agents; growth inhibitors; enzymes and fragments thereof; and fluorescent reporters.Proteins; antibiotics; toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including their fragments and / or variants; and various known antitumor or anticancer agents. In this document, the "percentage of identity (%)" of an amino acid sequence refers to the number of positions in the comparison window where the candidate sequence shares the same amino acid residues as the specific amino acid sequence, without considering any conserved substitutions as part of the sequence identity, after comparing the candidate sequence with the specific amino acid sequence in a comparison window, and, if necessary, introducing vacancies to achieve the maximum percentage of sequence identity, without considering any conserved substitutions as part of the sequence identity, and calculating its percentage of the total number of residues in the comparison window. Unless otherwise specified, the comparison window is the full length of the specific amino acid sequence. In this document, when referring to the constant domains CH2, CH3, and Fc, the term "wild-type" means that the constant domain has a sequence derived from the constant domain of a native immunoglobulin, or has no more than 1-5 amino acid residue changes compared to the native sequence (preferably no more than 1, 2, 3, 4, or 5 amino acid residue changes, more preferably conserved amino acid substitutions). For polypeptide sequences, “conservative modifications” include substitutions, deletions, or additions to the polypeptide sequence that result in the replacement of a certain amino acid with a chemically similar amino acid. A table providing a list of conserved substitutions for functionally similar amino acids is known in the art. The following eight groups contain JKRs with mutually conserved substitutions: 1) 75 BURA; HERG); 2) Aspartic acid (D), glutamic acid (E); 3) Asparagine (N), glutamic acid (Q); 449 BARR), lysine (K); DHS ARD, RRL; P RAM; MARV); 6) Propionic acid.(F), acetic acid (Y), chromoic acid (W); 7) serine (S), threonine (T); and 8) cysteine ​​(C), formic acid (M) (see, for example, Creighton, Proteins (1984)). In this document, the term “host cell” refers to a cell into which exogenous polynucleotides have been introduced, including progeny of such cells. Host cells include “transformers” and “transformed cells,” which include primary transformed cells and their derived progeny. Host cells are any type of cell system that can be used to produce the polypeptides or protein molecules of the present invention, including eukaryotic cells, such as mammalian cells, cytokinesiocytes, yeast cells; and prokaryotic cells, such as Escherichia coli cells. Host cells include cultured cells, as well as cells within transgenic animals, transgenic plants, or cultured plant or animal tissues. In this document, the term "expression vector" refers to a vector containing recombinant polynucleotides, which contains an expression control sequence that effectively links the nucleotide sequence to be expressed. The expression vector contains sufficient cis-acting elements for expression; other elements for expression may be provided by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, including but not limited to viscera, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses). In this document, the term "individual" refers to a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., human and non-human primates such as monkeys), rabbits, and hazelnuts (e.g., mice and ravens). In particular, the individual is a human. In this document, the term "treatment" refers to the intention to alter a disease in an individual receiving treatment.Clinical intervention of natural processes. Desired therapeutic effects include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or mitigating the disease state, and alleviating or improving prognosis. In this document, the terms “cancer” and “tumor” are used interchangeably to refer to or describe physiological disorders in mammals that are typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinomas, solid tumors, and liquid tumors. As shown in the examples, the HARA Fe scaffold, by introducing the specific Fc mutation combination of the present invention, after expression in cells and purification by affinity chromatography, produces a Fo heterodimer ratio of over 90%, which is higher than that of the control Fc protein containing only the tungsten-corresponding knob-into-hole mutation; and after further purification by ion exchange column, it can reach OBS UALR. HE, MET TIRE Fe EG. The purified Fo heterodimer has better thermal stability, wherein the Tm value of the CH3 domain determined by DSC method can reach over 74°C. In a first aspect, the present invention provides a heterodimer Fe scaffold that can be used to enhance the formation of a target heterodimer. By using the heterodimeric scaffold according to the present invention, the yield of the desired heteropolymorph product relative to unwanted heteropolymorph and homopolymorph impurities can be greatly improved, thereby increasing the production efficiency and cost of the desired heteropolymorph; simultaneously, the thermal stability of the desired heteropolymorph product is improved. The heterodimeric Fc scaffold according to the present invention comprises two paired and heterodimerized immunoglobulin Fc regions, wherein the two Fe regions distinguish the WEL CH3 domain. The heterodimeric Fo scaffold according to the present invention...The feature is that the CH3 AHP, RRS knob-into-hole (KIEHD) mutation, and also includes the following mutations: (a) the Fe region with the &4 hole mutation also includes the L351Y mutation; or (b) Fe Rik #4 L351Y with the 4 hole mutation and Fe KLM K409D with the D339R RE, HEL knob mutation. In this document, in some cases, for the sake of brevity, the above combination of mutations is also referred to as "the CH3 interface characteristic mutation of the present invention". Similarly, for the sake of brevity, the Fe region with the hole mutation and the polypeptide containing it are also referred to as the hole chain; and the Fo region with the corresponding knot mutation and the polypeptide containing it are referred to as the knot chain. Accordingly, when referring to other mutations introduced on the hole chain, the mutation is preceded by the above description "hole?" The term "hole" is used to specify that the mutation occurs on the hole chain; similarly, when referring to other mutations introduced on the knob chain, the expression "knob" or "*knob" is added before the mutation to specify that the mutation occurs on the knob chain. For example, hole L351Y refers to the substitution of a residue at the hole # Fe K L351 position; knob K409D refers to the substitution of a residue at the knob K409 position in the Fe region of the knob chain. Knob-into-hole (KIH) mutation techniques are known in the art. See, for example, US7951917B1; US ​​5,731,168; US 7,695,936, Ridgway et al., Prot Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001). Typically, this mutation technique involves the Fe of the first polypeptide. A protrusion (CFR knob FE) is introduced into the CH3 domain interface of the second polypeptide and the Fe region is then introduced.A corresponding cavity (hole mutation) is introduced into the CH3 domain interface, allowing the protrusion to be positioned within the cavity to promote the formation of heterodimers of the first and second polypeptides and inhibit homodimer formation. The protrusion can be constructed by replacing the small amino acid side chains from the interface of the first polypeptide with larger side chains (e.g., tyrosine or tryptophan). By replacing the large amino acid side chains with smaller amino acids (e.g., alanine or threonine), a complementary cavity of the same or similar size to the protrusion can be created at the interface of the second polypeptide. Such combinations of spatially and size-complementary protrusion and cavity mutations are referred to in the art as KIH mutations, wherein the protrusion mutation is called a knob mutation of the KIH mutation, and the cavity mutation is called a hole mutation of the KIH mutation. In one specific embodiment, the Fc scaffold according to the invention comprises a knock mutation in the CH3 domain of one of the first and second Fe regions of the Fe scaffold; the CH3 domain of the other of the first and second Fe regions of the Fe scaffold contains a complementary hole mutation. In a more specific embodiment, in the CH3 domain of one of the first and second Fo regions (e.g., the first Fe region) of the Fo scaffold according to the invention, an amino acid residue substitution is introduced, such that one or more amino acid residues are substituted with amino acid residues having a larger side chain volume, thereby creating a protrusion in the CH3 domain of the Fc region; simultaneously, in the CH3 domain of the other of the first and second Fc regions (e.g., the second Fe region) of the Fe scaffold according to the invention, an amino acid residue substitution is introduced, such that one or more amino acid residues are substituted with amino acid residues having a smaller side chain volume, thereby creating a protrusion in the CH3 domain of the Fc region.A cavity spatially complementary to the aforementioned protrusion is generated in the CH3 domain of the region, thereby allowing a protrusion present in the CH3 domain of one Fe region to be localized in a cavity present in the CH3 domain of another Fo region. The protrusion and cavity can be prepared by altering the nucleic acid encoding the polypeptide, for example, by site-specific induction or by peptide synthesis. In some more specific embodiments, the Fo scaffold according to the invention contains a KIH mutation, wherein the introduced knob mutation is T366W, and the introduced hole mutation is Y407V. In other more specific embodiments, the Fe scaffold according to the invention contains a KIH mutation, wherein the introduced knob mutation is T366W, and the introduced hole mutation is T366S-L368A-Y407V. In some embodiments, the Fe region of the heterodimeric Fc scaffold according to the invention is composed of or primarily of CH3 domains. In this embodiment, the invention thus provides a CH3 HORA according to the invention. In some embodiments, in addition to the CH3 domain, the heterodimorphic Fc scaffold according to the invention, or the heteropolymeric protein according to the invention comprising said scaffold, further comprises other immunoglobulin constant domains, for example, CH2 and / or homopolymer regions in the Fc region, and / or CH1 and / or CL domains linked to VH or VL in the target-binding domain. In such embodiments, these immunoglobulin constant domains may have the same or different species origin (but preferably all from humans), and / or have the same or different immunoglobulin types or subtypes (but preferably the same IgG type or IgG subtype). In one embodiment, the Fc scaffold according to the invention comprises CH2 and CH3 domains from IgG (especially human IgG) immunoglobulins. In one embodiment...In this embodiment, the Fo scaffold according to the invention further comprises a sharp-chain region and / or a CH1 domain from IgG (especially human IgG) immunoglobulin. As those skilled in the art will understand, reference to the species origin and type of a constant domain of immunoglobulin means that the constant domain comprises a natural and amino acid sequence or a variant sequence of immunoglobulin from the stated species and type, wherein amino acid variations in the variant sequence do not affect the determination of the species origin, type, or subtype of the constant domain. Typically, for the purposes of this invention, the variant sequence has a variation of no more than 10 amino acid residues relative to the corresponding natural sequence. As an example, the structural characteristics of various IgG subtypes can be found in the review by Gestur Vidasson et al. (IgG Subclasses and Allotypes: From Structure to Effector Functions, Front Immunol. 2014; $: S20, doi: 10.3389fimmu.2014.005$20), which is incorporated herein by reference in its entirety. In some embodiments, the immunoglobulin constant domain contained in the heterodimeric Fo scaffold or the heteropolymeric protein body of the present invention is a constant domain of IgG immunoglobulin. In some embodiments, the constant domain may be independently selected from different IgG subtypes, for example, IgG1, IgG2, IgG3 and IgG4 LA, KALA IgG subtypes or IgG4 subtype. In some embodiments, the constant domain is a human-derived IgG immunoglobulin constant domain. In some specific embodiments, the heterodimeric Pe scaffold according to the present invention comprises an IgG Fe region. In some more...In specific embodiments, the Fc region is an IgG1 Fc region. In other specific embodiments, the Fe region is an IgG4 Fc region. In some embodiments, the Fo region of the heterodimeric Fe scaffold according to the invention has an effector function. In some embodiments, the effector function is Foy receptor binding and / or FcRn receptor binding. In some embodiments, the Foy receptor is human FcyRIIa, FoyRI, and / or FeyRIIIa, especially FeyRI. In some embodiments, the heterodimeric Fo scaffold according to the invention (or binding molecules containing the heterodimeric Fo scaffold) has a binding affinity of not less than 60%, for example not less than 50%, 40%, 30%, 20%, 10%, HI Foy receptor binding affinity, compared to the natural IgG1-RAK Fe domain (or binding molecules containing the natural IgG1-RAK Fo domain). In some embodiments of the EP, the PR Foy receptor is FeyRI. In some embodiments, with regard to binding to the PRN receptor, the heterodimeric Fo scaffold (or binding molecules comprising said heterodimeric Fo scaffold) according to the invention exhibits an FcRn binding affinity substantially similar to that of the native IgG1 dimer Fe domain (or binding molecules comprising the native IgG1 RMR Fe domain), i.e., more than 70%, particularly more than 80%, and more particularly more than 90%, of the FcRn binding affinity of the native IgG1 dimer Fe domain. The Fe domain can confer favorable pharmacokinetic properties to heteropolymer proteins (e.g., bispecific antibodies) containing it, including prolonged serum half-life, contributing to good accumulation in target tissues and favorable tissue-blood partition ratios. However, the EFc domain may also cause heteropolymer proteins (e.g., bispecific antibodies) containing it to...For example, bispecific antibodies target cells expressing the Foy receptor more often than cells and / or tissues carrying the target. Furthermore, activation of the Fecy receptor signaling pathway can lead to cytokine release, resulting in severe side effects from heteropolymer proteins containing the Fe domain after systemic administration. In some embodiments, therefore, in addition to the CH3 interface characteristic mutation of the present invention, the RAW MHI Fo scaffold may contain or not contain other mutations as needed to maintain or affect its (one or more) effector functions. For example, it is known that the upper CH2 domain and sharp chain region of an antibody are involved in multiple effector functions, such as Foy LH binding and effector functions such as ADCC; some CH2 and CH3 interface residues of the antibody are involved in FcRn receptor binding. In some cases, the Fec region used in the heterodimeric scaffold of the present invention may be modified by additional mutations including the introduction of a CH2 domain and / or a homogeneous region (if present), RAE (ERA), or one or more desired effector functions, such as the binding affinity of the Fc region to the Foy receptor and / or FcRn. Amino acid modifications are known in the art, including, but not limited to, amino acid substitutions at one or more positions selected from E233, L234, L235, N297, P331, and P329, and more specifically, substitutions at one or more amino acids selected from E233P, L234A, L235A, L235E, N297A, N297D, and P331S. In some cases where the Fo scaffold has a human IgG1 Fe region, the binding of the Fe scaffold to the Foy receptor can be substantially reduced by including L234A-L235A mutations, L234A-L23SA-P329G mutations, and / or N297A mutations in the Fe region. In this invention, a Fo scaffold according to the invention is provided.Some exemplary sequences of the scaffold. In some embodiments, the heterodimeric Fo scaffold of the present invention comprises a first polypeptide and a second polypeptide, wherein the first polypeptide comprises a first Fe region and the second polypeptide comprises a second Fc region, wherein: (a) relative to the wild-type CH3 domain, the CH3 domains of the first and second Fe regions respectively, in addition to the characteristic CH3 interface mutation of the present invention described above, further comprise 0-3 amino acid residue changes; preferably, the wild-type CH3 domain is a natural IgG (especially IgG1 or IgG4, preferably human IgG1) immunoglobulin CH3 domain, more preferably comprising the amino acid sequence of SEQ ID NO: 19 or 20; (b) the first and second Fec regions comprise a natural IgG (especially IgG1 or IgG4, preferably human IgG1) immunoglobulin CH2 domain, or comprise the amino acid sequence shown in SEQ ID NO: 21 or a CH2 domain having 15 amino acid residue changes therewith; 11 WO 2025 / 092987 PCT / CN2024 / 129416 (c) &4 knob-mutated Fe region contains SEQ ID NO:1 &2HARBRPNRERAA EY 95%, 96%, 97%, 98% HK 99% 7] — EH R FF FI (d) &* hole-mutated Fe RL SEQ ID NO: 7 or 8 amino acid sequence or amino acid sequence having at least 95%, 96%, 97%, 98% or 99% double identity with it, and / or (e) the Fe scaffold does not form non-natural disulfide bonds between the first Fe region and the second Fe region. In some preferred embodiments, the knob-mutated Fe R&S SEQ ID NO: 1 or 2 and the CH3 domain containing the amino acid sequence shown or the amino acid sequence having at least 99% double identity with it; and the hole-mutated Fe R&S SEQ ID NO:The knockb-mutated Fe region comprises a CH3 domain of an amino acid sequence of 7 or 8 PARK or an amino acid sequence having at least 95% identity with it. In some embodiments, the knockb-mutated Fe region comprises a CH3 domain of the amino acid sequence shown in SEQ ID NO: 1 or 2, and the hole-mutated Fe region comprises a CH3 domain of the amino acid sequence shown in SEQ ID NO: 7 or 8. In some embodiments, the knockb-mutated Fe region comprises a CH3 domain of the amino acid sequence shown in SEQ ID NO: 1, and the hole-mutated Fe region comprises a CH3 domain of the amino acid sequence shown in SEQ ID NO: 7. In some embodiments, the knockb-mutated Fe region comprises a CH3 domain of the amino acid sequence shown in SEQ ID NO: 2, and the hole-mutated Fe region comprises a CH3 domain of the amino acid sequence shown in SEQ ID NO: 8. In any of the above embodiments, the Fo region containing the knob mutation may be the first Fe region and the Fc region containing the tungsten hole mutation may be the second Fc region, or vice versa. In some embodiments, the heterodimeric Fc scaffold according to the invention does not contain non-RRM, AMAA ROAR CE, ite, ARE DSC Me, CH3 domains with a Tm value of 705°C or higher that introduce the CH3 domain interface between the first and second Fo regions. In some embodiments, the heterodimeric Fe scaffold according to the invention has better thermal stability compared to the corresponding control Fc scaffold with the same KIH mutation. In this document, "corresponding control Fo scaffold" refers to a heterodimeric Fe scaffold that is identical in all sequence structures (including KIH RE) except for having unmutated natural residues at positions 1L351, D366, and K409, compared to the heterodimeric Fe XR of the invention.In some embodiments, the heterodimeric Fe scaffold according to the present invention has comparable purity to the corresponding control Fc scaffold having the same KIH mutation. In some embodiments, after expression, assembly, and purification from host cells under the same conditions, the purity of the heterodimeric Fo scaffold according to the present invention is at least 90%, 95%, 100%, 110%, or higher than that of the control Fo scaffold, and preferably greater than 90% as determined by SEC-HPLC. In a second aspect, the present invention provides a heteropolymer protein comprising a CH3 heterodimer according to the invention or a heterodimer Fc scaffold according to the invention. In some embodiments, the heteropolymer protein according to the invention comprises at least a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide respectively contain first and second tungsten CH3 domains, wherein the first and second CH3 domains in the first and second polypeptides meet at the interface and form a CH3 HOORE according to the invention. In some embodiments, the heteropolymer protein according to the invention comprises at least a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide respectively contain first and second tungsten Fe regions, wherein the first and second Fe regions in the first and second polypeptides meet at the interface and form an Fc scaffold according to the invention. In some embodiments, the heteropolymer protein is a single or multiple specific binding protein comprising at least one target-binding domain, wherein the at least one target-binding domain is attached to a CH3 heterodimer according to the invention or a heterodimer Fc scaffold according to the invention. In some embodiments, the heteropolymer protein according to the invention andThe white scaffold comprises one or more of the aforementioned Fo scaffolds. In some embodiments, the Fo scaffold is called an EK HRS RARE LER Feo scaffold. In this document, such single or multiple specific binding proteins of the Fo scaffolds of the present invention are also referred to as "Fo scaffold-based binding proteins". AF Fe SRWBAEA 12 WO 2025 / 092987 PCT / CN2024 / 129416 In some aspects, therefore, the present invention provides binding proteins based on the heterodimeric Fe scaffolds of the present invention. In some embodiments, the binding protein according to the present invention comprises at least one target-binding domain connected to the Fc scaffold. The type of target-binding domain used in the binding protein of the present invention is not specifically limited, as long as it has the ability to bind to a target. Such binding domains, as known in the art, can originate from binding pairs, including non-immunoglobulin-based binding pairs such as receptor / pair binding pairs, enzyme / substrate pairs, or immunoglobulin-based binding pairs such as antigen / antibody binding pairs, antibody / anti-idiotype antibodies. When the target binding domain originates from an immunoglobulin-based binding pair, the binding protein containing it can be an antibody, such as a mono / multispecific antibody. When the target binding domain originates from a non-immunoglobulin-based binding pair, the binding protein containing it can be an immunoadhesin. Furthermore, the invention also considers binding proteins containing binding domains from both of these different types of binding pairs; such antibody / immunoadhesin syncytiotroph proteins are sometimes referred to herein as antibodies. In some embodiments, the binding protein according to the invention comprises at least one antigen-binding domain. Antigens that can bind to the antigen-binding domain according to the invention can be selected from, for example, but not limited to: tumor-associated antigens, immunoassay antigens, and other antigens.Checkpoint molecules, angiogenic factors, or combinations thereof. Tumor-associated antigens may include, but are not limited to: MUCI-1, BCMA, CLDN18.2, HER2, BRAF, EGFR, CD20, CD38, FolR1, and CD5S2. Immune checkpoint molecules may include, but are not limited to: PD-L1, PD-1, PD-L2, CTLA-4, B7-H3, TIM3, LAG-3, VISTA, ICOS, 4-1BB, OX40, GITR, and CD40. Angiogenic factors may include, but are not limited to: basic FGE, HGE, Stie-2, Svegfr-1, Svegfr-2, EGF, IL-6, IL-8, PLGF, VEGF, PDGF-bb, ANG1, ANG2, SDF-1a, MDC, Galectin, TSP-1, Endocan, Enos, and HIF-1w. In some embodiments, the antigen is selected from tumor-associated antigens, such as EGFR and HER2. In some embodiments, the binding protein according to the invention encapsulates at least one non-immunoglobulin binding domain. In some implementations, examples of such non-immunoglobulin-binding domains that are available include, but are not limited to, ligand-binding domains from the following receptor proteins: 41BB; adrenocorticotropic hormone receptor; activin receptor; BLIR (4H = 3% B4 receptor); BMP receptor; C3a receptor; CSa tt: chemokine receptor; cytokine receptor; growth hormone receptor; BILA; interferon-α LH; FREP LA: FaR-γ CA: type I I-1 receptor; UII A; IL10 eA; IL-1L receptor; IL-12 receptor; BCMA; TACI; BAFF receptor; immunomodulatory signaling receptor CD72; Kaposi's sarcoma-associated herpesvirus GPCR; MA A4 receptor; lymphotoxin receptor; RON LA; SCE LA; AKAPR LA; TIUST2; TGF-β receptor; IP tumor necrosis factor receptor;TNFRSF19; erythropoietin receptor; leukemia inhibitory factor receptor; and C-kit receptor. In some implementations, examples of such non-immunoglobulin-binding domains available include, but are not limited to, the body-binding domains of ligands or the ligands themselves derived from: orMSH; 9E3 / cCAEF; adrenocorticotropic hormone; BER; AK155; angiogenesis inhibitors; Apo2L / TRAIL; BLR1 ligand / BCA-LUBLC / CXCL13; calcitonin gene-related peptide; CD27 ligand; CD30 ligand; CD40 ligand; endorphin; endostatin; erythropoietin; Fas ligand; Flt3 ligand; G-CSF; GCP-2 / CXCL6; GM-CSF; various cytokines, such as IFNa, IFNB; interferon Y; IL-1a; L-1p; IL-10; IL-11; LIL-12; IL-13; IL-15; IL-16; IL-2; IL-27; IL-3; IL-4; IL-5; IL-6; IL-7; IL-8 / CXCL8; IL-9; Somatostatin; Stem cell factor; Substance P; TARC / CCLI7; TCA3 / CCL1; TECK / CCL25; TGFB; Thrombopoietin; TNFα. In some embodiments, the non-immunoglobulin-binding domain is derived from proteins selected from: VEGE receptor; TNE 4A IL-1; lymphocyte function-associated antigen 3 (LFEA-3 / CDS8); lymphotoxin B receptor (LTBR); CTLA-4; IL-12; activin receptor; TACI; BR3; T cell receptor; CD4; L-selectin; piracetam receptor; CD44; NP LA; FAK yCA; 41BB fe IGE LK. In other embodiments, the non-immunoglobulin-binding domain is derived from the receptor-binding domain of a ligand selected from: IL-1, IL-12. Other available non-immunoglobulin-binding domains...Examples can also be found, for instance, US7951917B1 and US20220275048A1. Regarding the connection of the target-binding domains, in the binding protein according to the invention, the connection location of the target-binding domains on the Fc scaffold is not particularly limited. In some embodiments, the N-terminus or C-terminus of the first and / or second polypeptide of the scaffold is mentioned. In some embodiments, at least two or more, or all, of the at least one target-binding domains are optionally linked to each other via peptide linkers and thus to the heterodimeric Fc scaffold according to the invention. In other embodiments, at least two or more, or all, of the at least one target-binding domains are optionally separately linked to different ends of the Fo scaffold via peptide linkers. For example, when the binding protein contains two binding domains, one binding domain may be attached to the N-terminus of the first polypeptide of the Fo scaffold, while the other binding domain may be attached to a different end of the Fo scaffold, such as the C-terminus of the first polypeptide, the N-terminus of the second polypeptide, or the C-terminus of the second polypeptide. Therefore, in the binding protein according to the invention, at least one, two, three, or all of the four ends of the heterodimeric Fc scaffold (i.e., the N-terminus and C-terminus of the first and second polypeptides) may each be attached to at least one (preferably one or two) target binding domains. The at least one target binding domain contained in the binding protein may be the same as or different from each other. In one embodiment, at least two, more, or all of the at least one target binding domain are the same. In other embodiments, the at least one target binding domain is...At least two, more, or all of the target-binding domains are distinct from each other. In some embodiments, the binding protein is monospecific and has one or more binding domains for that specificity. In some embodiments, the binding protein is multispecific, and preferably, has one or more binding domains for each specificity. In some embodiments, the at least one target-binding domain is an antigen-binding domain independently selected from Fab, VHH, scFv, scFab, and crossFab. When the binding domain is Fab or crossFab, the Fab or crossFab can optionally be linked to another binding domain of the binding protein or an Fc scaffold, for example, to the N-terminus of the first or second polypeptide of the heterodimeric Fc scaffold according to the invention, via a peptide linker, through the C-terminus of a chain containing a VH domain or the C-terminus of a chain containing a VL domain. In some embodiments, at least one of the at least one target-binding domains is a VHH domain. In some embodiments, at least one of the at least one target-binding domains is a Fab domain. In other embodiments, at least one of the at least one target-binding domains is a VHH domain, and at least one of the at least one target-binding domains is a Fab domain. In other embodiments, at least two, more, or all of the at least one target-binding domains are Fab domains, and the Fab domains have the same or different VLCL light chains. In some embodiments, the binding protein is a single-specific monovalent protein containing a VHH domain, and preferably the VHH domain is linked to...The binding protein is attached to the N-terminus of the first or second polypeptide of the Fe scaffold. In some embodiments, the binding protein is a bispecific divalent binding protein containing VHH and / or Fab binding domains. In some embodiments, the binding protein is a bispecific antibody containing first and second Fab domains and an IgG conformation of the Fe scaffold, preferably, the first and second Fab domains are respectively linked to the C-terminus of the first and second polypeptides of the Fo scaffold via the C-terminus of their chains containing VH domains. In some embodiments, the binding protein is a bispecific antibody containing VHH and Fab in an IgG-like conformation, wherein the antibody consists of a heavy chain containing a VHH-Fc domain, a heavy chain containing a VH-Fc domain, and a light chain containing a VL-CL domain. In this document, the term "linkage" used when referring to different components in the scaffold or binding protein means that the components are fused or combined directly or via a suitable linker. Peptide linkers typically used for this purpose are short amino acid polypeptides with a certain degree of flexibility, generally between 1 and 50 amino acid residues in length, but not limited to this. In some cases, any chain of amino acid residues located between the two components can be considered a peptide linker, provided it does not affect the intended function of the two components being linked. Therefore, in some aspects, the heterodimer FeRR according to the invention, or the CH3 heterodimer according to the invention, linked to a polypeptide of interest, for example, to a target-binding domain according to the invention, or other functional polypeptide or protein domains, via a peptide linker, is within the scope of this invention. In the heteropolymeric protein according to the invention, those skilled in the art can readily determine the available [linkages] based on the components to be linked and the linking sites.Linker sequence. In some embodiments, the linker is used to connect the Fc scaffold according to the invention and the antigen-binding domain; and / or to connect two antigen-binding domains. In some embodiments, the available linker is a flexible linker peptide of $S-$0 amino acids, preferably containing glycosyl (G) and / or serine (S) and / or threoic acid residues (T). In some embodiments, the linker has an amino acid length of 5-30 amino acids, for example, 8, 10, 15, 20, 25 or 30 amino acid lengths, or has an amino acid length falling between any two integers. In some embodiments, the linker comprises an amino acid sequence (G4S)n, where n is an integer equal to or greater than 1, for example, n is an integer of 2, 3, 4, 5, 6 or 7. In a preferred embodiment, the linker consists of an amino acid sequence (G4S)n. In other embodiments, the linker is a homogeneous region of an immunoglobulin or a derivative thereof. Other examples of available linkers include, for example but not limited to, the following amino acid sequences: (GlysSer)>(SEQ ID NO: 22), (Gly4Ser)2 (SEQ ID NO: 12), (Gly3Ser)3 (SEQ ID NO: 24), (Gly,Ser)3 (SEQ ID NO: 25), (Gly3Ser)4 SEQ ID NO: 26), (Gly4Ser)4 (SEQ ID NO: 27), (Gly3Ser)s (SEQ ID NO: 28), (Gly4Ser)s (SEQ ID NO: 29), (Gly3Ser)s (SEQ ID NO: 30), (Gly4Ser)s (SEQ ID NO: 31), GGG (SEQ ID NO: 32), DGGGS (SEQ ID NO: 33), TGEKP (SEQ ID NO: 34), GGRR (SEQ ID NO: 35), EGKSSGSGSESK VD (SEQ ID NO: 36),KESGSVSSEQLAQERSLD (SEQ ID NO: 37), GGRRGGGS (SEQ ID NO: 38), LRQRDGERP (SEQ ID NO: 39), LRQKDGGGSERP (SEQ ID NO: 40), and GSTGSGSGKPGSGEGSTKG (SEQ ID NO: 23). Alternatively, if necessary, suitable flexible linker peptides can be rationally designed by simulating the three-dimensional structure of proteins and peptides using computer programs or by phage display methods. In a preferred embodiment, a peptide linker having the sequence shown in SEQ ID NO: 12 is used to link the Fe scaffold and its N-terminal antigen-binding domain, such as VHH or Fab. In some cases, chemically synthesized linkers can also be used to attach the binding domain to the Fe scaffold. In this case, the connection is also referred to as a "bridging". Examples of chemically synthesized connectors include: N-succinimide (NHS), disuccinimide succinate (DSS), bis(sulfosuccinimide succinate) (BS3), dithiobis(succinimide propionate) (DSP), dithiobis(sulfosuccinimide propionate) (DTSSP), ethylenedioxybis(succinimide succinate JEGS), ethylenedioxybis(sulfosuccinimide succinate) (Sulfo-EGS), disuccinimide tartrate (DSTD), disulfosuccinimide tartrate (Sulfo-DST), bis[2-(succinimide succinate)ethyl] bis(2-(sulfosuccinimide)ethyl) Sulfo-BSOCOES et al. When multiple adapters are used in the heteropolymeric protein of this invention, it is understood that these adapters may be the same as or different from each other. In some cases, chemically synthesized adapters may also be used to...Synthetically synthesized therapeutic agents (e.g., small toxin molecules) or diagnostic agents are incorporated into the heteropolymeric protein of the present invention, such as bispecific antibodies, for example, onto the glycan chain of the CH2 domain. Such slowed compounds are also present in the TEPER S RAE of the present invention. In some embodiments, the heteropolymeric protein of the present invention is a multispecific binding protein comprising binding to different antigenic epitopes, particularly a bispecific antibody. Bispecific antibodies having binding specificity against at least two different antigens or antigenic epitopes have broad clinical application potential, serving as targeted drugs for in vitro and in vivo immunodiagnostics and therapy, and for diagnostic immunoassays. See, for example, WO9850431A2. Bispecific antibodies can be used in in vitro assays to detect the functional properties of cell surface molecules and determine cytotoxicity mediated by different Fc receptors (Fanger et al., CritRevImmunol.12:101-124 (1992)); in enzyme-linked and immunoassay assays (Nolan et al., Biochem. Biophys. Acta. 1040:1-11 (1990); Hammerling et al., J. Exp. Med. 128: 1461-1473 (1968)); and for the immunodiagnosis of various diseases, such as cancer, in vitro or in vivo (Songsivilai et al., Clin. Exp. Immunol.79:315 (1990)). To facilitate the diagnostic use of BsAbs, one arm of a BsAb can bind to diseased tissue or cell surface antigens (e.g., tumor-associated antigens), while the other arm can bind to detectable markers, such as quinone binders that tightly bind radionuclides. In therapeutic applications, bispecific antibodies can be used to guide a patient's cellular immune defense mechanisms specifically against diseased tissues / cells, such as tumor cells, or pathogens. For this purpose, one arm of a BsAb can be linked to an immune system.One arm binds to surface antigens of cells (e.g., cellular, NK cells), while the other arm can bind to surface antigens of diseased tissues / cells. In some embodiments, the multispecific antibody according to the invention is a heterodimer consisting of or primarily of a heterodimer Fo scaffold according to the invention and at least one antigen-binding domain attached thereto, having a configuration selected from the following: -(antigen-binding domain)n-Fc / (antigen-binding domain)n-Fc, -(antigen-binding domain)n-Fc-(antigen-binding domain)ny(antigen-binding domain)n-Fc, -(antigen-binding domain)n-Fc-(antigen-binding domain)nmy(antigen-binding domain)n-Fc-(antigen-binding domain)m, wherein each of the above nm and α is independently selected from integers of 0, 1, and 2; 15 WO 2025 / 092987 PCT / CN2024 / 129416 Each of the above antigen-binding domains is independently selected from: Fv, scFv, Fab, scFab, crossFab, VHH, 8274 2% 4 The ligand-binding domain; the N-terminal linker of αFo"KAREFe; the C-terminal linker of RES"FoRPEFe; wherein the two monomers of the heterodimer are separated by the symbol " / "; wherein one of the two Fe is preferably a knob chain and the other is a hole chain. In some preferred embodiments, n is 1 and α is 0. In some preferred embodiments, n is 0 and α is 1. In some embodiments, n is 1 and α is 1. In some preferred embodiments, each of the above antigen-binding domains is independently selected from: scFvVHH, and Fab. In some specific embodiments, the multispecific heterodimer protein according to the invention is bispecific and has an IgG conformation or an IgG-like conformation. In some specific embodimentsIn this invention, the multispecific heterodimeric protein is a bispecific antibody and has a conformation selected from the following: -SCFEV-Fc / scEv-Fc, wherein two scFv domains for different specificities are respectively linked to the N-terminus of the Fc scaffold according to the invention; -Fab-FwWFab-FEc, wherein two Fab domains for different specificities are respectively linked to the N-terminus of the Fc scaffold according to the invention; -VHH-Fe / VHH-Fv, wherein two VHH domains for different specificities are respectively linked to the N-terminus of the Fe scaffold according to the invention; -scFv-Fe / Fab-Fc, wherein two scFv domains for different specificities and the Fab domain are respectively linked to the N-terminus of the Fe scaffold according to the invention; -SCFEWVHH-Fc, wherein two scFv domains for different specificities and the VHH domain are respectively linked to the N-terminus of the Fe scaffold according to the invention; -VHHVFab-FEc, wherein two scFv domains for different specificities and the Fab domain are respectively linked to the N-terminus of the Fe scaffold according to the invention; -VHHVFab-FEc, wherein two scFv domains for different specificities and the Fab domain are respectively linked to the N-terminus of the Fe scaffold according to the invention. The domains are respectively attached to the N-terminus of the Fc scaffold according to the invention. In embodiments of the heteropolymeric protein according to the invention comprising two or more Fab domains targeting different specificities, for example when the heteropolymeric protein is a bispecific antibody with a Fab-Fc / Fab-Fc configuration, light chain mismatch can be prevented by using a common light chain. See, for example, US7951917B. Alternatively, the first Fab-Fc hapten and the second Fab-Fc hapten can be generated separately, and then assembled in vitro to form the desired heterodimer by adding a suitable reducing agent to their mixture. In some embodiments, the heteropolymeric protein of the invention is a bispecific antibody comprising a VHH domain and a Fab domain that respectively bind to different antigens.In some embodiments, the bispecific antibody binds to HSA and B7-H3. In some embodiments, the VHH domain comprises the CDR1, CDR2, and CDR3 sequences of the VHH amino acid sequence shown in SEQ ID NO: 11; or comprises the amino acid sequence shown in SEQ ID NO: 11, or an amino acid sequence having at least 95% identity with it. In some embodiments, the Fab domain comprises VH and VL, wherein the VH comprises the VH shown in SEQ ID NO: 13 and the CDR1, CDR2, and CDR3 sequences of the amino acid sequence; or comprises the amino acid sequence shown in SEQ ID NO: 13, or an amino acid sequence having at least 95% identity with it; and wherein the VL #4 comprises the VL shown in SEQ ID NO: 14 and the CDR1, CDR2, and CDR3 sequences of the amino acid sequence; or comprises the amino acid sequence shown in SEQ ID NO: 14, or an amino acid sequence having at least 95% identity with it. In some embodiments, the VHH domain comprises the amino acid sequence of SEQ ID NO: 11; and the Fab comprises the VH of the sequence shown in SEQ ID NO: 13 and the VL of the sequence shown in SEQ ID NO: 14. In some embodiments, the bispecific antibody is a bivalent antibody. In some embodiments, the VHH and Fab domains are respectively linked to the N-terminus of the first and second polypeptides of the heterodimeric Fc scaffold according to the present invention. In some embodiments, the binding protein of the present invention is a bispecific antibody containing the first and second Fab domains of different antigens respectively. In some embodiments, the bispecific antibody binds EGFRVHER2. In some embodiments, the first Fab domain comprises VH and VL, wherein the VH comprises the VH and amino acid sequence shown in SEQ ID NO: 15.The CDR1, CDR2, and CDR3 sequences in SEQ ID NO: 15, or amino acid sequences having at least 95% identity with them; and wherein the CDR1, CDR2, and CDR3 sequences in the VL and amino acid sequences shown in SEQ ID NO: 16, or amino acid sequences having at least 95% identity with them; and wherein the VL #4 SEQ ID NO: 16 VL and amino acid sequences contain the CDR1, CDR2, and CDR3 sequences; or contain the amino acid sequences shown in SEQ ID NO: 16, or amino acid sequences having at least 95% identity with them. In some embodiments, the second Fab domain comprises VH and VL, wherein the VH comprises the CDR1, CDR2, and CDR3 sequences of the VH amino acid sequence shown in SEQ ID NO: 17; or comprises the amino acid sequence shown in SEQ ID NO: 17, or an amino acid sequence having at least 95% identity with it; and wherein the VL comprises the CDR1, CDR2, and CDR3 sequences of the VL amino acid sequence shown in SEQ ID NO: 18; or comprises the amino acid sequence shown in SEQ ID NO: 18, or an amino acid sequence having at least 95% identity with it. In some embodiments, the first Fab domain comprises the VH sequence shown in SEQ ID NO: 15 and the VL sequence shown in SEQ ID NO: 16; and the second Fab comprises the VH sequence shown in SEQ ID NO: 17 and the VL sequence shown in SEQ ID NO: 18. In some embodiments, the bispecific antibody is a bivalent antibody. In some embodiments, the first and second Fab domains are respectively linked to the N-terminus of the first and second polypeptides of the heterodimeric Fo scaffold according to the invention. IV. SRR. BA, BE In some aspects, the present invention provides one or more CHB HORA, HORA RR, HRERKEGR binding proteins according to the invention.A polynucleotide; one or more vectors containing said one or more polynucleotides, wherein said one or more polynucleotides may be present in a single vector or in multiple separate vectors. In other aspects, the invention also provides host cells containing said one or more nucleic acids or said one or more vectors. Depending on the circumstances, a single or multiple vectors containing said one or more nucleotides may be introduced into the same host cell to express the desired product in one host cell; or a single or multiple vectors containing said one or more polynucleotides may be introduced separately into different host cells to express intermediates that produce different chains or combinations of different chains (e.g., different monomeric proteins) of the heteropolymer according to the invention in different host cells, and, under suitable conditions for assembling the heteropolymer according to the invention, the heteropolymer according to the invention is produced by mixing said intermediates. In the invention, there are no particular limitations on the expression vectors that can be used, including but not limited to viruses, plasmids, viscera, bacteriophages, or yeast artificial chromosomes (YAC). In the invention, there are no particular limitations on the host cells that can be used. Suitable host cells include prokaryotic microorganisms such as *Escherichia coli*, eukaryotic microorganisms such as filamentous fungi or yeast, or various eukaryotic cells such as mammalian host cells, cytokine cells, etc. Examples of useful mammalian host cell lines include SV40-transformed monkey kidney CV1A (COS-7), ABEK (HEK293 or 293F 20 2), 44He (BHR), monkey bone cells (CV1), African green monkey bone cells (VERO-76), human cervical cancer cells (HELA), canine kidney cells (MDCK), Buffalo rat liver cells (BRL 3A), and so on.Human lung cells (W138), human liver cells (HepG2), CHO cells, NSO4, YO2, NSO4, P3X63, and Sp2 / O, etc. In some embodiments, mammalian cell lines suitable for catalytic culture can be used. In some preferred embodiments, the host cells are CHO or HEK293 cells. V. Production and Purification of the Heteromeric Protein of the Invention In another aspect, the present invention provides a method for producing the heteromeric protein of the present invention. To produce the heteromeric protein of the present invention, the polypeptide chain of the heteromeric protein of the present invention can be obtained, for example, by solid-state peptide synthesis (e.g., Merrifield solid-phase synthesis) or recombinant production, and assembled under suitable conditions. As mentioned above, in the case of recombinant production, the assembly can occur for expressing the heteromeric protein SANG EMP, or, if necessary, the assembly of the heteromeric protein can be performed in vitro after obtaining the intermediate polypeptide chain for expression from the host cells. In one embodiment, the present invention provides a method for producing the heteropolymeric protein of the present invention, the method comprising: culturing a host cell containing a polypeptide chain encoding the polypeptide chain under conditions suitable for expressing the heteropolymeric protein; and assembling the polypeptide chain to produce the heteropolymeric protein under conditions suitable for the assembly of the polypeptide chain into the heteropolymeric protein. In some embodiments, the method comprises: culturing a host cell containing a polypeptide chain encoding the polypeptide chain under conditions suitable for expressing the heteropolymeric protein; and recovering the heteropolymeric protein produced by the host cell from the cell culture.Preferably, the purity of the heteropolymeric protein according to the present invention, as determined by SEC-HPLC after protein A affinity chromatography, is greater than 80%, preferably greater than 85%, and more preferably greater than 90%. In other embodiments, the method includes: culturing host cells containing a polypeptide chain encoding the heteropolymeric protein under conditions suitable for expressing the polypeptide chain; and obtaining an intermediate of the heteropolymeric protein from the host cells in the cell culture; mixing the intermediate under conditions suitable for assembling the heteropolymeric protein, and recovering the resulting heteropolymeric protein. In the case of heterodimeric proteins, monomers containing fnob chains and monomers containing hole chains are expressed in different host cells, and the heteropolymeric protein is generated and recovered from the mixture of the monomers under conditions suitable for assembly. Preferably, after ion exchange chromatography, the recovered product, determined by SEC-HPLC, has a purity greater than 80%, preferably greater than 85%, and more preferably greater than 90% for the heteropolymeric protein according to the present invention. The antibody prepared by the method described herein can be purified using known prior art techniques such as high-performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, etc. After purification, the purity of the heteropolymeric protein of the present invention can be determined by any of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high-performance liquid chromatography, etc. The physical / chemical properties and / or biological activity of the heteropolymeric protein provided herein can be identified, selected, or characterized by various assays known in the art. VI. GroupCompositions and Uses This invention also relates to a composition comprising the heteropolymeric protein of the invention (e.g., a binding protein according to the invention). In embodiments where the composition is a pharmaceutical composition, the composition further comprises a pharmaceutically acceptable carrier. As used herein, “pharmaceutically acceptable carrier” includes any and all physiologically compatible solvents, dispersion media, isotonic agents, and absorption delay agents, etc. In this invention, a pharmaceutical composition generally refers to a drug for the treatment or prevention of a disease or for the examination or diagnosis of a disease. In some preferred embodiments relating to the pharmaceutical compositions of the invention, the heteropolymeric protein of the invention is a bispecific antibody, for example, a bispecific antibody specifically targeting a disease-associated antigen. In some embodiments, the heteropolymeric protein of the invention is the sole active ingredient in the pharmaceutical composition. In other embodiments, the pharmaceutical composition may comprise the heteropolymeric protein described herein with one or more other therapeutic agents. The pharmaceutical compositions of the invention can be formulated according to methods well known to those skilled in the art. Furthermore, pharmaceutically acceptable carriers or media, such as sterile water or physiological saline, vegetable oils, emulsifiers, flocculating agents, surfactants, stabilizers, flavoring agents, excipients, solvents, preservatives, binders, etc., can be appropriately combined with the heteropolymer protein of the present invention to formulate a unit dose form suitable for drug administration. For example, a pharmaceutically acceptable carrier can be used to prepare a sterile solution or suspension for non-gastrointestinal administration in the form of an injection. The amount of active ingredient in the formulation will be set to produce an effective amount within a predetermined range after administration to an individual. The pharmaceutical compositions of the present invention are suitable for various...Various routes of administration, including but not limited to intravenous, intramuscular, subcutaneous, parenteral, rectal, intravenous, or epidermal administration (e.g., by injection or infusion), are permitted. Accordingly, the pharmaceutical compositions of the present invention can be formulated into dosage forms for administration via the intended route, such as injectable, nasal, pulmonary, or transdermal dosage forms. For injectable dosage forms, routes of administration may include, but are not limited to, intravenous, intramuscular, intraperitoneal, subcutaneous, or intratumoral administration, either systemically or locally. Furthermore, the specific method of administration of the pharmaceutical compositions of the present invention may be appropriately selected based on the patient's age and symptoms. Additionally, the detection or diagnostic reagents for heterologous multisomal proteins described herein are also within the scope of the present invention. The reagent may include one or more other elements, such as: instructions for use; other reagents, such as markers or reagents for conjugation; a pharmaceutically acceptable carrier; and a device or other material for administration to a subject. The following examples are described to aid in understanding the invention. It is not intended and should not be construed in any way as limiting the scope of protection of the present invention. 18 WO 2025 / 092987 PCT / CN2024 / 129416 Examples Example 1 Design Fe mutant candidate combination sequences Search for knob-into-hole (KIHD) Fe heterodimer homopolymer structures (e.g., 4NQS and SID8) from protein databases (www.pdb.org) and compare them with wild-type IgG1 Fe crystal structures (e.g., 1HZH) to analyze the reasons for the reduced thermal stability of the KIH model. The KIH mutation reduces the number and strength of amino acids in contact between the two CH3 domains. In wild-type IgG1 Fe, some key amino acids at the CH3-CH3 domain contact interface form a downholeThe core, including L351, L368, Y407, etc., covers a large surface area (see Figure 1A). However, in the KIH model, because L368A and YY407V mutate into small side chains RAM, BARR SH Ro A Bae) CORA 1B). In addition, the contact between some amino acids near the core is also affected. For example, in wild-type IgG1 Fe, the K409 side chain of one CH3 domain is in direct contact with the amino acids L368, K370, D399, F405, and Y407 of another CH3 domain; in particular, K409 forms a complex hydrogen bond network with D399, S364, T411, and a water molecule, while preventing electrostatic clash between K409 and K370. However, in the KIH structure, the K409WHS to T366W mutation in the Knob chain results in the loss of direct contact with 1L368A and Y407V, and also the loss of stable water molecules and their mediated hydrogen bond network. To enhance the CH3 interaction strength and improve thermal stability, we selected amino acids near the Fc contact interface in the KI model as mutation targets, and finally designed a series of Fo heterodimer mutation combinations. The specific amino acid mutation information is shown in Table 1. The KIH combination is the handle-hole mutation combination disclosed in patent US7951917B1. M1-M7 are newly designed candidate mutation combinations. Table 1. Design of Fo heterodimer mutant combinations, where chain A is the chain containing the knob mutation and chain B is the chain containing the corresponding hole mutation. | are ea Y-D399R a ee ee nee i See Oc a baa K Y-D399K Example 2 VHH-Fe / Fe heterodimer expression and purification In this example, to verify the effect of the Feo mutant combination on the table in promoting heterodimer formation, a VHH-Fe fusion molecule with chain A and a Fo-100 chain with chain B were first designed.Based on this, VHH-Fe and Fe genes were synthesized, expression plasmids were constructed, and the culture supernatant was collected after transfection of 293F cells. The supernatant was purified using Protein A affinity, and the purified product was analyzed using SDS-PAGE and SEC-HPLC methods to determine the content of heterodimers and other impurities, thus identifying the effects of different mutation combinations on the expression yield and purity of VHH-Fc / Fc heterodimers. The expression vector was constructed based on the constant region amino acid sequence (POLST) of human immunoglobulin IgG from the Uniprot protein database, including the ABP, CER, CH2, and CH3 domains. Then, according to Table 1, a total of 10 Fc REKAKM FF (3. SEQ ID NO:1 2 SEQ ID NO:10) were designed. A VHH fragment (derived from the Ozoralizumab antibody, see SEQ ID NO:11) and a flexible linker polypeptide CL (SEQ ID NO:12) were introduced to the N-terminus of the @ A ## Fe fragment to construct the A-chain VHH-Fc fusion molecule; simultaneously, the B-chain Fc fragment remained unchanged. The molecular weight difference between VHH-Fc and Fc is beneficial for distinguishing the proportions of AB FPR-FR AK, AA / BB homodimers, and A / B EARARD TERK (HMWS) formed during recombinant expression. Using codons optimized for mammalian / human expression, the A-chain VHH-Fc and B-chain Fc DNA encoding genes were designed, cloned into the expression vector pcDNA3.4 (Invitrogem), and plasmids for expressing VHH-Fc and Fe were obtained. Gene synthesis and plasmid construction were performed by Genwiz Suzhou Co., Ltd. For 293F cell transfection, VHH-Fc and Fe plasmids were mixed at a molar ratio of 1:1 or 0.6:1, and then L / 10 transfection volume of OPM-CD Trans293 medium was added.Optimum Biotech, P82019). FectoPRO transfection reagent (PolyPlus, PT-116-010) was added to the culture medium at a ratio of 2 ml / 1 h cells. The culture medium containing the plasmid and transfection reagent was mixed and allowed to stand for 10 min. The mixture was then added to Expi293F cells (Invitrogen, A14635) and cultured with shaking at 437°C and 8% CO2. 16 h after transfection, 10 D KK-volume feed (Optimum Biotech, F081918-001) was added, and the cells were cultured for another 6 days. Protein A was purified by equilibrating 1 ml Protein A Diamo d chromatography column (Borglon, AA0273) with 5 column volumes of equilibration solution (20 mM sodium phosphate, pH 7.4). Heqing's cell culture medium was loaded onto the chromatography column at a flow rate of 0.5 column volumes / min. The packing material was then rinsed sequentially with 5 column volumes of equilibration buffer (20 mM sodium phosphate, pH 7.4) and pre-elution buffer (20 mM acetate-sodium acetate, pH 2). Finally, elution was performed with 10 column volumes of eluent (20 mM acetate-sodium acetate, pH 3.2), and the collected eluent was adjusted to pH 6.0 with 1.5 SM Tris base. For SDS-PAGE analysis, the purified product from the Sug Protein A column was mixed with non-reducing loading buffer (DTT-free) and run on 10% SurePAGE Fh BK (Genscript, M00666). After electrophoresis, the gel was stained with Coomassie Brilliant Blue and destained before being photographed using a gel imaging system. The results are shown in Figure 2. The co-expression products of VHH-Fc and Fo plasmids mainly contained five proteins of different molecular weights: AHIR monomer, AA homodimer, BB homodimer, A monomer, and BB monomer. Under a plasmid ratio of 1:1, the KIH control, M1, M2, M5S, and M6 combinations showed higher heterodimer content (greater than 90%), and M3 showed 127% heterodimer content.[M4, M7 BAW HIRAS ERIK, £ 80-90% if]. Under a plasmid ratio of 0.6:1, the heterodimer content of combinations M1, M3, MS, M6, and M7 was higher, greater than 90%, while the heterodimer content of combinations KIH, M2, and M4 was lower, between 80-90%. The results indicate that combinations MI1 and M6, compared to KIH, have a higher ability to promote AB heterodimer formation and inhibit AA / BB homodimer formation. Combination M2, compared to the KIH control, has a similar ability to promote AB heterodimer and AA / BB homodimer formation. Combination M3, compared to the KIH combination, has a similar ability to promote AB heterodimer formation, but combination M3 induces more AA homodimers and fewer BB homodimers. SEC-HPLC analysis was performed on a Theromo Vanquish Core high-performance liquid chromatograph. Protein A column purified at 1 mg / ml was analyzed using SEC Mix. The column was a Waters BioResolve SEC mAb (2.5µm, 7.8x300mm). Isocratic elution was performed using VA PBS as the mobile phase for 30 minutes at a flow rate of 0.5 ml / min. The detection wavelength was 280 nm, the column temperature was 20 °C, the injection volume was 10 μL, and the temperature control plate temperature was 5 °C. The results are shown in Table 2. Overall, the dimer proportions calculated by SEC-HPLC analysis were similar to those obtained by SDS-PAGE analysis. Under a plasmid ratio of 1:1, the heterodimer content of the KIH control, M1, M2, MS, and M6 combinations was high, greater than 90%, while the heterodimer content of the M3, M4, and M7 combinations was low, between 80-90%. Under a plasmid ratio of 0.6:1, the heterodimer content of the M1, M3, MS, M6, and M7 combinations was...The heterodimer content was high, greater than 90%, while the heterodimer content of the 122% KIH, M2, and M4 combination was low, between 80-90%. # 2 VHH-Fe / Fe mutant group was analyzed by SEC-HPLC using the following plasmid ratios: RAF | SEC purity | Plasmid ratio expression level | SEC purity (mg / ml) (%) (mg / ml) (%). Based on the above results, it can be seen that introducing the porous 1L3S1Y RE on the basis of KIH does not significantly change the heterodimer and homodimer formation ratio. Introducing the 409D / D399R double mutant on the basis of KIH-L351Y does not significantly change the heterodimer ratio, but induces more AA MOR and fewer BB homodimers. Kl 3 VHH-Fe / Fe heterodimer thermal stability analysis. Heterodimer purification: 1 ml Mono S ion exchange column <Cytiva, 17516801) Wash with equilibration buffer (20 mM sodium phosphate, pH 6.0) for 10 column volumes or to baseline. Protein A affinity purified samples were adjusted to pH 2-6.0 with 1.5 mM Tris base or 50 mM sodium acetate (pH 3.2), and loaded onto a Mono 5 column at a flow rate of 0.5 column volumes / min. Then wash with equilibration buffer for 10 column volumes. Finally, elute with elution buffer (20 mM sodium phosphate + 1 mH L sodium acetate, pH 6.0) in a 0-30% gradient, collecting the eluent in separate tubes. The heterodimer purity of each tube was determined by SEC-HPLC, and samples with a purity greater than 98% were combined. The thermal stability of the samples was analyzed using a Microcal PEAQ-DSC. The method was as follows: Replace the protein sample with buffered PBS, #482 lmg / mL. The detection temperature range was set to 20-100°C, with a heating rate of 90 Hz. PBS was added to the assay cell as a blank buffer, and temperature scanning was performed. Then, the solution in the sample cell was removed, and the sample to be tested was added.Temperature scanning was performed. The experimental data were processed using the instrument's built-in analysis software to calculate the Tm values. The Tm measurement results for the VHH-Fe / Fe mutant combinations are shown in Table 3 and Figure 3. The KIH control had at least two Tm values. The first Tm value (Tm1) was 65.68°C, reflecting the thermal stability of the VHH fragment and CH2 domain, while the second Tm value (Tm2) was 71.48°C, reflecting the thermal stability of the CH3 domain. The Tm values ​​for the M1 and M6 mutant combinations were only 67.56°C and 66.31°C, respectively, significantly lower than the KIH control. These results indicate that introducing electrostatic redirection design based on the KIH structure unexpectedly reduces the thermal stability of the CH3 domain. Interestingly, the CH3 domain Tm value of the M2 mutant combination reached approximately 74.03 sC, an increase of about 25°C compared to the KIH control; while the CH3 domain Tm value of the combination with the full M3 mutant was even higher, reaching approximately 75.84°C. The single amino acid mutation AREA, BH Fc L3S1Y can improve the stability of the CH3 domain in the KIH form; furthermore, this mutation has a synergistic enhancing effect with A-chain FcK409D and B-chain FcD399R AAMR EMS, further improving the stability of the CH3 domain. Other mutant combinations, including M4, MS5, and M7, showed poor thermostability, with Tm values ​​all less than 70*C. These results indicate that introducing A-chain L351Y or B-chain L351Y-D399K into the KIH base does not improve the thermostability of the CH3 domain. #& 3 VHH-Fe / Fe mutant combination Tm value Example 4 VHH-Fc / Fab-Fe heterodimer expression and purification To verify the universality of the mutant combination in Example 2 across different antibody sequences and molecular forms, VHH-Fc / Fab-Fc heterodimer was also constructed.The fusion protein consists of an A-chain VHH-Fc fusion protein. A VHH antibody fragment (amino acid sequence see SEQ ID NO:11) and a flexible linker polypeptide CR (SEQ ID NO:12) are introduced at the human end of the first Fc fragment to form an A-chain VHH-Fc fusion protein. A Fab antibody fragment is introduced at the N-terminus of the second Fc fragment to form a Fab-Fc half-antibody. The Fab amino acid sequence is derived from the B7-H3 DRE antibody obtained via hybridoma technology and contains a light chain domain VL-CL (RAM FIL SEQ ID NO:14) and a heavy chain domain VH-CHI (amino acid sequence see SEQ ID NO:13). The heavy chain of the Fab-Fc antibody is named the B chain, and the light chain is named the C chain. The molecular weight difference between the VHH-Fc fusion protein and the Fab-Fe half antibody is beneficial for distinguishing various protein components formed during recombinant expression, including ABC HORA, AA MORK, BBP dimer, BBCC homodimer, A monomer, B monomer, and C monomer. The target bispecific antibody is ABC HORA, HMA. Similar to Example 2, A, B, and C chain genes were synthesized, plasmids expressing A, B, and C chains were constructed, transfected into 293F cells, and the culture supernatant was collected. Protein A affinity purification was performed, and the purified products were analyzed using SDS-PAGE and SEC-HPLC methods to determine the content of ABC heterodimers and other impurities. The SDS-PAGE analysis results are shown in Figure 4. Two different plasmid transfection ratios (A chain plasmid: B chain plasmid: C chain plasmid) were used: HAL: 1:1.5 or 0.6:1:13. In the KIH control, ABC heterodimer purity was below 90% under both conditions. Other mutants, gag, M1, M2, M3, M5, M6, and M7, showed ABC heterodimer purity above 90% under at least one condition. The M4 mutant... (12%)The combined effects were poor; the purity of ABC heterodimers was lower than that of the KIH control under both conditions. SEC-HPLC analysis results are shown in Table 4. Under both ABC plasmid ratios of 1:1:1.5 and 470.6:1:15, the expression levels of the M1-M7 mutant combination (22 WO 2025 / 092987 PCT / CN2024 / 129416) were greater than 200 mg / L, similar to or higher than the KIH control. The proportions of ABC heterodimers produced by each mutant combination calculated by SEC-HPLC, compared with the SDS-PAGE 34728 RS AAW KIN control under both conditions, showed that ABC HORA AMAT 90% was higher than 90% under the ABC plasmid ratio of 1:1:1.5. In the MIL-M7 mutant combination, the proportions of MI, M2, M3, M5, and M7 heterodimers were higher than 90%. Under the condition of ABC plasmid ratio of 0.6:1:1.5, the proportion of heterodiomers in the M1, M6, and M7 combinations was higher than that in the KIH control, and greater than 90%. Table 4 VHH-Fe / Fab-Fe mutant combination SEC-HPLC analysis mg / l mg / l Example 5 S Fab-Fe half antibody expression and purification To verify the universality of the mutant combination in Example 2 in different antibody sequences and molecular forms, two Fab fragments binding to different antigens were also linked in the Fo region to construct two different Fab-Fc half antibody mutants. The A chain Fc designed in Table 1 of Example 1 was linked to the first Fab at the N-terminus to obtain the A half antibody, wherein the Fab was derived from the anti-EGFR monoclonal antibody Zalutumumab and contained the heavy chain domain VH-CHI (amino acid sequence see SEQ ID NO:15) and the light chain domain VL-CL (amino acid sequence see SEQ ID NO:16). The Fc fragment of the y-chain designed in Table 1 of Example 1 was linked to a second Fab fragment at the N-terminus to obtain a y-half antibody, wherein the Fab fragment is derived from the antibody.The HER2 monoclonal antibody Pertuzumab contains a heavy chain domain VH-CHI (α28RF5]IL SEQ ID NO:17) and a light chain domain VL-CL (amino acid sequence see SEQ ID NO:18). Similar to Example 2, the light and heavy chain genes of the A and B haptens were synthesized, plasmids expressing these genes were constructed, and 293F cells were transfected to express the A and B haptens, respectively. The culture supernatant was collected, purified by Protein A affinity, and the purified products were analyzed using SDS-PAGE and SEC-HPLC methods. The SDS-PAGE analysis results are shown in Figure 5S. For all tested hapten mutants, the 293F cell expression products mainly formed a 75 kDa hapten monomer and a 150 kDa homodimer, as well as a small amount of a 2 SKDa light chain and a 500 kDa heavy chain. Among them, the Fab-Fc mutants KO, K2, K7, HO, H2, and H7 had a monomer content greater than 80%; mutants K9 and H8 had a monomer content less than 50%. SEC-HPLC analysis results are shown in Table $. All half-antibody mutants expressed at levels greater than 100 mg / L in 293 cells. The proportions of half-antibody monomers, homodimers, and polymers in the expression products of 293 cells were calculated using the built-in analysis software of SEC-HPLC. The sum of monomer and dimer contents produced by all Fab-Fc mutants was greater than 95%, and the polymer content was less than 5%. KOPF AT produced 46.27% dimer, and K2 produced 56.17% dimer, which should be gold-encapsulated non-covalent dimers and disulfide-linked covalent dimers. These results suggest that the K409D mutation has essentially no effect on the homodimerization of the CH3 domain. K9 Fat A 86.2% dimer. K9 produced significantly more dimers compared to KO.The L3S1Y mutation promoted homodimerization of the CH3 domain. K7 produced 26.51% dimer and 73.24% monomer. H2 produced 14.38% dimer and H7 produced 15.05% RK. H2 and H7 produced significantly fewer homodimers than H0O, indicating that the D399R mutation prevented homodimerization of the CH3 domain through electrostatic repulsion. H8 produced 64.71% dimer. HS8 produced significantly more dimers than HO, indicating that the L3S1Y mutation promoted homodimerization of the CH3 domain. Table 5 SEC-HPLC Analysis Results of Fab-Fe Haptops we | im | sso] ass | 0 mw | | sao] isos | 0 _—* Example 6 Preparation and Purification of Fab-Fe / Fab-Fe Heterodimers In Vitro Assembly The hapten solutions obtained by affinity chromatography in Example $ were mixed in equimolar ratios (1:1) as shown in Table 6. Then, 1.5M Tris base was added to neutralize to pH 8-8.2. The total protein content of the reaction system was 2 mg. After standing at room temperature for 30 DAG, 200x molar ratio GSH %i& (Sigma, SO073) was added. The reaction samples were incubated at 37°C for 4 hours, and samples were taken at 0.5, 1, 2, and 4 hours to immediately determine the in vitro assembly efficiency. The reaction samples were then transferred to a 42°C refrigerator and incubated overnight. Purity was analyzed by HIC-HPLC (High Performance Liquid Chromatography for Water-to-Effect Interaction). Only HIC testing was performed using a Theromo Vanquish Core HPLC system with a TOSOH TSKgel Bulty-NPR column (2.5hm, 4.6x100mm) and a 1.3SM eRe Fe 20mm BRA filter.SIR (pH 7.0) was used as mobile phase A, and 20 mM phosphoric acid 3h YER (pH 7.0) was used as mobile phase B for gradient elution (0 - 15 min 0%B - 100%B, 15.5 - 22 min 0%B - 0%B), the flow rate was 0.6 ml / min, the detection wavelength was 280 nm, the column temperature was 23 °C, the injection volume was 10 μl, and the temperature of the temperature control plate was 5 °C. The HIC-HPLC chromatogram of the half-antibody and the bispecific antibody reacted for 4 hours is shown in Figure 6. This method can sensitively detect the assembly efficiency of bispecific antibody heterodimer molecules. Because the monomers or homodimers formed by the half-antibody have hydrophobicity differences, resulting in different peak elution times in the HIC chromatogram. The peak elution times of the bispecific antibody molecules generated by the in vitro assembly of two Fab-Fc are also different from those of the half-antibody. The HIC-HPLC analysis results are shown in Table 6. Under the conditions of 37 °C and 0.5 hours of HFA, the proportion of AB heterodimers generated by the combination of KIH, M1, M2 and M3 mutants has been greater than 90%. Under the conditions of 37 °C, 2 hours and 4 hours of HFA, the proportion of AB heterodimers generated by the combination of KIH, M1, M2 and M3 mutants increased to approximately 93%. Table 6 HIC-HPLC analysis of bispecific antibody assembly efficiency 24 WO 2025 / 092987 PCT / CN2024 / 129416 Heterodimer purification The 1 ml Capto S ImpAct ion exchange column (Cytiva, 17371751) was rinsed with 5 column volumes of the equilibration solution (20 mM sodium phosphate, pH 6.0) or until the baseline. The in vitro assembly sample was adjusted to pH 5.8 - 6.2 with 1M acetic acid, then diluted 1 - 2 times with ultrapure water, and filtered through a 0.22 μm PES membrane. The sample was loaded onto the Capto S column at a flow rate of 0.5 column volume / minute. Then it was rinsed with the equilibration solution for 10 CV. Finally, it was eluted with the elution solution (20 mM sodium phosphate + 1M sodium chloride, pH 6.0) according to a 0 - 30% gradient, and the elution solution was collected in fractions. SEC-HPLC detectionDimer peak purity of each sample tube was recorded, and samples with a purity greater than 98% were combined. SEC-HPLC analysis results are shown in Table 7. Under 37°C CEA 4 hours conditions, the combined KIH, M1, M2, and M3 mutants produced 96% RAMP AF, primarily composed of AB heterodimers, with minor amounts of AA or BB homodimers. Other impurities included A or B monomers, or high molecular weight aggregates, all less than 2%. Table 7: Purity of bispecific antibodies produced by SEC-HPLC 3-47 37°C CEA 4h. Example 7: Bispecific antibody-antigen binding activity test. Example 6: Binding activity of purified bispecific antibodies to antigen. Recombinant human EGFR protein (ACRO Biosystems, EGR-HS222) and HER2 protein (ACRO Biosystems, HE2-H5225) were diluted to 1 μg / mL with coating buffer (Solarbio, C1055). 100 μL of 4-HT was added to each well, and the plate was incubated at 4°C for 16 hours. Blocking agent was prepared by adding 3% BSA (AL, 9048-46-8) and 0.05% Tween 20 (4 I, A600560-0500) to PBS solution. After washing the plate three times with 0.05% PBST, 300 μL of blocking agent was added to each well, and the plate was incubated at room temperature for 2 hours. The penicillin antibody was stably released using OS% BSA - 0.05% PBST blocking agent, with a maximum concentration of 1 Sug / mL, following a 3-fold sequential gradient. After washing the plate three times with 0.05% PBST, add 100 μL of double antibody solution to each well and incubate at room temperature for 1 hour. After washing three times with 0.05% PBST, add 100 μL of HRP-labeled secondary antibody (Bethyl, A80-304P, 1:5000 dilution) to each well and incubate at room temperature for 1 hour. After washing three times with 0.0% PBST, add 100 μL of chromogenic reagent (Solarbio, PR1210) to each well and incubate at room temperature in the dark for 10 minutes. Add 100 μL of stop solution (Solarbio, C1058) to each well and read the plate using a multi-plate reader (Tecan, Spark).The absorbance at OD450 was recorded. The ELISA results are shown in Figure 7. The bispecific antibodies produced by combinations of KIH, M1, M2, and M3 mutants exhibited very similar antigen-binding curves. The IC50 values ​​for EGFR binding were between 17.0 and 24.9 ng / ml, and for HER2 binding were between 12.8 and 14.6 ng / ml. Mutations in the CH3 domain of the Fe region of the RGA did not affect the antigen-binding ability of the bispecific antibodies. Example 8. The binding activity of the bispecific antibody to the Fe receptor was determined using thin-layer interferometry (BLD). The affinity of the purified bispecific antibody from Example 6 for binding to human FoyRI receptor and human FcRn was measured. The equilibrium dissociation constant (KD) was calculated using a Fortebio Octet Red 96 instrument. A column of HISIK (Fortebio, Cat 18-S120) sensors was immersed in a standard buffer (1xPBS, pH 7.4 with 0.1% BSA, 0.02% Tween-20). After the sensor was equilibrated in the standard buffer, Sug / ml recombinant human FeyRI protein (ACRO Biosystems, Cat FCA-H52H1) was immobilized, and the antibody bound and dissociated in the standard buffer. Instrument operation steps: Baseline 1 (60 s), Loading (~15 s, 0.5nm), Baseline2 (60 s), Association (60 s) and Dissociation (120 s), 4324 1000rpm, i A 30°C. Take a column of SA (Fortebio, Cat 18-5019) & Siz iz F pH6.0 buffer (1xPBS, pH 6.0, with 0.1% BSA, 0.02% Tween-20). 4&8 After equilibration in pH6.0 buffer, take 200ul of pH6.0 buffer, 100nM antibody and human FeRn and add them to 96 JLB ERR CT bi HK ILA (Greiner, 655209)?Detection was performed using a Fortebio Octet Red96 instrument. The sensor location was selected based on the sample placement. After equilibration in pH 6.0 buffer, 2 μg / ml of recombinant human FcRn protein (ACROBiosystems Cat FCM-H8286) was immobilized, followed by binding with 100 nM antibody and dissociation in pH 6.0 buffer. Instrument operation steps: Baseline 1 (60 s), Loading (~100 s, 3.0 nm), Baseline 2 (60 s), Association (60 s), and Dissociation (60 s), at a rotation speed of 1000 rpm and a temperature of 30°C. The Fortebio experimental results are shown in Figure 8. The affinity of the control antibody IgG1 45A for FoyRI was 6.33 x 10°M; the affinity of the KIH, M1, M2, and M3 mutant combinations for human FecyRI ranged from 1.01 x 10°M to 1.03 x 1035M. The affinity of the control antibody IgG1 for human FcRn was A 141 x 10°M; the affinity of the KIH, M1, M2, and M3 mutant combinations for FcRn was slightly lower, ranging from 1.44 x 10°M to 1.85 x 10°M. The affinity values ​​were within the range of instrument detection error. Therefore, the Fe mutant combinations obtained in this study essentially maintained binding to both human FcRn and human FoyRI. Moreover, the affinity values ​​of the M1, M2, and M3 mutant combinations were very similar to those of the KIH control. Example 9: Accelerated Stability Test of Bispecific Antibody. The purified bispecific antibody from Example 6 was replaced with a 20 mM solution of ketamine and diluted to 1 mg / ml, then filtered through a 0.22 μm PES filter. Samples were placed in a 425°C oven, and samples were taken on days 7 and 14 for purity analysis by SEC-HPLC and SDS-PAGE. The SEC-HPLC results are shown in Table 9. The purity of each mutant combination only slightly decreased after storage at 42°C for 7 days and 14 days.In the study, the purity of the KIH, M1, M2, and M3 mutant combinations decreased by 0.21%, 0.73%, 0.47%, and 0.57% on day 7, respectively; and by 1.43%, 1.50%, 1.46%, and 0.84% ​​on day 14, respectively. These results indicate that the newly designed Fe mutant combinations exhibit good stability under high-temperature storage conditions. SDS-PAGE analysis also showed that after storage at 42°C for 14 days, the proportions of protein bands of different molecular weights remained essentially unchanged compared to storage for 0 days. Table 9. Discussion of Storage Stability of Bispecific Antibodies in Accelerated Experiments by SEC-HPLC Analysis. By analyzing the reasons for the decreased thermal stability of the handle-pore model, we selected amino acids near the CH3 contact interface of the Fe fragment as mutation targets to investigate the effects of different mutation combinations on the production performance, physicochemical properties, and biological functions of Fo-based IgG-like single / multispecific antibodies. Unwilling to be bound by theory, we believe that in wild-type IgG1 Fe, amino acid 1351 in one chain directly contacts 1351, P352, P353, S354, and T366 in the opposite side chain; while in the KIH structure, 351 in the pore chain directly contacts L351, P352, P353, S354, and T366 in the handle chain, but with increased contact distance, the interaction force is expected to weaken. Mutating L351 of the pore chain, for example, to form a ketone acid with a larger side chain (L35S1Y), is presumably intended to increase the contact between the L351 residues and the handle chain amino acids P352, P353, S354, and TIOOW, thereby producing a stronger water retention effect (see Figure 1C). It also suggests the formation of new contacts with other handle chain amino acids, such as #E&(OH) in L351Y forming new combinations with T366W and E357.Ammonia bond network. Based on the L351 mutation of the porous chain, a combination of mutations of chain K409D and porous chain D399R is further introduced. Not wanting to be bound by theory, it is speculated that this mutation combination can lead to a pair of salt bridge connections similar to those in IgG1Fo; while, porous chain D399R will form new contacts with the amino acids of chain K392, T411, and T366W, and the F409D mutation of the chain will retain direct contact with porous chain F405 (COLA 1D). The experimental results of the above embodiments show that combining the above mutations in the KIH structure, i.e., porous chain 1L351Y or porous chain L3S1Y + chain K409D-D399R RE, WHR Fe scaffold and the production and physicochemical properties of mono / multispecific proteins (especially bispecific antibodies) containing it, can impart a good promoting effect. After expression in cells and purification by affinity chromatography, the Fec heterodimer scaffold protein and protein of the mutant combination described by Bao Renjin produced a Fo heterodimer ratio of over 90%, which is higher than the control Fc EO with only Bao Renjin corresponding to the knob-into-hole mutation; and after further purification by ion exchange column, the purity can reach over 98%. Furthermore, compared to the control Fc protein, the purified Fe heterodimeric scaffold protein exhibits better thermal stability, with the CH3 domain Tm value measured by DSC reaching 74°C. Further, the experimental results of the above examples also show that combining the above mutations in the KIH structure, namely the L351Y or L351Y+40-F 4% K409D-D399R mutation, has virtually no impact on the antigen-binding function of the heterodimeric Fe scaffold and the single / multi-specific proteins (especially bispecific antibodies) containing it, as well as the FcRn and FoyR receptor binding functions. These results combined indicate that, based on KIM...Introducing the L351Y mutation or a combination of the L351YD399R mutation and the K409D mutation will be beneficial to the production and stability of heterodimeric target antibodies. 27 WO 2025 / 092987 PCT / CN2024 / 129416 Sequence Listing SEQ ID | Sequence Description / Name BIRR ARF RR hoe 1 Fe KO ® & (knob) | DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS (T366W) HEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ (EU 221-447) DWLNGKEYKCK VSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG SFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 2 Fe K2 MutationDKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVV VDVS (K409D) HEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCK VSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG SFFLY SDLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPGK 3 Fe K9 MutationDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS (L351Y) HEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTY PPSREE MTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG SFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 4 Fe K7 MutationDKTHTCPPCPAPELLGGPSVELFPPKPKDTLMISRTPEVTCVVVDVS (L351Y-K409D) HEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTY PPSREE MTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG SFFLY SDLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPGK 5 Fe HO mutation (hole) | DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS (T366S-L368A-Y40_ | HEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ TV) DWLNGKEYKCK VSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG SFFLVSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPGK Fe H2 mutation DKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVV VDVS (D399R) HEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCK VSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTIPPVLRSDG SFFLVSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPGK 7 Fe H8 mutation DKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVV VDVS (L351Y) HEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTY PPSREE MTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPGK Fe H7 Mutation DKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVS (L351 Y-D399R) HEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTY PPSREE MTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTIPPVLRSDG SFFLVSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPGK [9 | Fe2-D399K _| DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDYS | 28 WO 2025 / 092987 PCT / CN2024 / 129416 HEDPEVKFNW Y VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPOQVY TLPPSREE MTKNQVSLSCAVKGFY PSDIAVEWESNGQPENNYKTTPPVLKSDG SFFLVSKLTVDKSRW QOGNVEFSCS VMHEALHNHY TOKSLSLSPGK 10 Fe H7-D399K Mutation DKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVS (L351 Y-D399K) HEDPEVKFNW Y VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY TY PPSREE MTKNQVSLSCAVKGFY PSDIAVEWESNGQPENNYKTTPPVLKSDG SFFLVSKLTVDKSRW QOGNVEFSCS VMHEALHNHY TOKSLSLSPGK 11 Ozoralizuamab VHH | EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMS W VRQAP KGL EWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTA VYYCTIGGSLSRSSQGTLVTVSS 13 hz32F8.9- VH+CH1 | QVOLVQSGAEVKKPGSSVKVSCKTSGYTFTDY VITW VRQAPGQG LEWMGETYPRSDRTY YNENFKGRVTITADKSTNTAY MELSSLRSED TAVY YCARWDNYYFDYWGQGTLVTVSSASTKGPS VFPLAPSSKST SGGTAALGCLVKD YFPEPVTVSWNSGALTSGVHTFPAVLOSSGLYS LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC 14 hz32F8.9-VL+CL DVVMTQSPLSLPVTLGQPASISCRSSQSLVHSNAITY LWY QQRPG QSPRLLIY KVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVY CSQTTHVPWTFGGGTK VEIKRTVAAPS VFIFPPSDEQLKSGTAS VVC LLNNFYPREAK VOQWKVDNALOSGNSQESVTEQDSKDSTYSLSSTL TLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 15 Zalutumumab-VH+ | QVQLVESGGGVVQPGRSLRLSCAASGFTFSTY GMHW VRQAPGKG CH1 LEW VAVIWDDGSYKY YGDSVKGRFTISRDNSKNTLYLQMNSLRAE DTAVY YCARDGITMVRGVMKDYFDYWGQGTLVTVSSASTKGPSV FPLAPSSKSTSGGTAALGCLVKD YFPEPVTVSWNSGALTSGVHTFP AVLQOSSGLYSLSSVVTVPSSSLGTQTYICN VNHKPSNTKVDKKVEP KSC 16 Zalutumumab- AIQLTQSPSSLSASVGDRVTITCRASQDISSALVWY QQKPGKAPKLL VL+CL TYDASSLESGVPSRFSGGTSESDFTLTISSLQPEDFATY YCQQFNS YP LTFGGGTKVEIKRTVAAPS VFIFFPSDEQLKSGTAS V VCLLNNFY PREAKVQWKVDNALOSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE KHKVYACEVTHQGLSSPVTKSFNRGEC 17 Pertuzumab-VH+C EVQLVESGGGLVQPGGSLRLSCAASGFTFTD Y TMD W VRQAPGKG H1 LEW VAD VNPNSGGSIYNQRFKGRFTLS VDRSKNTLYLQMNSLRAE DTAVY YCARNLGPSFYFDY WGQGTLVTVSSASTKGPSVFPLAPSSK STSGGTAALGCLVKD YFPEPVTVSWNSGALTSGVHTFPAVLOSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC 18 Pertuzumab- DIQMTQSPSSLSASVGDRVTITCK A QDVSIGVAW YQQKPGKAP L VL+CL LIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATY YCQQYYT PYTFGQGTKVEIKRTVAAPS VFIFPPSDEQLKSGTAS VVCLLNNFYP REAKVQWKVDNALOQOSGNSQES VTEQDSKDSTYSLSSTLTLSKADY EKHKVYACEVTHQGLSSPVTKSFNRGEC 29 WO 2025 / 092987 PCT / CN2024 / 129416 (EU 341-447) PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQOQGNVFSCSVMHEA Se [Enwoeuuroe 20 Wild-type CH3 domain | GQPREPOVY TLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ Without K447 residue PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQOQGNVFSCSVMHEA [Gerke [eames 21 Sh 4 CH2 domain | APELLGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFEN (EU 231-340) WY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY a 30 WO 2025 / 092987 PCT / CN2024 / 129416 PA BR1. A heterodimeric Fc scaffold comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises a first Fc region, the second polypeptide comprises a second Fc region, wherein the first Fe region comprises a first CH3 domain, and the second Fe region comprises a second CH3 domain, wherein, relative to the wild-type CH3 domain, the first Fe region and the second Fe region respectively comprise a KIH-mutated knob mutation and a hole mutation in their CH3 domains, and wherein (a) the Fe region containing the hole mutation further comprises an L351Y mutation; or (b) the Fe region containing the hole mutation further comprises L351Y and the Fe region containing the D339R RK, and the Fe region containing the LAS knob mutation further comprises K409D, wherein the first polypeptide and the second polypeptide pair and heterodimerize through the first and second Fe regions to form the Fe scaffold, wherein the amino acid residues are numbered according to the EU index in Kabat. 2. The heterodimeric Fe scaffold of claim 1, wherein the first and second Fe RLS CH2 and CH3 2573, AALS chain regions, CH2 and CH3 structural domains. 3. The heterodimeric Fc scaffold of any one of claims 1-2, wherein the first and second Fe regions are of the IgG type, such as IgG1, IgG2, IgG3 or IgG4 subtypes, preferably, the first and second Fe regions are human IgG1 BLA. 4. The heterodimeric Fe scaffold of any one of claims 1-3, wherein the knob mutation is T366W; and the hole mutation is Y407V or T366S-L368A-Y407V, preferably, the first Fc region contains T366W, and the second Fc region contains T366S-L368A-Y407V-L351Y; or the first Fe region contains T366W-K409D, and the second Fe region contains...T366S-L368A-Y407V-L351Y-D399R. 5. The heterodimeric Fe scaffold of any one of claims 1-4, wherein, (a) relative to the wild-type CH3 domain, the first and second CH3 domains respectively further comprise 0-3 PRAMRAKE, Ub, wherein the wild-type CH3 domain is the CH3 domain of natural human IgG1 immunoglobulin, more preferably comprising the amino acid sequence of SEQ ID NO: 19 or 20; (b) the first and second Fo regions comprise the CH2 domain of natural human IgG1 immunoglobulin, or comprise the SEQ ID NO: 21 PRAMRAKE, or a CH2 domain having a 1-5 amino acid residue change therewith; (c) the Fe region with the &4 knob mutation comprises the amino acid sequence of SEQ ID NO: 1 or 2 or an amino acid sequence having at least 93%, 96%, 97%, 98%, or 99% identity therewith; (d) the Fe region containing the hole mutation comprises the amino acid sequence of SEQ ID NO: 7 or 8 or an amino acid sequence having at least 95%, 96%, or 96% identity therewith. 97%, 98%, 99% identical amino acid sequences, and / or (e) the Fe scaffold does not form non-natural disulfide bonds between the first Fe region and the second Fe region. 6. The heterodimer FeRR,FP ATA Fe scaffold of any one of claims 1-5, having increased thermal stability compared to the corresponding control Fe scaffold containing only the KIH mutation, and preferably, as determined by DSC, the CH3 domain of the Fe scaffold has a Tm value of approximately 70 °C or higher. 7. A CH3 heterodimer comprising a heterodimerized first immunoglobulin CH3 domain and a second immunoglobulin CH3 domain, KP: relative to the wild-type CH3 domain, the first CH3 domain and the second CH3 domain respectively contain the knob mutation and the hole mutation in the KIH mutation, and wherein(a) The CH3 domain containing the hole mutation also contains the L3S1Y mutation; or (b) The CH3 domain containing the hole mutation also contains the L351Y and D339R mutations, and the CH3 domain containing the knob mutation contains the K409D mutation, wherein the amino acid residues are numbered according to the EU index in Kabat. Preferably, the first and second CH3 domains are of the IgG type, such as IgG1, IgG2, IgG3 or IgG4 subtypes. Preferably, the wild-type CH3 domain is the natural human IgG1 RARE A CH3 domain. More preferably, relative to the wild-type CH3 domain, the first and second CH3 domains have 0-3 amino acid residues modified. 8. A heteropolymeric protein comprising claims 1-6 9. A binding protein comprising the heterodimeric Fe scaffold of any one of claims 1-6 and at least one target-binding domain connected thereto. 10. The binding protein of claim 9, wherein the at least one target-binding domain is independently selected from: a ligand, a receptor-binding domain of a ligand, a ligand-binding domain of a receptor, and an antigen-binding domain of an antibody. 11. The binding protein of any one of claims 9-10, wherein the binding protein is single-specific or bispecific. 12. The binding protein of any one of claims 9-11, wherein the binding protein comprises at least one target-binding domain independently selected from Fv, scFv, Fab, scFab, crossFab, VHH, and a ligand, preferably 1-6, more preferably 1, 2, or 3 of the target-binding domains. 13. Claims 9-1214. The binding protein of any one of claims 9-13, wherein the binding protein comprises only one of the heterodimeric Fc scaffolds. Preferably, the binding protein has an IgG conformation or IgG HHA, and preferably, the binding protein has a conformation selected from: scFv-Fe / scFv-Fe, Fab-Fe / Fab-Fe, WHH-Fce / VHH-Fc, VHH-Fc / Fab-Fc, VHH-Fe / scFv-Fe, WHH-Fe / Fc, scFv-Fe / Fe, Fab-Fc / Ec, ligand-EFc / ligand-Fc, ligand-Fc / scFv-EFc, ligand-Fc / VHH-Fc, ligand-Fc / Fab-Fc, ligand-Fc / EFc. 15. The binding protein of any one of claims 9-14, wherein the binding protein is bispecific and comprises: (a) two different Fab domains respectively attached to the N-terminus of the first and second Fe regions of the heterodimeric Fo scaffold, wherein the Fab domains have the same or different VLCL light chains; or (b) a VHH domain and a Fab domain respectively attached to the N-terminus of the first and second Fc regions of the heterodimeric Fc scaffold. 16. The binding protein of any one of claims 9-15, having one or more of the following properties: - thermal stability, wherein, as determined by DSC, the CH3 domain has a Tm value greater than or equal to 74°C; - purity of 90% or higher; - binding affinity to FcRn receptor, not less than 80%, preferably not less than 85%, 90%, or 95%; - binding affinity to FeyR1 receptor, not less than 509% of the binding affinity to the natural IgG1 FeyR1 receptor; - storage stability, wherein, after storage at 425°C for 2 weeks, as determined by SEC-HPLC, the purity is...The degree of change is less than 2%. 17. A polynucleotide encoding a heterodimeric Fe scaffold of any one of claims 1-6, a CH3 heterodimer of claim 7, a heteromultimeric protein of claim 8, or a binding protein of any one of claims 9-16. 32 WO 2025 / 092987 PCT / CN2024 / 129416 18. A polynucleotide as claimed in claim 17. 1 SR RAR HS A 19. A composition comprising the heteromultimeric protein of claim 8 or a binding protein of any one of claims 9-16. 20. A conjugate comprising a therapeutic agent conjugated to the heteromultimeric protein of claim 8 or a binding protein of any one of claims 9-16. 21. Use of the composition of claim 19 or the conjugate of claim 20 as a medicament or in the preparation of a medicament, preferably, the medicament is used for cancer treatment. 33 WO 2025 / 092987 PCT / CN2024 / 129416 nN i ee PR gery —— Cora 6 sy AN NA Qe cw Nn Ne | ET mA a | CA a Ve | ~~ 可 1 N ~~ ro | RE SR SN S AS. BS SI NSRas9 本 SS Ke Se €. %& ae AN ae SR RAR Qf EP occas Be NR ge PE eg GP NA RER We < 3 人 & LO ww | a | ON ee | SR Figure 1 1 / 6 WO 2025 / 092987 PCT / CN2024 / 129416 Plasmid ratio 1 Plasmid ratio 0.6:1 Region KR Ma M2 MS Ma MS Ma My Se By ee SS SESS SSeS s Se BB | a. 2 ASB mc iieieIeé#=\yy».. . .«§ Figure 2 2 / 6 : SE8 i aS Sat eat * RS H RY C23 RS AR | 3 Xx CN Py H N han IE 下 . 1 i i Ss gad i VRS ey boi rR N SS,fest all 2 4 £ ogat :, Le San fo SB ; S is By fooy x down 2 x ty and : s oh BY : ‘ beaD Smale z 3 N gad : x ra METS i R Xe i 5 it 4 : : SF SH : x Cs N : : 1 ‘ H cat : up aah : N 1 SN RB BY : : \ : at 0 A, t : 1 HR ws.. : : people DNSONDENNNNNNONEENNNEOE i EN AN 853 | Skin: eae att ; bureau H AINE Goh ANY i \ | four Poy : : KN.. : EN : x IAS : Yao Pou ON : PSA ge NE : SX 1 Owe? 3 Dal : 1 person “ol six four an j : Po Me : into fh Wes z %. Otic’ over x ve BY : x hom 2 : ‘ Bal way weve ag ae Te : i af a %. ‘ SSNS Nene ~s SE SAY H Get ova, we ened 8 > : 3 ER - a By mh 5 a i in st < xt th a Be ot es this x oe a oy 8 & Ww Ry Fey ahh 3 i Ny SS ad Ye SEEMING AN 1 SED SES LS :, = : fe wen 1 QS HSS 3 89,283 : t four SS : axe, fy Bet SR SS PY et 4 ran : 3 34.87 1 see! 3 : inl agag > go : and ay 2 py lage! 2 1 ie A Qo six 3 : 5 Ley : x Np Ss : B ees i 3 Soe ns 3 y por : : Lt a 3 x Ine WY : < Mos ; x ‘ wan : : a a x 1 RSSony six < ees | ei ws NON : xg 3 : : SRG aes BSL | eae SF 6Y Ws in | sete x a fy H in : pny ' SEN pt Sans! pos bON spa 3 It seems the original text contains some garbled or unclear characters which make it difficult to provide a more meaningful and accurate translation. Please check and correct the original text if possible.= H : i oe Nou ; g ~ Be A 1 iO 1 : 所 3 : 和 * i By Taaapesegesepennnpesegeangnenep eae gennapene aan gens enapann enapanapanageens | Nanepeeepnagnonypapnnnpanngnnnananegnangnn ann seannnnp n nys SAMBA AR AARg RAR! Ray RN au RSS 8 x 好 x» ws: 8 38 3s ou x RY x % ws WO 2025 / 092987 PCT / CN2024 / 129416 UH Ma M2 ME M4 M5 Mo M4? SEH MI MY MS M4 M5 MG M7 Pees 270 eS ee AvBee | RN aa ee =. i iee=:= i... <= Peer’ — | |... i |. 3 _ =; Figure 4 KO K2 K9 K7 HO H2 H8 H7 see 130 — ©. ss sw NN GK aoe Fe iS SE F~ 8 .§..... Figure $ 4 / 6 “ KW heteradiner a Mi. heterodimer ood Me heterodimer fom M3 heterodimer 3 PEs COORG BY pooNe Ke anomer Py MH? homodimer Re Re WII S HER? <3 te 8g £8 < ee & eae 8 he A x a 3 RQ 站 RS $ CAB GyWRY ead Urey NESRNRANRSRSNNSERRSS AR NSRESSSSS NS 汪汪 5 / 6 / 129416 we ‘ . 2 ae *s . see SIR e Se .. SEM & 8a if See 7 : F NBs. . . i 内 ye awe xis 1 ss wos 8S and sees ime isan} 286 SES see ee eens a3 mn 8% ong *R | ~ \ eon $2 we NY wane: 和 Ka aaa s ‘EOwUMS Ege fly ! | RE... Lets SEG, LRA . a og“ager” SHE Lees roe (aan) {Aste hw SS A REAS 6 / 6 INTERNATIONAL SEARCH REPORT International application No. PCT / CN2024 / 129416 A. CLASSIFICATION OF SUBJECT MATTER C07K16 / 00(2006.01)i; A61K39 / 395(2006.01)i; C12N15 / 13(2006.01)i; A61P37 / 02(2006.01)i; A61P35 / 00(2006.01)i; CO7K 16 / 46(2006.01)i According to International Patent Classification (IPC) or to both national classification and IPC B. FIELDS SEARCHED Minimum documentation searched (classification system followed by classification symbols) IPC:C07K,A61K,C12N,A61P Documentation searched other than minimum documentation to the extent that such documents are included in the fields searched Electronic data base consulted during the international search (name of data base and, where practicable, search terms used) NTXT, ENTXTC, VCN VEN, PUBMED, NCBI, EBL, ISI Web of Science, CNKI, BAIDU, BING: Fe, heterodimer, FR body, — 38K, KIH, knob, hole, L351Y, D339R, K409D, L351, D339, K409, thermal stability Cc. DOCUMENTS CONSIDERED TO BE RELEVANT Citation ofdocument, with indication, where appropriate, of the relevant passages Relevant to claim No. Xx CN 109790215 A (BEIJING HANMI PHARM. CO., LTD.) 21 May 2019 (2019-05-21) 1-21 claims 1-53, and description, paragraphs 397 and 439 Xx CN 104080811 A (ZYMEWORKS INC.) 01 October 2014 (2014-10-01) 1-21 claims 43-61, and description, paragraph 162 Xx CN 109897103 A (ZYMEWORKS INC.) 18 June 2019 (2019-06-18) 1-21 claims 1-44, and description, paragraphs 13 and 170 Xx CN 116096353 A (DRAGONFLY THERAPEUTICS, INC.) 09 May 2023 (2023-05-09) 1-21 description, paragraphs 177 and 238, and table 11 Xx US 2013336973 Al (ZYMEWORKS INC.) 19 December 2013 (2013-12-19) 1-21 claims 1-62 A CN 116888149 A (INNOVENT BIOLOGICS (SUZHOU) CO., LTD.) 13 October 2023 1-21 (2023-10-13) entire document [| Further documents are listed in the continuation of Box C. See patent family annex. * Special categories of cited documents: «T” later document published after the international filing date or priority “a” documentdefining the general state of the art which is not considered date and not in conflict with the application but cited to understand the to be of particular relevance principle or theory underlying the invention “PD” document cited by the applicant in the international application “x” document of particular relevance; the claimed invention cannot be “f” earlier application or patent but published on or after the international considered novel or cannot be considered to involve an inventive step filing date when the document is taken alone “{ document which may throw doubts on priority claim(s) or which is “Y” document of particular relevance; the claimed invention cannot be cited to establish the publication date of another citation or other considered to involve an inventive step when the document is special reason (as specified) combined with one or more other such documents, such combination “OQ” document referring to an oral disclosure, use, exhibition or other being obvious to aperson skilled in the art means “&” document member of the same patent family «p” document published prior to the international filing date but later than the priority date claimed Date of the actual completion of the international search Date of mailing of the international search report 16 January 2025 22 January 2025 Name and mailing address of the ISA / CN Authorized officer China National Intellectual Property Administration (ISA / CN) China No. 6, Xitucheng Road, Jimenqiao, Haidian District, Beijing 100088 Telephone No. Form PCT / ISA / 210 (second sheet) (July 2022) INTERNATIONAL SEARCH REPORT International application No. PCT / CN2024 / 129416 Box No. I Nucleotide and / or amino acid sequence(s) (Continuation of item 1.c of the first sheet) 1. With regard to any nucleotide and / or amino acid sequence disclosed in the international application, the international search was carried out on the basis of a sequence listing: a. forming part of the international application as filed. b. [|furnished subsequent to the international filing date for the purposes of international search (Rule 13zer.1(a)), 六] accompanied by a statement to the effect that the sequence jlisting does not go beyond the disclosure in the international application as filed. 2. [| With regard to any nucleotide and / or amino acid sequence disclosed in the international application, this report has been established to the extent that a meaningful search could be carried out without a WIPO Standard ST.26 compliant sequence listing. 3. Additional comments: Form PCT / ISA / 210 (continuation of first sheet) July 2022) INTERNATIONAL SEARCH REPORT International application No. PCT / CN2024 / 129416 Box No. II Observations where certain claims were found unsearchable (Continuation of item 2 of first sheet) This international search report has not been established in respect of certain claims under Article 17(2)(a) for the following reasons: 1. Claims Nos.: 21 (in part) because they relate to subject matter notrequired to be searched by this Authority, namely: Claim 21 (in part) relates to the use of the composition of claim 19 or the conjugate of claim 20 as a drug, which belongs to a method for treatment of diseases as defined in PCT Rule 39.1(iv). The international search and written opinion are provided on the basis of a corresponding pharmaceutical use. 2. [| Claims Nos.: because they relate to parts of the international application that do not comply with the prescribed requirements to such an extent that no meaningful international search can be carried out, specifically: 3. [| Claims Nos.: because they are dependent claims and are not drafted in accordance with the second and third sentences of Rule 6.4(a). Form PCT / ISA / 210 (continuation of first sheet) July 2022) INTERNATIONAL SEARCH REPORT International application No. Information on patent family members PCT / CN2024 / 129416 cited in search report (day / month / year) (day / month / year) CN 109790215 A 21 May 2019 NZ 750566 A 26 April 2024ECSP 19022190 A 31 May 2019 MY 194994 A 30 December 2022 CL 2019000836 To 02 August 2019 BR 112019006074 A2 18 June 2019 AU 2017 6828 February = 2019 AU 2017336867 B2 14 March 2024 IL 265605 A 30 May 2019 IL 265605 Bl 01 February 2024 IL 265605 B2 O1 June 2024 CA 30356 1 Al 18 DOP 2035 2019000079 A November 15, 2019 WO 2018059502 Al 05 April 2018 US 2019284299 = Al 19 September 2019 US 11498977, B2 November 15, 2022 CO 20190576 Al 22 April 2019 EA 201990285 =Al 30 December 2019 JP 2019535650 A 12 December 2019 JP 7424727 “”B2 30 January 2024 KR May 2019 519 A 2530 2019 KR 102242990 “Bl 22 April 2021 TN 2019000055 Al 15 July 2020 SG 11201901307, VA 28 March 2019 ZA 201901708 B 30 November 2022 Mx 2013 Ax 2019 12 August 2019 SA 519401441 ”Bl 27 December 2022 PH 12019500655 Al 05 August 2019 EP 3504234 Al 03 July 2019 EP 3504234 =A4 02 December 2020 SG 192013 30 March 2020 CN 104080811 A 01 October 2014 ES 2899956 = T3 15 March 2022 HK1200465 Al 07 August 2015 HRP 20211773 T1 04 March 2022 AU 2012332021 Al 19 June 2014 AU 2012332021 B2 20 July 2017 AU 2012332021 A8 12 October 2017 CA 2854233 Al 10 May 2013 CA 2854233 C 12 May 2020 HUE 056462 T2 28 February 2022 RU 2014121832 A 10 December 2015 RU 2675319 = C2 18 December 2018 DK 2773671 =T3 15 November 2021 PL 2773671 =T3 24 January 2022 PT 2773671 T 14 December 2021 KR 20140092370 A 23 July 2014 KR 102052774 ~=Bl 04 December 2019 WO 2013063702 Al 10 May 2013 CY 1124872 T1 25 November 2022 US 2015284470 = Al 08 October 2015 Form PCT / ISA / 210 (patent family annex) (July 2022) INTERNATIONAL SEARCH REPORT International application No. Information on patent family members PCT / CN2024 / 129416 cited in search report (day / month / year) (day / month / year) US 9732155 B2 15 August 2017 Mx 2014005348 A 12 November 2014 Mx 358862 B 06 September 2018 US 2020087414 Al 19 March 2020 AU 2017245451 = Al 02 November 2017 AU 2017245451 B2 22 August 2019 AU 2017245451 B9 29 August 2019 BR112014010580 A2 02 May 2017 BR 112014010580 = B1 12 January 2021 JP 2014533243 A 11 December 2014 JP 6326371 B2 16 May 2018 US 2018 Al 2010 = Al February 2018 US 9988460 B2 05 June 2018 US 2018016347 Al 18 January 2018 US 10457742 ~=B2 29 October 2019 US 2013195849 = Al 01 2013 US 2013 February 2013 2017 EP 2773671 = Al 10 September 2014 EP 2773671 A4 03 June 2015 EP 2773671 Bl 15 September 2021 RS 62689 —s BI 31 December 2021 RU 2018 2018 March 2012 CN 109897103 A 18 June 2019 None CN 116096353 A 09 May 2023 Mx 2022013112 A 16 January 2023 EP 4138778 To 01 March 2023 WO 2016 2016 October 2016 US 2023272041 To 31 August 2023 CA 3173809 To 28 October 2021 IL 297495 A 01 December 2022 KR 20230004746 A 06 January 2023 JP 2023 AU 2023 June 2023 2021260960 = Al 24 November 2022 US 2013336973 = Al 19 December 2013 WO 2013166594 Al 14 November 2013 WO 2013166594 A8 06 February 2014 JP 2015 August 2525 A 2015 JP 6351572 = B2 04 July 2018 CA 2872540 Al 14November 2013 US 2016257763 Al 08 September 2016 AU 2013258834 Al 27 November 2014 AU 2013258834 B2 07 September 2017 EP 2847230 = Al 18 March 2015 EP 2847230 8 A4 16 December 2015 EP 2847230 ”Bl 12 August 2020 ES 2843054 ”IT3 15 July 2021 CN 116888149 A 13 October 2023 TW 202233693 A 01 September 2022 TWI 825544 B 11 December 2023 US 2024034795 Al 01 February 2024 EP 4273161 = A1 08 November 2023 WO 2022143912 A1 07 July 2022 Form PCT / ISA / 210 (patent family annex) (July 2022) International Search Report International Application No. PCT / CN2024 / 129416 A. Subject Classification C07K16 / 00(2006.01)i;, A61K397 / 395(2006.01Dii C12N15 / 13(2006.01D)ii A61P37 / 02(2006.01TD)ij A61P35 / 00(2006.01)i; CO7K 16 / 46(2006.01)i According to the International Patent Classification (PC) or simultaneously according to the National Classification and IPC Minimum literature for both classification searches (indicate classification system and classification number): IPC: C07K, A61K, C12N, A61P. Electronic databases consulted during international searches for literature in the search field other than the minimum literature (database name and search terms used <if applicable>): NTXT, ENTXTC, VCN, VEN, PubMed, NCBLEBLISI. Web of Science: CNKLBAIDUBING: Ec. (Note: The last part, "heterodimer," appears to be a typo and is left untranslated.),hole, L351Y, D339R, K409D, L351, D339, K409, thermal stability C. Related documents Referenced documents, where necessary, indicate the relevant paragraphs Related claims x CN 109790215 A CLE ERSE 54h BIR ZA A) 2019465 H21 (2019-05-21) 1-21 Claims 1-53 and paragraphs 397, 439 of the specification x | CN 104080811 A (Benhuo Co., Ltd.) 20144210 A148 (2014-10-01) | 1-21 | BIBER OU x | CN 109897103 A (Benhuo Co., Ltd.) 2019466 A18H (2019-06-18) | 1-21 WL BIRR VIO x | CN 116096353 A GEMET TERRIA BBR ZA) 2023465 HOA (2023-05-09) | 1-21 Paragraphs 177, 238, Table 11 | US 2013336973 A1 (ZYMEWORKS INC.) 201341219 (2013-12-19) | 1-21 | Claims [62 A Jon 116888149 A (Innovent Biologics (Suzhou) Co., Ltd.) October 13, 2023 (2023-10-13) | 1-21 [] The remaining documents are listed on the next page in column C. See the patent family appendix. * Specific type of cited document: "T" Documents published after the application date or priority date, not in conflict with the application, but considered not particularly relevant for understanding purposes; "A" Documents indicating a general state of the prior art; "D" Documents cited by the applicant in an international application; "x" Documents deemed not to be protected; "e" Documents indicating that the claimed invention is not... <p 可 能 对 优 求 构 成 怀疑 的 文 ， 或 关 定 另一 篇 sh, 主公 个 水 4 elf i trad us mv ea heer ree pra ea ee aaem ar, sore vutb ge” 同族 专利 文件 "0” 涉 及 口头 公开 、 使 用、 展 览 其 他 方式 bo eer his "p” 公布 日 先 于 国际 申请 但 迟 所 要 优先 权 检索 实际 完成 期 报告 邮寄 2025 年1 月 16 22 isa xcn 名 称 和 地 址 受权 官员 中 国 家 知识 产权 局 管 冰 北京 市 海淀 区 门 桥 西 土 城 路 6 号 100088 a 电话 码 (+86) 010-53961945 pct 210 表 ( 第 2 页 ) (2022 年7 cn2024 129416 [ 栏 核 音 酸 氨基 序列 续 1 1.c 项 关于 任何 并 ,国际 是 基于 下 列 进 行 : a. 作为 一 部 分 提交>». (] For the purpose of international search, it shall be filed after the international filing date (Rules 13.3.10) ), []). Accompanied by a statement indicating that the sequence listing does not exceed the scope of disclosure of the filed international application. 2. [] The non-report was made within the scope of a meaningful search, taking into account any nucleotide and / or amino acid sequences disclosed in the international application without receiving a sequence listing conforming to WIPO's "ST.26" standard. PCTVISA / 210 Table (Continued from page 1) (July 2022) International Search Report International Application No. PCT / CN2024 / 129416 Section II [Comment that certain claims are considered unsearchable (Continued from page 1, item 2) Pursuant to Article 17(2)(a) of the Treaty, the reasons for not making an international search report for certain claims are as follows: 1. Claims 21 (partial) because they relate to a subject matter for which the entity is not required to conduct a search, namely: Claim 21 (partial) relates to the use of the composition of claim 19 or the compound of claim 20 as a pharmaceutical method for the treatment of diseases specified in PCT Rule 39.1 Civ). The international search and written opinion are based on the corresponding pharmaceutical use. 2. [L] Claims; Because they involve parts of the international application that do not meet the required requirements, no meaningful international search can be conducted, specifically: 3. L] claims; because they are dependent claims and not written in accordance with the requirements of the second and third persons in Rule 6.4. PCT / ISA / 210 Form (Continued on page 1) (July 2022) International Search Report International Application No. AT FAMERS ARSE, PCT / CN2024 / 129416 CN 109790215 A 2019465 A214 NZ 750566 A 2024464 A268 ECSP 19022190 A 2019465 A314 MY 194994 A20224F12 A304 CL 2019000836 Al August 2, 2019 BR 112019006074 = A2 June 18, 2019 AU 2017336867 Al February 28, 2019 AU 2017336867 -B2 March 14, 2024 IL 265605 A 2019465 A308 IL 265605 Bl February 1, 2024 IL 265605 B2 June 1, 2024 CA 3035681 Al April 5, 2018 DOP 2019000079 A November 15, 2019 WO 2018059502 Al April 5, 2018 US 2019284299 Al September 19, 2019 US 11498977. B2 20224F:11 A154 CO 2019003133 A2 April 12, 2019 PE 20190576 Al April 22, 2019 EA 201990285 Al December 30, 2019 Je 2019535650 A 20194612 A124 Je 7424727 = -B2 2024461 A308 KR 20190055813 A 2019465 A230 KR 102242990 Bl 2021464 A220 TN 2019000055 Al 2020467 A154 SG 11201901307 VA 2019563 A288 ZA 201901708 B 20224F:11 A304 MX 2019003563 A 201948 A124 SA 519401441 Bl 20224612 A274 PH 12019500655 Al August 5, 2019 EP 3504234 Al July 3, 2019 EP 3504234 Ad December 2, 2020 SG 10201913263. SA 2020463 A308 CN 104080811 A October 1, 2014 ES 2899956 = T3 March 15, 2022 HK 1200465 Al; August 7, 2015 HRP 20211773 TI1; March 4, 2022 AU2012332021 Al June 19, 2014 AU 2012332021 B2 July 20, 2017 AU 2012332021 A8 October 12, 2017 CA 2854233 ”Al May 10, 2013 CA 2854233 C May 12, 2020 HUE 056462 = I 2 February 28, 2022 RU 2014121832 A December 18, 2015 A108 RU 2675319 C2 June 12, 2018 A184 DK 2773671 = T3 November 15, 2021 PL 2773671 = T3 April 6, 2022 A244 PT 2773671 T November 12, 2021 A144 KR 20140092370 A July 23, 2014 A238 KR 102052774 Bl December 4, 2019 WO 2013063702 Al May 10, 2013 CY 1124872 -T1 April 6, 2022 A254 US 2015284470 Al October 8, 2015 PCT / ISA / 210 Form (Family Patent Annex) (July 2022) International Search Report International Application No. AT FAMERS ARSE, PCT / CN2024 / 129416 US 9732155 B2 August 15, 201January 18, 2018 US 10457742 B2 October 29, 2019 US 2013195849 A1 August 1, 2013 US 9574010 B2 February 21, 2017 EP 2773671 A September 10, 2014 EP 2773671 = A4 June 3, 2015 EP 2773671 Bi September 15, 2021 RS 62689 B1 December 31, 2021 RU 2018142832 A 2019563 A214 CN 109897103 A 2019466 A184 No CN 116096353 A 2023465 AOA MX 2022013112 A January 16, 2023 EP 4138778 Al March 1, 2023 WO 2021216916 Al October 28, 2021 US 2023272041 Al August 31, 2023 CA 3175809 Al October 28, 2021 IL 297495 A 20224612 A148 KR 20230004746 A January 6, 2023 JP 2023522972 A June 1, 2023 AU 2021260960 Al November 24, 2022 US 2013336973 Al December 19, 2013 WO 2013166594 Al November 14, 2013 WO 2013166594 AS February 6, 2014 JP 2015522525 A August 6, 2015 JP 6351572 B2 July 4, 2018 CA 2872540 Al November 14, 2013 US 2016257763 Al September 8, 2016 AU 2013258834 Al November 27, 2014 AU 2013258834 B2 September 7, 2017 EP 2847230 Al March 18, 2015 EP 2847230 = A4 December 16, 2015 EP 2847230 ”Bi August 12, 2020 ES 2843054 13 July 15, 2021 CN116888149 A October 13, 2023 TW 202233693 A September 1, 2022 TWI 825544 B December 11, 2023 US 2024034795 Al February 1, 2024 EP 4273161 Al November 8, 2023 WO 2022143912 ”Al July 7, 2022 PCT / ISA / 210 Form (Patent Family Annex) (July 2022) (12) International Applications Published Under the Patent Cooperation Treaty ~~ jo p=Ry 4 Zo > (19) ata LUAU AA AC AA A YN JA — enn — (10) International Publication Number (43) International Publication WO 2025 / 092987 A9 2025 4E 5 8 H (08.05.2025) WIPO! PCT 51) International Patent Classification Numbers: IR, IS, IT, JM, JO, JP, KE, KG, KH, KN, KP, KR, KW, KZ, CO7K 16 / 00 (2006.01) _ A61P.37 / 02 (2006.01) LA, LC, LK, LR, LS, LU, LY, MA, MD, MG, MK, MN, A61K 39 / 395 (2006.01) A6LP 35 / 00 (2006.01) MU, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM, PA, C12N 15 / 13 (2006.01) CO7K 16 / 46 (2006.01) PE, PG, PH, PL, PT QA, RO, RS, RU, RW, SA, SC, SD, (2) International application: ALE UA, UG, US, UZ, VC, VN, WS, ZA, ZM, ZW. International application date: November 1, 2024 (01.11.2024) 、 、 eee” (84) Designated countries, unless otherwise specified, require each available region (25) Application language: Chinese protection: ARIPO (BW, CV, GH, GM, KE, LR, LS, MW, MZ, (26) Publication language: Chinese NA, RW, SC, SD, SL, ST, SZ, TZ,UG, ZM, ZW), Eurasia, (AM, AZ, BY, KG, KZ, RU, TJ, TM), European training (AL, AT, BE, (30) RIAL: BG, CH, CY, CZ, DE, DK, EE, ES, FI, FR, GB, GR, HR, PCT / CN2023 / 129691 HU, IE, IS, IT, LT, LU, LV, MC, ME, MK, MT, NL, NO, 20234F-11 437 (03.11.2023) CN PL, PT, RO, RS, SE, SI, SK, SM, TR), OAPI (BF, BJ, CF, CD Applicant: Tuoji Pharmaceutical (Suzhou) Co., Ltd. CG, CI, CM, GA, GN, GQ, GW, KM, ML, MR, NE, SN, (PHRONTLINE BIOPHARMA (SUZHOU) CO., LTD) TD, TG). — [CN / CN]; FA EE) YT. is 4 oe M0 TH SR FB DX 8 AR TG 7 19th Floor, Building 1, Tianyun Plaza, No. 111 Wusongjiang Avenue” This international publication: . = 1915 % 215124 (CN). Shanghai Tuoji Pharmaceutical Co., Ltd. MEMTRI SRS CRAIN ARG). == Company GHANGHAIPHRONTLINE BIOPHARMA including the sequence listing portion of the instruction manual, as outlined in Section 5.2(a). — CO., LTD) [CN / CN]; HE] THAR BR 48) Publication date of the revised version: = TABS EK EATS 3 965 2830 1 ZF 201203 (CN). 2026 4-3 A 26 A (26.03.2026) mee (72) 2H A: EVI CAV, Siyi); Suzhou City, Jiangsu Province, China (15) Correction: > See the announcement published on March 26, 2026 (26.03.2026) at Tianyun Plaza, No. 111 Wusongjiang Avenue, Guobu Street, Zhongqu District — 1 ta 1-5 PE 19 21915 2 215124 (CN). Chen Zhaoyuan == (CHEN,Zhaoyuan; [EYL 9} A KIN TH HXBB <No. 111, Wusongjiang Tiandao, Yibujie Street, Tianyun Square, Building 1, Room 19219153215124 (CN). Huang, Mao; — Room 301, No. 396, Li Shizhen Road, Free Trade Pilot Zone, Pudong New Area, Shanghai, China 201203 (CN). SS (74) Agent: Zhongzi Law Office, Beijing (ZHONGZLLAW OFFICE); No. 26, Ping'anli Xidajie, Xicheng District, Beijing, China 100034 (CN). == (81) HERCR DATS, Beier pele = Paula): AE, AG, AL, AM, AO, AT, AU, AZ, BA, BB, BG, = BH, BN, BR, BW, BY, BZ, CA, CH, CL, CN, CO, CR, CU, = CV, CZ, DE, DJ, DK, DM, DO, DZ, EC, EE, EG, ES, FI, = GB, GD, GE, GH, GM, GT, HN, HR, HU, ID, IL, IN, IQ, = (54) Title: FC REGION-BASED HETERODIMER MOLECULE AND USE THEREOF N g (54) Invention Name: Fc Region-Based Heterodimer Molecule and Its Use = (57) Abstract: Provided are an immunoglobulin Fc region-based heterodimeric Fc stent, a heterologous multimeric protein containing a the heterodimeric Fc stent, in particular a single or multispecific binding protein, and a composition comprising same and a use. a (57) Abstract: HEARSE FP REPREARKIN ROR ARORA. ASA ARR OR ARR S RB fo) Heterologous multimeric protein, especially a single or multispecific binding protein, and a composition comprising the same andCompositions and uses thereof. WO 2025 / 092987 PCT / CN2024 / 129416 Heterodimeric molecules based on the Fe region and their uses Technical Field This invention relates to immunoglobulin Fe RH HORA Fe scaffolds, heterodimeric multimeric proteins comprising said heterodimeric Fe scaffolds, especially single or multispecific binding proteins, and compositions comprising the same and their uses. Background Art Bispecific antipodies are a rapidly developing class of novel antibody drugs in recent years. These antibodies can simultaneously bind to two different antigens or two different epitopes on the same antigen, thus offering and promising additional clinical benefits for the treatment of complex diseases. Currently, bispecific antibodies have many different structural forms, including non-IgG-like forms based on the fusion of antibody fragments such as scFv, Fab, VH, and VL, and IgG-like forms based on the Fo domain. Compared to non-IgG-like forms, bispecific antibodies with structures similar to conventional IgG exhibit better physicochemical properties, stability, and pharmacokinetic characteristics, showing broad application prospects. The antibody Fo region is known to have dimerizing function, playing a crucial role in maintaining antibody function in vivo. Several heterodimerization Fc techniques have been reported for assisting bispecific antibody production (Moore GL, Methods 2019, 154: 38-50). For example, Carter et al. invented the handle-hole model, successfully achieving bispecific antibody preparation (patent US7951917B1). In this method, specific small side-chain amino acids located at the CH3 region interface of the first heavy chain are mutated into large side chains and amino acids (e.g., T366Y), and specific amino acids located at the CH3 region interface of the second heavy chain are mutated into small side chains and amino acids (e.g., Y407T), thereby leading to...The mutated residues at the interface are spatially complementary, thereby promoting the formation of Fo heterodimers. Besides the example mutations mentioned above, more widely used mutation combinations in this method include the handle-hole T366W and hole-hole T366S, and the L368A and Y407V mutation combinations, which can produce Fc HARKs at a rate of 80-90%. However, the handle-hole model's ability to inhibit the formation of homodimer (Chomodimer) impurities is still insufficient. Electrostatic reversal is also widely used to promote Fc heterodimer formation. This method mutates a specific amino acid in one heavy chain CHB to a positively charged αAAR (Lys or Arg), and a specific amino acid in another heavy chain CH3 to a negatively charged amino acid (Asp or Glu). Through electrostatic attraction between the mutated residues, it promotes the formation of Po heterodimers. For example, Kannam et al. generated a high proportion of heterodimers by introducing mutations D399K and E3S6K into the CH3 region of the first heavy chain and mutations K409D and K392D into the CH3 region of the second heavy chain (Patent US8592562B2). Igawa et al. also promoted Fo heterodimer formation by introducing mutations E356K and D399K into the CH3 region of the first heavy chain and mutations K439E and K409D into the CH3 region of the second heavy chain (Patent US10011858B2). Genmab invented the DuoBody technology. This technology involves introducing the F40SL mutation into the CH3 region of the heavy chain of one antibody and the CH3 K409R mutation into the heavy chain of another antibody. By expressing and purifying the two antibodies separately, and then mixing them in the presence of a reducing agent (such as 2-MEA), a controllable Fab arm exchange between the two antibodies can be achieved. This technique produces Fo HORA KB 90-95% (patent US9150663B2). Fab arm exchange technology (SEED) is also used to promote Fo FF —HAA Ga (Muda et al. Proteins Eng. Des. Sel. 2011, 24: 447-454). This technology utilizes the sequence differences between IgA and IgG in the CH3 domain, generating Po HARA through the pairing of complementary CH3 domains, thus avoiding homodimer formation. Zymeworks reported a scaffold structure to improve the stability of Fo heterodimers (patent US20120149876A1). For example, one scaffold design includes a heavy chain CH3 region with T366L K392M, T394W mutations and another heavy chain CH3 region with F40SA, Y407A mutations, achieving a Tm value of 74sC. Additionally, one scaffold design 2a includes an L3S$1YY047A mutation in the CH3 region of one heavy chain and a T366VK409F mutation in the CH3 region of another heavy chain. This scaffold achieves a Tm value of 75.5°C. The proportion of Fo heterodimers reaches over 90%. Although various strategies for Fo heterodimer formation have been proposed, these strategies have shown certain limitations in application. Bispecific antibodies against Fo from Bio and BREA have homology mismatch and reduced thermal stability issues, thus affecting the druggability of antibody drugs. For example, it has been found that most Fo monoclonal antibodies formed using existing technologies have poor thermal stability. The Tm value of the CH3 domain of wild-type IgG1 monoclonal antibodies is generally greater than 80°C (Ionescu RM, J Pharm Sci. 2008, 97: 1414-1426). However, mutations in the CH3 and amino acids introduced through Fe heterodimerization technology can significantly reduce the melting temperature (Tm) of the CH3 domain. For example, the Tm value of CH3 HIRAM produced by handle-well 154% B1 (T366W / T366S, L368A, Y407V) is significantly lower.The value is 69.4°C (Atwell S, J. Mol. Biol. 1997, 270: 26-35). The CH3 HRA produced by (D399K, E356K / K409D, K392D) has a Tm of 124 at 68.8°C (Gunasekaran K, J. Biol. Chem. 2010, 285: 19637-19646). For structurally complex antibody drugs, lower Tm values ​​are desirable. This affects drug manufacturing and storage. While introducing non-natural disulfide bonds (such as Y349C and Y354C) into the handle-hole model can improve hetero-Fc dimer formation efficiency and thermal stability, for some drugs, such as antibody-drug conjugates (ADCs), introducing additional disulfide bonds can adversely affect product quality. Furthermore, the Fo produced by existing technologies... The heterodimer content is generally between 80-95%, with other impurities including Fe BAR, Fe homodimers, and high molecular weight polymers. These impurities need to be further removed by other methods such as ion exchange chromatography, especially the removal of homologous heavy chain mismatch impurities, which poses a challenge to downstream antibody purification processes. Moreover, the higher the impurity content, the lower the recovery rate of the purified product. This also means that, given the wide application prospects of IgG-like bispecific antibodies, there is still a need in the field to develop Fo heterodimerization technology to solve the chain mismatch and thermal stability problems of bispecific antibodies, in order to improve the druggability of bispecific antibodies. Summary of the Invention The inventors have discovered that, in a scaffold structure based on a dimerized Fo region, by introducing a specific CH3 domain interface mutation in addition to the introduction of a knock-in-hole (KIH) mutation, antibodies coated with this FeO scaffold can exhibit superior thermal stability compared to corresponding antibodies coated only with a KIH mutation; while maintaining the advantages brought by the KIH mutation (especially promoting the heavy chain of the target heterodimer).(Advantages of pairing), and maintains biological effects comparable to natural antibodies, including antigen-specific binding and FcRn LAR FeyR receptor binding. Based on this finding, the inventors have proposed a Fo heterodimerization mutant combination that can reduce the chain mismatch ratio and increase the stability of the target antibody product. Applying the mutant combination of the present invention can effectively improve the druggability and stability of bispecific antibodies. In a first aspect, the present invention provides a CH3 heterodimer containing the mutant combination of the present invention and a heterodimer Fc scaffold. In a second aspect, the present invention provides the use of the heterodimer Fc scaffold and the CH3 heterodimer of the present invention as components for constructing heteropolymeric proteins, especially single / multispecific binding proteins, and corresponding heteropolymeric proteins comprising the Fe scaffold or CH3 heterodimer. In a third aspect, the present invention particularly provides binding proteins comprising the heterodimer Fc scaffold according to the present invention. Fourthly, the present invention provides isolated polynucleotides encoding heterodimeric Fe scaffolds, CH3 HORA, and FRA RAE AR binding proteins according to the present invention; vectors (especially expression vectors) containing isolated polynucleotides of the present invention; and host cells containing isolated polynucleotides or vectors (especially expression vectors) of the present invention. The present invention also provides uses and methods for producing heterodimeric Fo scaffolds, CH3 heterodimers, heteropolymeric proteins, and binding molecules of the present invention using the polynucleotides, vectors, or host cells of the present invention. 2 WO 2025 / 092987 PCT / CN2024 / 129416 In a fifth aspect, the present invention provides compositions containing heteropolymeric proteins or binding proteins according to the present invention, and compositions containing heteropolymeric proteins or binding proteins according to the present invention.Conjugated therapeutic agents. In some embodiments, the composition is a pharmaceutical composition comprising the OHRERAEA of the present invention (especially the multispecific binding protein of the present invention) and a pharmaceutically acceptable carrier. In a sixth aspect, the present invention also provides methods of using the heteropolymer protein, binding protein, composition, and sustained agent according to the present invention, and their uses therein. In some embodiments, the present invention provides the use of the heteropolymer protein, binding protein, composition, or sustained agent according to the present invention as a medicament. In some embodiments, the present invention provides the use of the heteropolymer protein, binding protein, composition, or sustained agent according to the present invention for treating a disease in an individual in need. In a specific embodiment, the disease is cancer. The present invention also provides the FE of the heteropolymer protein, binding protein, composition, or sustained agent according to the present invention in the preparation of a medicament for treating a disease in an individual in need, and a method of treating an individual disease using the heteropolymer protein, binding protein, composition, or sustained agent according to the present invention, wherein the method comprises administering a therapeutically effective amount of the heteropolymer protein, binding protein, composition, or sustained agent according to the present invention to the individual. In some embodiments, the heteropolymeric protein, binding protein, composition, or slowed compound is formulated into a pharmaceutically acceptable form. In some embodiments, the disease is cancer. In some embodiments, the individual is preferably a mammal, particularly a human. Brief Description of the Drawings Figure 1 shows a schematic diagram of a partial crystal structure of the CH3-CH3 region of the Fe dimer. Figure 1A is a hydrophobic representation of the CH3-CH3 interface of wild-type IgG1 (PDB: 1HZH).Interactions. Figure 1B shows the hydrophobic interactions at the CH3-CH3 interface in the handle-hole model (PDB:4NQS). The left side of the figure represents the handle (knob) chain, and the right side represents the hole (hole) chain. Figure 1C shows the 3D structure of the CH3-CH3 region based on computer simulation of the M2 mutant combination. The hole L3S1Y mutation interacts with the handle T366W and E357 RRR Ra A CRRA, respectively. Figure 1D shows the 3D structure of the CH3-CH3 region based on computer simulation of the M3 mutant combination. The 10-F K409D and hole D399R mutations form new hydrogen bond networks (indicated by dashed lines) with multiple amino acids at the CH3-CH3 interface. Figure 2 shows the non-reducing SDS-PAGE electrophoresis diagram of the Ska VHH-Fe / Fe mutant combination. In Figure 2A, the transfection ratio of VHH-Fc and Fec plasmids is 1:1. Figure 2B shows the VHH-Fe Fe plasmid transfection ratio at 0.6:1. For each mutant combination shown, the heterodimer (AB) and other impurities (LISA ROR AFH HBA) contained in the purified protein A product exhibit different migration distances due to differences in molecular weight. Figure 3 shows the Tm value of the VHH-Fc / Fc mutant combination determined by DSC. Analysis of sample Tm fi. HANK EA SARE Cp value changes using Microcal PEAQ-DSC indicated one to three Tm values, with the highest Tm value reflecting the stability of the CH3 domain. Figure 4 shows the non-reducing SDS-PAGE electrophoresis image of the VHH-Fc / Fab-Fc mutant combination. For each mutant combination shown, the heterodimer (ABC) and other impurities (homodimemers, haptens, free monomers) contained in the purified protein A product exhibit different migration distances due to differences in molecular weight. Figure 5 shows the non-reducing SDS-PAGE electrophoresis pattern of the Fab-Fc mutant. For the shown...For each mutant, the heavy chain to light chain plasmid transfection ratio was 1:1.3. For each mutation shown, the half-antibody monomers, disulfide-linked homodimers, and other impurities (free light chains and free heavy chains) contained in the protein A purification product exhibited different migration distances due to differences in molecular weight. Figure 6 shows the in vitro assembly effect of the Fab-Fc mutant combination detected by HIC-HPLC. The half-antibody purified by protein A affinity showed two main peaks, representing either the monomer or the homodimer. The bispecific antibody generated in vitro showed a heterodimer main peak, with other smaller peaks representing half-antibody monomers or homodimer impurities. Figure 7 shows the binding activity of the bispecific antibody to the antigen as determined by ELISA. Figure 8 shows the binding affinity constant of the bispecific antibody to FcRn and FoFoyRI as determined by Fortebio. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. Furthermore, the materials, methods, and examples described herein are illustrative only and are not intended to be limiting. Other features, objects, and advantages of the invention will be apparent from this specification and the accompanying drawings and from the appended claims. The term "about" when used in conjunction with a numerical value means to encompass a range of numerical values ​​having a lower limit of less than a specified numerical value and an upper limit of more than a specified numerical value. In this document, the terms "comprising" or "including" mean including the stated elements, integers, or steps, but do not exclude any other elements.Integers or steps. For example, when referring to the Fc RAS CH domain, it encompasses the Fc region consisting of or primarily consisting of the CH3 domain, as well as the Fe region which also contains other constant domains such as CH2. In this document, RIE Fo RRA is a scaffold protein dimer consisting of or primarily consisting of paired and dimerized immunoglobulin Fc regions. This dimer structure acts as a scaffold, at which other functional molecules, including, but not limited to, binding domains, biologically active peptides or fragments thereof, and toxins, can be linked or ligated to its N-terminus or C-terminus. In some respects, this scaffold can be used in particular as a component for constructing multispecific (e.g., bispecific) antibodies or immunoadhesives. In this document, the term "heteromeric protein" refers to a molecule containing at least a first polypeptide and a second polypeptide, wherein the second polypeptide differs from the first polypeptide in its amino acid sequence by at least one amino acid residue. Preferably, the heteromeric protein is a single-specific or multispecific binding protein containing at least one target-binding domain. In some aspects, the first and second polypeptides of the heteropolymeric protein include polymerizing domains (e.g., the first and second CHB domains of the CH3 heterodimer according to the invention; the first and second Fo regions of the RARE AR A ROR MA Fo scaffold), wherein the polymerizing domains facilitate the interaction of the first and second polypeptides at the interface and the formation of a "heterodimer". In some aspects, the heteropolymeric protein may include a "heterodimer" formed by the first and second polypeptides, or other polypeptides may be present in addition to the first and second polypeptides to form a higher-order tertiary structure. Examples of heteropolymeric proteins include, but are not limited to, single-specific or multi-specific binding proteins, such as...Bispecific antibodies, bispecific immunoadhesins, or antibody / immunoadhesin complexes. As used herein, the terms "binding protein" and "binding molecule" are used interchangeably and, in their broadest sense, refer to a protein molecule that specifically binds to at least one target. An example of a binding protein is an antibody and an immunoadhesin. "ETP, RIK Fo scaffold binding protein" refers to a binding protein comprising a Fo scaffold and at least one target-binding domain connected thereto, or consisting primarily of a Fo scaffold and at least one target-binding domain connected thereto. In some embodiments, the binding protein comprises one (and only one) Fc scaffold. In other embodiments, the binding protein may comprise more than one Fc scaffold. In this document, the term "single-specific" means that the binding molecule is capable of specifically binding to one and only one specific target (e.g., one epitope). The term "multi-specific" means that the binding molecule is capable of specifically binding to at least two different targets (e.g., at least two different epitopes). Typically, a multi-specific binding molecule comprises at least two target-binding domains that are specific to different targets. In some embodiments, the different targets may be polypeptides or proteins or portions thereof located on the same cell, different cells, the same antigen, or different antigens, such as receptors, ligands, or antigenic epitopes. In some embodiments, the multi-specific binding molecule is bispecific, capable of simultaneously binding to two different targets, such as two different epitopes, for example, Two different epitopes expressed on two different cells, or on two different antigens, or on the same antigen. In this paper, the term "valence" indicates the binding of epitopes to cells.A specific number of target-binding domains exist in a binding molecule. Therefore, the term "monovalent" in relation to a binding molecule indicates the presence of one (and no more than one) target-specific binding domain in the binding molecule. Correspondingly, the term "multivalent" indicates the presence of multiple target-specific binding domains in the binding molecule. For example, a "tetravalent" binding molecule means that the binding molecule contains a total of four target-specific binding domains, regardless of whether the targets targeted by the binding domains are the same. In this document, the term "target-binding domain" refers to a portion of a polypeptide or protein that provides interaction with a target. Exemplary binding domains include the antigen-binding domain of an antibody, a ligand, a receptor-binding domain of a ligand, and a ligand-binding domain of a receptor. In some aspects of the invention, a preferred target-binding domain is the antigen-binding domain of an antibody. The antigen-binding domain of an antibody typically contains amino acid residues from the complementarity-determining region (CDR) of the antibody. Natural immunoglobulin molecules typically have two antigen-binding domains, while Fab molecules typically have a single antigen-binding domain. In this document, the term "antigen-binding domain" refers to an antibody or fragment thereof that specifically binds to an antigenic epitope. In some specific embodiments, the antigen-binding domain comprises an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL), or, when the antibody is a heavy chain antibody, a single heavy chain variable region (VHHD). In some embodiments, the antigen-binding domain may also include an antibody constant region. Available heavy chain constant regions include any one of the following five types: o, β, s, γ, or he. Available light chain constant regions include the following two...Any of the following types: K and λ. Therefore, in some aspects, the specific antigen-binding domain may be selected from, including, but not limited to: antibody fragments containing the heavy chain variable region (VHD) and the light chain variable region (VD) of the antibody, such as Fv, scFv, Fab, scFab, crossFab, and antibody fragments containing the heavy chain variable region of the heavy chain antibody, such as VHH. In this document, the terms "antigen determination" and "epitope" are used interchangeably and refer to a site on a macromolecule such as a polypeptide or protein that interacts with the antigen-binding domain and forms a complex. In some aspects, an epitope may be, for example, a linear epitope consisting of consecutive amino acid segments, or a conformational configuration consisting of discontinuous amino acid residues located in different regions. According to the invention, useful antigen determinations may be, for example, on the surface of tumor cells, the surface of virus-infected cells, the surface of other diseased cells, the surface of immune cells, or present in free macromolecules and / or the extracellular matrix (ECM) in body fluids. In this document, the term "antigen" refers to, Large molecules that can elicit an immune response in mammals, thereby producing specific antibodies against them. In some aspects, antigens useful according to the invention are polypeptides or proteins, including, but not limited to, naturally occurring forms of proteins derived from vertebrates and their variants, fragments, or derivatives. The vertebrates mentioned include mammals, such as primates, for example, humans. Hereinafter, the term "immunoglobulin" refers to a protein having a structure that produces naturally occurring antibodies. For example, IgG immunoglobulins are heterotetrameric glycoproteins of about 150,000 Daltons, composed of two light chains and two heavy chains linked by disulfide bonds. Each immunoglobulin heavy chain...Each immunoglobulin light chain has a heavy chain variable region (VH), also called the heavy chain variable domain, followed by three heavy chain constant domains (CH1, CH2, and CH3), also called the heavy chain constant domain. Similarly, from the N-terminus to the C-terminus, each immunoglobulin light chain has a light chain variable region (VL), also called the light chain variable domain, followed by a light chain constant domain (CL), also called the light chain constant domain. In IgG antibody molecules, the VH-CH1 of the heavy chain typically pairs with the VL-CL of the light chain to form a Fab fragment that specifically binds to the antigen. Therefore, an IgG immunoglobulin is essentially composed of a Fab molecule and two dimerized Fc regions. The heavy chains of immunoglobulins can be classified into one of three types based on the type of their constant domains, referred to as a(IgA), S(gD), e(IgE), yUgG), and yUgG), yUgM, some of which can be further subdivided into subtypes, such as yIl(gG1), yIl(gG2), yIl(gG3), yIl(gG4), yIl(gG1), yIl(gG2), yIl(gG2). The light chains of immunoglobulins can also be classified into one of two types based on the amino acid sequence of their constant domains, referred to as K and human. In this document, the term "antibody" refers to a polypeptide containing one or more domains that bind to epitopes on antigens of interest, wherein the binding domain has a variable region sequence derived from or sequence-identical to that of immunoglobulins. This term encompasses a wide range of antibody structures, including but not limited to, single-chain antibodies, mono / multispecific antibodies, mismatched antibodies, humanized antibodies, human sequence antibodies, full-length antibodies, and antibody fragments, as well as antigen-binding compounds and antibodies assembled from them as components, provided that they exhibit the desired antigen-binding activity. Typically, antibodies are variable via their heavy chains.Three complementarity-determining regions (HCDR1-3) in the variable region of the heavy chain (VH) and three complementarity-determining regions (LLCDR1-3) in the variable region of the light chain (VDL) form an antigen-binding domain on the surface of the VH-VL dimer. The six CDRs confer specific binding of the antibody to the antigen. However, in the case of heavy-chain antibodies such as those from camelid heavy-chain antibodies, the antibody can confer specific binding of the antibody to the antigen through three complementarity-determining regions (CDR1-3) in its single VH domain (also referred to as the VHH domain in this article). The VHH domain, like the variable regions of the heavy and light chains of conventional IgG antibodies, contains four conserved framework regions (FR) and three complementarity-determining regions (CDR), and is arranged in the order of FR1-CDR1-FR2-CDR2-FR3-CD3-FR4. In this article, "complementarity-determining region" or "CDR region" or "<CDR" or "hypervariable region" is the region in the variable region of the antibody that is highly variable in sequence and forms a structurally defined loop (<hypervariable loop>] and / or contains antigen-contact residues (:antigen contact points four). CDRs are mainly responsible for binding to the antigen epitope. In the VHH domain and VHVVL domain of the antibody of the present invention, the CDRs are sequentially numbered starting from the N-terminus and are usually referred to as CDR1, CDR2, and CDR3. The CDR sequences in a defined VHH domain and a defined VHVAVL domain can be determined using methods well known in the art. Those skilled in the art can easily determine the CDR sequence ranges of the amino acid sequences of any given antibody variable region on http: / / www.abysis.org / abysis / , including the region ranges and their combined ranges of CDRs defined by the Kabat, AbM, Chothia, Contact, and IMGT methods. Unless otherwise stated, and otherwise in this inventionIn this document, the term "CDR" or "CDR sequence" encompasses a CDR sequence determined in any of the foregoing methods and combinations thereof, but preferably a CDR sequence defined according to the Kabat scheme. In this document, the term "antibody fragment" refers to a non-complete portion of an antibody containing an antigen-binding domain. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab'):, diabody, linear antibodies, single-chain antibodies (e.g., scFv, scFab), and single-domain antibodies (sdAb). The amino acid sequence of VH or VL in an antibody fragment can be modified by substitution, deletion, addition, and / or insertion, as long as the antigen-binding energy is maintained. Furthermore, variable regions of antibody fragments can be conjugated and humanized. In this document, "Fv domain" refers to the smallest antibody fragment including both the complete antigen recognition and binding sites. "Fw" is a dimer formed by a VH and a VL bond strongly linked by a non-covalent bond (VH-VL = RA). In this paper, "scFv domain" refers to a single-chain polypeptide in which two variable regions (typically a VH and a VL) are linked via a linker to form an antigen-binding domain essential for antigen binding. In this paper, the term "Fab BHAA, AMP RIL eG" refers to an antigen-binding domain in antibodies formed by the pairing of the heavy chain variable region VH and the heavy chain constant region CHI (VH-CHI1) with the complementary light chain variable region VL and the light chain constant region CL (VL-CL). In this paper, the term "crossFab" or "crossFab domain" refers to a Fab domain where CHI and CL exchange, i.e., an antigen-binding domain formed by the pairing of VH-CL and VL-CHI1. In this paper, the term "ScFab" refers to a Fab domain linked as a single polypeptide chain via an artificial linker. In thisIn this document, the term "VHH" or "VHH domain?" refers to a heavy chain variable domain derived from a heavy chain antibody lacking a light chain. Therefore, VHHs differ from the conventional VHs of four-chain immunoglobulins in that they do not require pairing with a light chain variable domain to form an antigen-binding domain. Such VHH molecules can be derived from antibodies produced in camelid species (e.g., camels, alpacas, dromedaries, llamas, and guanacos). Other species besides camelids may also produce naturally occurring heavy chain antibodies lacking a light chain, and these VHHs are also within the scope of this invention. In some cases, for the therapeutic application of VHHs, it is desirable to reduce their immunogenicity. Therefore, preferably, the VHH domain used in the binding protein of this invention comprises a humanized sequence. In this document, "immunoglobulin constant domain" refers to a constant domain derived from or obtained from the heavy chain (e.g., human IgG1 heavy chain) or light chain of immunoglobulins, including heavy chain constant domains CH1, CH2, CH3, and optionally CH4; and light chain constant domain CL. This term includes both native and variant sequence constant domains. In this document, the "type" or "subtype" of an immunoglobulin constant domain refers to the type or subtype determined based on its amino acid sequence. Heavy chain constant domains can be classified into five distinct types based on their sequence: IgA, IgD, IgE, IgG, and IgM, or further into subtypes (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. Therefore, in this paper, when referring to the IgG1 Fe region or CH3 domain, it means that the Fe region or CH3 domain, based on its amino acid sequence, can be classified as IgG type, and further classified as IgG1 subtype. Similarly, the light chain constant domain can be classified according to its sequence.The KK and CH2 and CH3 domains are included. Those skilled in the art can readily determine the type or subtype of the constant domain by comparing the amino acid sequence of the constant domain with the sequences of the corresponding constant domains of different types or subtypes of natural immunoglobulins. In this document, when the heterodimeric Fo scaffold or heteropolymeric protein molecule of the present invention contains multiple immunoglobulin heavy chain constant domains, it should be understood that they can be selected independently of each other according to the intended function or use of the molecule. As an example, for the heteropolymer of the present invention containing the CHI1 domain and the CH2 and CH3 domains, all three constant domains can be of the IgG1 subtype, such as human IgG1, or only the CH3 domain or the CH2 and CH3 domains can be of the IgG1 subtype, such as human IgG1. In this paper, "IgG conformation" refers to an antibody conformation identical to that of IgG immunoglobulin, essentially consisting of two Fab domains linked to the N-terminus of a dimerized Fe domain via immunoglobulin and sclerochain regions (or, where appropriate, via flexible linker peptides). Thus, in a typical case, an IgG conformation antibody consists of two heavy chains and two light chains, where each heavy chain has VH, CH1, CH2, and CH3 domains at its ANSI C-terminus; and each light chain has VL and CL domains from its N-terminus to its C-terminus. In this paper, "IgG-like conformation" refers to an antibody that retains the same stereotyped structural features as IgG immunoglobulin, but with one Fab domain missing, and / or at least one Fab domain replaced by a binding domain of a different conformation (e.g., scFv, VHH, ligand, or receptor ligand-binding domain).Such IgG-like conformation antibodies can be described by specifying the type of binding domain attached to the Fo dimer scaffold. For example, VHH-Fc / Fc is a monovalent IgG-like conformation antibody composed of the VHH-EFc 4 Jk Fe Fe peptide; VHH-Fc / Fab-Fc antibody is a divalent IgG-like conformation antibody composed of the VHH-Fc peptide and the Fab-Fc peptide. In this document, the term "immunoadhesin" refers to an antibody-like protein molecule formed by the fusion of a non-immunoglobulin binding domain with desired binding specificity (e.g., a binding domain derived from a cell surface receptor or ligand, or the ligand itself) with an immunoglobulin constant domain (e.g., the Fo region or CH3 domain). The immunoglobulin constant domain can be derived from any immunoglobulin, such as IgG, especially IgG1, IgG2, IgG3, or IgG4. In this paper, the term "Fc MBCA Fo region" is used to define the immunoglobulin and albumin heavy chain constant region portion containing the CH3 domain or a fragment thereof. In some cases, this immunoglobulin and albumin portion may also contain one or more other immunoglobulin constant domains or fragments thereof, including the 46 244 KY CH or CH2 domains. In this paper, when referring to the amino acids of the Fc region and constant domains, the EU numbering system (also known as the EU index) described in Kabat et al., Sequences of Proteins of Immunological Interes, 1991, Public Health Service, National Institutes of Health, Bethesda, MD, 1991 is used. When referring to the human IgG Fo region, the EU numbering of the amino acid residues can also be found in the IMGT Scientific chart.(https: / / www.imgtore / IMGTScientificCharVNumbering / Hu_IGHGnberphtoml), which is hereby incorporated by reference. According to this numbering, in the human IgG1 immunoglobulin heavy chain, amino acids 118-215 are CH2 domains, amino acids 216-230 are CH2 domains, and amino acids 231-340 are CH3 domains. In some cases, the CH3 domain (C Kae BER <Lys447) may be absent. Those skilled in the art can readily determine the constant domains contained in the heterodimeric Fc region and binding protein according to the invention, their types / subtypes, and species origins, by sequence alignment with the published constant regions of native immunoglobulins. In some embodiments, the first and second Fe regions of the heterodimeric Fc scaffold according to the invention contain or are composed of CH3 domains. In some embodiments, the Fo region further comprises a CH2 domain. In other embodiments, the Fo region further comprises a homogeneous region or a partially sharp-chain region, for example, including the lower sharp-chain portion of the tungsten sequence "CPPCP". As will be understood by those skilled in the art, the CHB heterodimer of the present invention is a specific form of the heterodimer Fc scaffold according to the present invention, in which the first and second Fe regions of the Fo scaffold are composed of or substantially composed of CH3 domains. Therefore, unless explicitly stated otherwise, the description of the heterodimer Fc scaffold of the present invention is equally applicable to the CH3 heterodimer according to the present invention. In this document, the term "Fc CEH BOA Fe region" encompasses both native sequence Fc regions and variant sequence Fc regions. In this document, the term "native sequence Fo region" encompasses various naturally occurring immunoglobulin Fe region sequences, such as various Ig isotypes.The Fc region sequence of IgG and its allotypes (Gestur Vidarsson et al., IgG subclasses and allotypes: from structure to effector functions, 20 October 2014, doi: 10.3389 / fimmu.2014.00520.) refers to the Fc region polypeptide relative to the native sequence FeO RSMAS MH. The modification can be the addition, deletion, or substitution of amino acid residues. Substitution can include both naturally occurring and non-natural amino acids. The purpose of the modification can be to alter the physicochemical properties of the Fe region, such as thermal stability and heterodimerization tendency, and / or the binding of the Fc region to its receptor and its effector functions. In this paper, "Fo scaffold" is also referred to as "Fec scaffold dimer" or "dimer Fo RR". ALP, RIE RO RG' Fe scaffold 7 WO 2025 / 092987 PCT / CN2024 / 129416 refers to the fact that the first and second Fc regions constituting the Fo scaffold are identical in sequence. Correspondingly, a "heterodimer" Fec scaffold refers to the fact that the first and second Fo regions constituting the Fc scaffold are identical in sequence. In this paper, the term "CH3 dimer" refers to a pair of paired and dimerized CH3 domains. The term "CH3 MARA, Am" refers to a dimer in which the first and second CD3 domains are identical in sequence. Correspondingly, "CH3 heterodimer" refers to a dimer in which the first and second CD3 domains differ in sequence by at least one amino acid residue. In this paper, when a Fo-based heteropolymeric protein (e.g., a mono / multispecific binding protein) encapsulates only one Fc scaffold, such a polymeric protein can be considered as composed of two Fc regions based on the two Fo regions of the scaffold.A dimer consists of one Fo member of the Fe scaffold (COPPA), MARMARA, AP, or FOR, and a polypeptide (e.g., a target-binding domain, which may be 0, 1, or more) attached to it, constituting one protein unit (i.e., monomer). Another Fe member of the Fe scaffold and a polypeptide (e.g., a target-binding domain, which may be 0, 1, or more) attached to it constitute another protein unit (i.e., monomer) of the dimer. In this case, it should be understood that each monomer constituting the dimer can be a single or multiple polypeptide chains. When two monomers are identical in sequence structure, i.e., they are copies of each other, the dimer is referred to herein as a homodimer. For example, native IgG immunoglobulin is a typical example of this homodimer. When two monomers are different, for example, differing in the Fo sequence and / or the sequence of the binding domain attached to it, the dimer is referred to herein as a heterodimer. For example, bispecific IgG antibodies or IgG-like antibodies are typical examples of this type of heterodimer. Mono / multispecific antibodies based on heterodimeric Fe scaffolds are another typical example of such heterodimers. In this document, the term "purity" in relation to the heteropolymeric protein according to the invention (e.g., the binding protein according to the invention) refers to the ratio of the heteropolymeric protein to the total protein in the purified product after expression and purification from host cells. The purity of the target product can be determined by measuring the ratio of the target heteropolymeric protein product to various chain mismatch products in the purified product using SEC-HPLC. Preferably, the purity is greater than 90%. In this document, the term "purity" in relation to the heteropolymeric protein according to the invention (e.g., the binding protein according to the invention) refers to the heteropolymeric protein according to the invention (e.g., the binding protein according to the invention)The term "thermal stability" related to the protein (as defined by DSC) refers to, for example, the protein exhibiting a CH3 Tm value greater than 705°C; or, when the protein is an antibody of IgG conformation, exhibiting a CH3 Tm value similar to that of natural human IgG1 immunoglobulin (e.g., +2°C, preferably +1°C). The heterodimeric multimeric protein of the present invention, in the HEAT HORA Fe scaffold, preferably has its Tm value determined without introducing non-natural diacid bonds into the heterodimeric Fo scaffold. In this document, when referring to a single mutation, it is described by the position of the amino acid residue at which the mutation occurs and the amino acid residues before and after the mutation, denoted as [original and amino acid residues] mutated residue position [mutated amino acid residues]. For example, the substitution of threonine at position 366 of the Fc region with chromoic acid is denoted as T366W; the substitution of uric acid at position 409 of the Fe region with chromoic acid is denoted as Y407V. When referring to multiple mutation combinations, mutation combinations located on the same polypeptide chain are connected by the symbol "", for example, the mutation combination occurring in hole #14) L351Y and D399R can be represented as "L351Y-D399R"; for mutation combinations located on different polypeptide chains, the symbol "1" is used to separate them, for example, the combination of the L351Y and D399R mutation occurring on the hole chain and the region 409D mutation occurring on the knob chain can be represented as "hole L351Y-D399R / knob K409D". In this document, the terms "flexible linker" or "peptide linker" are used interchangeably and refer to a short amino acid sequence consisting of amino acids, such as glycosyl (G) and / or serine (S) and / or threonine residues (T) used alone or in combination, or homogeneous regions or modified forms of immunoglobulins. In this document, the term "flexible linker" is used interchangeably with "peptide linker".The term "modified compound" refers to a protein-binding modification or derivative formed by covalently linking or modifying other molecules (e.g., therapeutic or diagnostic molecules) to a target protein (e.g., the heteropolymeric protein or binding protein according to the invention). Examples of such other molecules include, but are not limited to, proteins / peptides / peptides, markers, drugs, and cytotoxic agents, such as: radioisotopes; chemotherapeutic agents; growth inhibitors; enzymes and fragments thereof; fluorescent reporter proteins; antibiotics; toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant, or animal origin, including fragments and / or variants thereof; and various known antitumor or anticancer agents. In this document, the "percentage of identity (%)" of an amino acid sequence refers to the number of positions in the comparison window where the candidate sequence shares the same amino acid residues as the specific amino acid sequence, without considering any conserved substitutions as part of the sequence identity. This is achieved by comparing the candidate sequence with the specific amino acid sequence within a comparison window, and, if necessary, introducing vacancies to achieve the maximum percentage of sequence identity, without considering any conserved substitutions as part of the sequence identity. Unless otherwise specified, the comparison window is the full length of the specific amino acid sequence. In this document, when referring to the constant domains CH2, CH3, and Fc, the term "wild-type" means that the constant domain has a sequence derived from the constant domain of a native immunoglobulin, or has no more than 1-5 amino acid residue changes compared to the native sequence (preferably no more than 1, 2, 3, 4, or 5 amino acid residue changes, more preferably conserved amino acid substitutions). For polypeptide sequences,"Conservative modifications" include substitutions, deletions, or additions to the polypeptide sequence that result in the replacement of a certain amino acid with a chemically similar amino acid. A table providing a list of conserved substitutions for functionally similar amino acids is known in the art. The following eight groups contain JKRs with mutually conserved substitutions: 1) 75 BURA; HERG); 2) Aspartic acid (D), glutamic acid (E); 3) Asparagine (N), glutamic acid (Q); 449 BARR), lysine (K); DHS ARD, RRL; P RAM; MARV); 6) Theopropylamine (F), glutamic acid (Y), cromoglycine (W); 7) Seric acid (S), threonine (T); and 8) Cysteine ​​(C), formic acid (M) (see, for example, Creighton, Proteins (1984)). In this document, the term "host cell" refers to a cell into which a foreign polynucleotide has been introduced, including progeny of such cells. Host cells include "transformers" and "transformed cells," which include primary transformed cells and their derived progeny. Host cells are any type of cell system that can be used to produce the polypeptides or protein molecules of the present invention, including eukaryotic cells, such as mammalian cells, cytokines, yeast cells; and prokaryotic cells, such as *E. coli* cells. Host cells include cultured cells, as well as cells within transgenic animals, transgenic plants, or cultured plant or animal tissues. In this document, the term "expression vector" refers to a vector containing recombinant polynucleotides, which contains an expression control sequence that effectively links the nucleotide sequence to be expressed. The expression vector contains sufficient cis-acting elements for expression; other elements for expression may be provided by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, including but not limited to granules, plasmids (e.g.,...Viruses (naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses). In this document, the term "individual" refers to a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and semi-humans (e.g., mice and rodents). Specifically, the individual is a human. In this document, the term "treatment" refers to a clinical intervention intended to alter the natural process of a disease in an individual receiving treatment. Desired therapeutic effects include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or mitigating the disease state, and alleviating or improving prognosis. In this document, the terms "tumor" and "tumor" are used interchangeably to refer to or describe a physiological disorder in mammals characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, solid tumors, and liquid tumors. I. HARA Fe scaffold: As shown in the examples, by introducing the specific Fc mutation combination of the present invention, the heterodimeric Fc scaffold according to the present invention, after expression in cells and purification by affinity chromatography, produces a Fo heterodimer ratio of over 90%, higher than the control Fc protein containing only the tungsten-corresponding knob-into-hole mutation; and after further purification by ion exchange column, it can reach OBS UALR. HE, MET TIRE Fe EG. The purified Fo heterodimer has better thermal stability, wherein the Tm value of the CH3 domain determined by DSC can reach over 74°C. In a first aspect, therefore, the present invention provides a heterodimeric Fe scaffold that can be used to enhance the formation of desired heterodimers.By using the heterodimeric scaffold according to the present invention, the yield of the desired heterodimeric product relative to unwanted heterodimeric and homodimeric impurities can be greatly improved, thereby increasing the production efficiency and cost of the desired heterodimeric product; at the same time, the thermal stability of the desired heterodimeric product is improved. The heterodimeric Fc scaffold according to the present invention comprises two paired and heterodimeric immunoglobulin Fec regions, wherein the two Fec regions distinguish the WEL CH3 domain. The heterodimeric Fo scaffold according to the present invention is characterized in that, in the CH3 AHP, RRS knob-into-hole (KIEHD) mutation, and further comprising the following mutations: (a) the &4 hole mutation Fe region further comprising the L351Y mutation; or (b) the hole mutation-containing Fo region further comprising the L351Y and D399R mutations, and comprising the knob mutation Fe RiL&* K409D. In this document, in some cases, for the sake of brevity, the above combination of mutations is also referred to as "the CH3 interface characteristic mutation of the present invention". Similarly, for the sake of brevity, the Fe region having the hole mutation and the polypeptide containing it are also referred to as the hole chain; and the Fo region having the corresponding knob mutation is also referred to as the hole chain. The region and the polypeptide containing it are called the knot chain. Accordingly, when referring to other mutations introduced on the hole chain, the expression "hole?" or "hole" is added before the mutation to clarify that the mutation occurs on the hole chain; similarly, when referring to other mutations introduced on the knot chain, the expression "knob?" or "*knob" is added before the mutation to clarify that the mutation occurs on the knot chain. For example, holeL351Y refers to the protein introduced into the hole chain at the FcK L351 position.The knob K409D refers to the substitution of a D residue at the K409 position in the Fe region of the knob chain. Knob-into-hole (KIH) mutation techniques are known in the art. See, for example, US7951917B1; US ​​5.731,168; US 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001). Typically, this mutagenesis technique involves introducing a protrusion (a "handle" or "knob" mutation) at the Fo region CH3 domain interface of the first polypeptide and a corresponding cavity (a "hole" or "hole mutation") at the Fe region CH3 domain interface of the second polypeptide, allowing the protrusion to be positioned within the cavity to promote the formation of a heterodimer between the first and second polypeptides and inhibit the formation of a homodimer. The protrusion can be constructed by replacing a small amino acid side chain from the interface of the first polypeptide with a larger side chain (e.g., acetic acid or tryptophan). By replacing the large amino acid side chain with a smaller amino acid (e.g., propionic acid or threonine), a complementary cavity of the same or similar size to the protrusion can be created at the interface of the second polypeptide. Such combinations of complementary protrusions and cavities in spatial location and size are referred to in the art as KIH mutations, wherein the protrusion mutation is referred to as the knot of the KIH mutation. In one specific embodiment, therefore, the Fe stent according to the invention comprises a KIH mutation, wherein the CH3 domain of one of the first and second Fe regions of the Fe stent comprises a knot RB; the CH3 domain of the other of the first and second Fe regions of the Fo stent comprises a complementary corresponding hole mutation. In a more specific embodiment, in the first and second Fe regions of the Fo stent according to the inventionIn the CH3 domain of one of the Fo regions (e.g., the first Fc region), amino acid residues are introduced for substitution, such that one or more amino acid residues are replaced by amino acid residues with larger side chain volumes, thereby creating a protrusion in the CH3 domain of that Fe region. Simultaneously, in the CH3 domain of the other of the first and second Fo regions of the Fo scaffold according to the invention (e.g., the second Fe region), amino acid residues are introduced for substitution, such that one or more amino acid residues are replaced by amino acid residues with smaller side chain volumes, creating a cavity in the CH3 domain of that Fc region that is spatially complementary to the aforementioned protrusion. Thus, a protrusion present in the CH3 domain of one Fe region can be localized in a cavity present in the CH3 domain of the other Fe region. The protrusion and cavity can be prepared by altering the nucleic acid encoding the polypeptide, for example, through site-specific mutagenesis or peptide synthesis. In some more specific embodiments, the Fo scaffold according to the invention comprises a KIH mutation, wherein the introduced knob mutation is T366W and the introduced hole mutation is Y407V. In other more specific embodiments, the Fo scaffold according to the invention comprises a KIH mutation, wherein the introduced knob mutation is T366W and the introduced hole mutation is T366S-L368A-Y407V. In some embodiments, the Fe region of the heterodimeric Fo scaffold according to the invention is composed of or primarily of a CH3 domain. In this embodiment, the invention thereby provides a CH3 HORA according to the invention. In some embodiments, in addition to the CH3 domain, the heterodimeric Fc scaffold according to the invention or the heteropolymeric protein according to the invention comprising said scaffold also comprises other immunoglobulin and protein constant domains, such as CH2 and / or homopolymers in the Fo region.The region, and / or the target-binding domain includes CH1 and / or CL domains linked to VH or VL. In such embodiments, these immunoglobulin constant domains may have the same or different species origin (but preferably all from humans), and / or have the same or different immunoglobulin types or subtypes (but preferably the same IgG type or IgG subtype). In one embodiment, the Fc scaffold according to the invention includes CH2 and CH3 domains from IgG (especially human IgG). In one embodiment, the Fo scaffold according to the invention also includes a CH1 domain from IgG (especially human IgG). As those skilled in the art will understand, when referring to the species origin and type of the immunoglobulin constant domain, it means that the constant domain contains a natural amino acid sequence or 10 amino acid residues from the immunoglobulins of that species and type. For the purposes of this invention, the variant sequence has a variation of no more than 10 amino acid residues relative to the corresponding natural sequence. As an example, the structural features of various IgG subtypes can be found in the review by Gestur Vidasson et al. (IgG Subclasses and Allotypes: From Structure to Effector Functions, Front Immunol. 2014; $: S20, doi: 10.3389fimmu.2014.005$20), which is incorporated herein by reference in its entirety. In some embodiments, the immunoglobulin constant domain contained in the heterodimeric Fo scaffold or the heteromultimeric protein body of the present invention is the constant domain of IgG immunoglobulin.In some embodiments, the constant domains may be independently selected from different IgG1 subtypes, such as IgG1, IgG2, IgG3, and IgG4 LA, KALA IgG1 subtypes or IgG4 subtype. In some embodiments, the constant domains are human-derived IgG immunoglobulin constant domains. In some specific embodiments, the heterodimeric Fe scaffold according to the invention comprises an IgG Fe region. In some more specific embodiments, the Fc region is an IgG1 Fc region. In other specific embodiments, the Fc region is an IgG4 Fc region. In some embodiments, the Fo region of the heterodimeric Fe scaffold according to the invention has an effector function. In some embodiments, the effector function is Foy receptor binding and / or FcRn receptor binding. In some embodiments, the Foy receptor is human FcyRIIa, FoyRI, and / or FeyRIIIIa, especially FeyRI. In some embodiments, the heterodimeric Fo scaffold (or binding molecules containing the heterodimeric Fo scaffold) according to the invention has a binding affinity of not less than 60%, for example not less than 50%, 40%, 30%, 20%, 10%, 10% for the IgG1-R1 Fe domain (or binding molecules containing the IgG1-R1 Fe domain), compared to the binding affinity of the natural IgG1-R1 Fe domain. In some embodiments, the IgG1-R1 Fe domain has a binding affinity of not less than 60%, for example not less than 50%, 40%, 30%, 20%, 1 ...The binding affinity is approximately 70% or more, particularly approximately 80% or more, and even more specifically approximately 90% or more. The Fe domain can confer favorable pharmacokinetic properties to heteropolymers (e.g., bispecific antibodies) containing it, including prolonged serum half-life, facilitating good accumulation in target tissues, and favorable tissue-blood partition ratios. However, the EFc domain may also cause heteropolymers (e.g., bispecific antibodies) containing it to target cells expressing the Foy receptor more often than the desired cells and / or tissues carrying the target. Furthermore, activation of the Fecy receptor signaling pathway may lead to cytokine release, resulting in serious side effects following systemic administration of heteropolymers containing the Fe domain. In some embodiments, therefore, in addition to the CH3 interface characteristic mutation Koh, RAW MHI Fo scaffold of the present invention, other mutations may be included or excluded as needed to maintain or affect its (one or more) effector functions. For example, it is known that the upper CH2 domain and the homogeneous region of an antibody are involved in a variety of effector functions, such as Foy LH binding and effector functions such as ADCC; some CH2 and CH3 interface residues of the antibody are involved in FcRn receptor binding. In some cases, the Fec region used in the heterodimeric scaffold of the present invention can be modified by including additional mutations that introduce the CH2 domain and / or homogeneous region (if present), RAE (ERA), one or more desired effector functions, such as the binding affinity of the Fc region to the Foy receptor and / or FcRn. Amino acid modifications are known in the art, including, but not limited to, amino acid substitutions at one or more positions selected from E233, L234, L235, N297, P331, and P329, more specifically,One or more amino acid substitutions selected from E233P, L234A, L235A, L235E, N297A, N297D, and P331S. In some cases where the Fo scaffold has a human IgG1 Fe region, the binding of the Fe scaffold to the Foy receptor can be substantially reduced by including L234A-L235A mutations, L234A-L23SA-P329G mutations, and / or N297A mutations in the Fe region. In this invention, some exemplary sequences of the Fo scaffold according to the invention are provided. In some embodiments, the heterodimeric Fo scaffold of the present invention comprises a first polypeptide and a second polypeptide, wherein the first polypeptide comprises a first Fe region and the second polypeptide comprises a second Fc region, wherein: (a) relative to the wild-type CH3 domain, the CH3 domains of the first and second Fe regions, in addition to the characteristic CH3 interface mutation of the present invention described above, respectively comprise 0-3 amino acid residue changes; preferably, the wild-type CH3 domain is a natural IgG (especially IgG1 or IgG4, preferably human IgG1) immunoglobulin CH3 domain, more preferably comprising the amino acid sequence of SEQ ID NO: 19 or 20; (b) the first and second Fc regions comprise a natural IgG (especially IgG1 or IgG4, preferably human IgG1) immunoglobulin CH2 domain, or comprise the amino acid sequence shown in SEQ ID NO: 21 or a CH2 domain having 15 amino acid residue changes therewith; 11 WO 2025 / 092987 PCT / CN2024 / 129416 (c) &4 knob mutant Fe region contains SEQ ID NO:1 &2HARBRPNRERAA EY 95%, 96%. 97%, 98% HK 99% 7] — EH R FF FI (d) &* hole mutant Fe RL SEQ ID NO: 7 or 8 amino acid sequence or having at least 95%, 96%,The Fe scaffold contains an amino acid sequence with 97%, 98%, or 99% double identity, and / or (e) the Fe scaffold does not form non-natural disulfide bonds between the first and second Fe regions. In some preferred embodiments, the Fe scaffold contains a CH3 domain of the amino acid sequence shown in SEQ ID NO: 1 or 2 with a knockb mutation, or an amino acid sequence having at least 95% identity with it; and contains a CH3 domain of the amino acid sequence shown in SEQ ID NO: 7 or 8 with a hole mutation. In some embodiments, the knockb-mutated Fe region contains a CH3 domain of the amino acid sequence shown in SEQ ID NO: 1 or 2, and the hole-mutated Fe region contains a CH3 domain of the amino acid sequence shown in SEQ ID NO: 7 or 8. In some embodiments, the knockb-mutated Fe region comprises a CH3 domain of the amino acid sequence shown in SEQ ID NO: 1, and the hole-mutated Fe region comprises a CH3 domain of the amino acid sequence shown in SEQ ID NO: 7. In some embodiments, the knockb-mutated Fe region comprises a CH3 domain of the amino acid sequence shown in SEQ ID NO: 2, and the hole-mutated Fe region comprises a CH3 domain of the amino acid sequence shown in SEQ ID NO: 8. In any of the above embodiments, the knockb-mutated Fo region may be a first Fe region and the hole-mutated Fc region may be a second Fc region, or vice versa. In some embodiments, the heterodimeric Fo scaffold according to the present invention does not contain non-RRM, AMAA ROAR CE, ite, ARE DSC Me, CH3 domains with a Tm value of 705C or higher that introduce the interface between the first and second Fo region CH3 domains. In some implementation schemesAccording to the present invention, the heterodimeric Fe scaffold exhibits better thermal stability compared to the corresponding control Fc scaffold with the same KIH mutation. Hereinafter, "corresponding control Fc scaffold" refers to a heterodimeric Fe scaffold identical in all sequence structures (including KIH RR) except for the presence of unmutated native residues at positions 1L351, D366, and K409, compared to the heterodimeric Fe XR of the present invention. In some embodiments, the heterodimeric Fe scaffold of the present invention has comparable purity to the corresponding control Fc scaffold with the same KIH mutation. In some embodiments, after expression, assembly, and purification from host cells under the same conditions, the purity of the heterodimeric Fe scaffold of the present invention is at least 90%, 95%, 100%, 110%, or higher than the purity of the control Fe scaffold, and preferably greater than 90% as determined by SEC-HPLC. In a second aspect, the present invention provides a heteropolymeric protein comprising a CH3 heterodimer according to the invention or a heterodimeric Fe scaffold according to the invention. In some embodiments, the heteropolymeric protein according to the invention comprises at least a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide respectively contain first and second CH3 domains, wherein the first and second CH3 domains in the first and second polypeptides meet at the interface and form a CH3 HOORE according to the invention. In some embodiments, the heteropolymeric protein according to the invention comprises at least a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide respectively contain first and second Fc regions, wherein the first and second Fe regions in the first and second polypeptides respectively contain first and second Fec regions.The regions meet at the interface and form the Fc scaffold according to the invention. In some embodiments, the heteropolymeric protein is a single or multiple specific binding protein containing at least one target-binding domain, wherein the at least one target-binding domain is attached to the CH3 heterodimer or the heterodimeric Fc scaffold according to the invention. In some embodiments, the heteropolymeric protein according to the invention contains more than one of the Fo scaffolds. In some embodiments, the Fc scaffold is called an EK HRS RARE LER Feo scaffold. In this document, such single or multiple specific binding proteins of the Fo scaffold of the present invention are also referred to as "Fo scaffold-based binding proteins". AF Fe SRWBAEA 12 WO 2025 / 092987 PCT / CN2024 / 129416 In some aspects, therefore, the present invention provides binding proteins based on the heterodimeric Fe scaffold of the present invention. In some embodiments, the binding protein according to the invention contains at least one target-binding domain attached to the Fc scaffold. The type of target-binding domain used in the binding protein of this invention is not specifically limited, as long as it has the ability to bind to the target. Such binding domains, as known in the art, can originate from binding pairs, including non-immunoglobulin-based binding pairs such as receptor / pair binding pairs, enzyme / substrate pairs, or immunoglobulin-based binding pairs such as antigen / antibody binding pairs, antibody / anti-idiotype antibodies. When the target-binding domain originates from an immunoglobulin-based binding pair, the binding protein containing it can be an antibody, such as a mono / multispecific antibody. When the target-binding domain originates from a non-immunoglobulin-based binding pair, the binding protein containing it can be an immunostriptin.Furthermore, the present invention also considers binding proteins comprising binding domains from both different binding pairs; such antibody / immunoadhesin syncytiotroph proteins are sometimes referred to herein as antibodies. In some embodiments, the binding protein according to the invention comprises at least one antigen-binding domain. Antigens that can bind to the antigen-binding domain according to the invention may be selected from, for example, but not limited to: tumor-associated antigens, immune checkpoint molecules, angiogenic factors, or combinations thereof. Tumor-associated antigens may include, but are not limited to: MUCI-1, BCMA, CLDN18.2, HER2, BRAF, EGFR, CD20, CD38, FolR1, and CD5S2. Immune checkpoint molecules may include, but are not limited to: PD-L1, PD-1, PD-L2, CTLA-4, B7-H3, TIM3, LAG-3, VISTA, ICOS, 4-1BB, OX40, GITR, and CD40. Angiogenic factors may include, but are not limited to: basic FGE, HGE, Stie-2, Svegfr-1, Svegfr-2, EGF, IL-6, IL-8, PLGF, VEGF, PDGF-bb, ANG1, ANG2, SDF-1a, MDC, Galectin, TSP-1, Endocan, Enos, HIF-1w. In some embodiments, the antigen is selected from tumor-associated antigens, such as EGFR and HER2. In some embodiments, the binding protein according to the invention encapsulates at least one non-immunoglobulin binding domain. In some embodiments, examples of such non-immunoglobulin binding domains available include, but are not limited to, ligand-binding domains from the following receptor proteins: 41BB; adrenocorticotropic hormone receptor; activin receptor; BLIR (4H = 3% B4 receptor); BMP receptor; C3a receptor; CSa tt: chemokine receptor; cytokine receptor; growth hormone receptor;BILA; Interferon-α LH; FREP LA; FaR-γ CA; I-1 receptor; UII A; IL10 eA; IL-1L receptor; IL-12 receptor; BCMA; TACI; BAFF receptor; CD72 immunomodulatory signaling receptor; Kaposi's sarcoma-associated herpesvirus GPCR; MA A4 receptor; Lymphotoxin receptor; RON LA; SCE LA; AKAPR LA; TIUST2; TGF-β receptor; IP tumor necrosis factor receptor; TNFRSF19; erythropoietin receptor; and leukemia inhibitory factor receptor; and C-kit CH. In some embodiments, examples of such non-immunoglobulin-binding domains available include, but are not limited to, the ionotropic binding domains of ligands or the ligands themselves derived from: orMSH; 9E3 / cCAEF; adrenocorticotropic hormone; BER; AK155; Angiogenesis inhibitors; Apo2L / TRAIL; BLR1 ligand / BCA-LUBLC / CXCL13; Calcitonin gene-related peptide; CD27 ligand; CD30 ligand; CD40 ligand; Endorphins; Endostatin; Erythropoietin; Fas ligand; Flt3 ligand; G-CSF; GCP-2 / CXCL6; GM-CSF; Various cytokines, such as IFNa, IFNB; Interferon Y; IL-1a; L-1p; IL-10; IL-11; LIL-12; IL-13; IL-15; IL-16; IL-2; IL-27; IL-3; IL-4; IL-5; IL-6; IL-7; IL-8 / CXCL8; IL-9; Somatostatin; Stem cell factor; Substance P; TARC / CCLI7: TCA3 / CCL1; TECK / CCL25; TGFB; thrombopoietin; TNFα. In some embodiments, the non-immunoglobulin-binding domain is derived from proteins selected from: VEGE receptor; TNE 4A IL-1; lymphocyte function-associated antigen 3 (LFEA-3 / CDS8); lymphotoxin B receptor (LTBR);CTLA-4; IL-12; activin receptor; TACI; BR3; T cell receptor; CD4; L-selectin; piracetam receptor; CD44; NP LA; FAK y CA; 41BB fe IGE LK. In other embodiments, the non-immunoglobulin binding domain is derived from the receptor binding domain of a ligand selected from IL-1, IL-12. Examples of other available non-immunoglobulin binding domains can also be found, for example, US7951917B1 and US20220275048A1. The connection mode of the target binding domain in the binding protein according to the invention is not particularly limited in terms of the connection location of the target binding domain on the Fc scaffold. In some embodiments, the first and / or second polypeptide of the Fo scaffold is attached to the N-terminus or C-terminus. In some embodiments, at least two or more or all of the at least one target-binding domains are optionally linked to each other via peptide linkers and thus to the heterodimeric Fc scaffold according to the invention. In other embodiments, at least two or more or all of the at least one target-binding domains are optionally separately linked to different ends of the Fo scaffold via peptide linkers. For example, when the binding protein comprises two binding domains, one binding domain may be attached to the N-terminus of the first polypeptide of the Fo scaffold, while the other binding domain is attached to a different end of the Fo scaffold, for example, the C-terminus of the first polypeptide, the N-terminus of the second polypeptide, or the C-terminus of the second polypeptide. Therefore, in the binding protein according to the invention, the four ends of the heterodimeric Fc scaffold (i.e., the N-terminus of the first and second polypeptides and...)At least one, two, three, or all of the C-terminal regions may each be linked to at least one (preferably one or two) target-binding domains. The at least one target-binding domain contained in the binding protein may be the same or different from each other. In one embodiment, at least two, more, or all of the at least one target-binding domains are the same. In other embodiments, at least two, more, or all of the at least one target-binding domains are different from each other. In some embodiments, the binding protein is monospecific and has one or more binding domains for that specificity. In some embodiments, the binding protein is multispecific, and preferably, has one or more binding domains for each specificity. In some embodiments, the at least one target-binding domain is independently selected from Fab, VHH, scFv, scFab, and crossFab antigen-binding domains. When the binding domain is Fab or crossFab, the Fab or crossFab can optionally be linked via a peptide linker to another binding domain of the binding protein or to an Fc scaffold, for example, to the N-terminus of the first or second polypeptide of the heterodimeric Fc scaffold according to the invention, via the C-terminus of a chain containing a VH domain or a chain containing a VL domain. In some embodiments, at least one of the at least target-binding domains is a VHH domain. In other embodiments, at least one of the at least target-binding domains is a Fab domain. In still other embodiments, at least one of the at least target-binding domains is a VHH domain, and the at leastAt least one of the target-binding domains is a Fab domain. In other embodiments, at least two, more, or all of the at least one target-binding domains are Fab domains, and the Fab domains have the same or different VLCL light chains. In some embodiments, the binding protein is a single-specific monovalent protein containing a VHH domain, and preferably the VHH domain is linked to the N-terminus of the first or second polypeptide of the Fe scaffold. In some embodiments, the binding protein is a bispecific bivalent binding protein containing VHH and / or Fab binding domains. In some embodiments, the binding protein is a bispecific antibody containing first and second Fab domains and an IgG conformation of the Fe scaffold, preferably, the first and second Fab domains are linked to the C-terminus of the first and second polypeptides of the Fo scaffold via the C-terminus of their VH domain-containing chains, respectively. In some embodiments, the binding protein is a bispecific antibody with an IgG-like conformation comprising VHH and Fab, wherein the antibody consists of a heavy chain containing a VHH-Fc domain and a light chain containing a VL-CL domain. In this document, the term "linkage" as used when referring to different components in a scaffold or binding protein means the fusion or combination of the components directly or via a suitable linker. Peptide linkers typically used for this purpose are short amino acid polypeptides with a degree of flexibility, generally between 1 and 50 amino acid residues in length, but not limited to this. In some cases, any chain of amino acid residues located between the two components can be considered a peptide linker, provided it does not affect the intended function of the two components being linked. Therefore, in some…Regarding the heterodimer FeRR according to the invention, or the CH3 heterodimer according to the invention, which is linked to a polypeptide of interest, such as the target-binding domain according to the invention, or other functional polypeptide or protein domains, via a peptide linker, is all within the scope of this invention. In the heteropolymeric protein according to the invention, those skilled in the art can readily determine the available linker sequences based on the components to be linked and the linking sites. In some embodiments, the linker is used to link the Fc scaffold and the antigen-binding domain according to the invention; and / or to link two antigen-binding domains. In some embodiments, the available linker is a flexible linker peptide of $S-$0 amino acids, preferably containing glycosyl (G) and / or serine (S) and / or threoic acid residues (T). In some embodiments, the linker has an amino acid length of 5-30 amino acids, for example, 8, 10, 15, 20, 25, or 30 amino acid lengths, or an amino acid length falling between any two integers. In some embodiments, the linker comprises an amino acid sequence (G4S)n, where n is an integer equal to or greater than 1, for example, n is an integer of 2, 3, 4, 5, 6, or 7. In a preferred embodiment, the linker consists of an amino acid sequence (G4S)n. In other embodiments, the linker is a homogeneous region of an immunoglobulin or a derivative thereof. Examples of other available linkers include, for example, but not limited to, the following amino acid sequences: (GlysSer)n (SEQ ID NO: 22), (Gly4Ser)2 (SEQ ID NO: 12), (Gly3Ser)3 (SEQ ID NO: 24), (Gly,Ser)3 (SEQ ID NO: 25), (Gly3Ser)4 (SEQ ID NO: 26), (Gly4Ser)4 (SEQ ID NO: 26).NO: 27), (Gly3Ser)s (SEQ ID NO: 28), (Gly4Ser)s (SEQ ID NO: 29), (Gly3Ser)s (SEQ ID NO: 30), (Gly4Ser)s (SEQ ID NO: 31), GGG (SEQ ID NO: 32), DGGGS (SEQ ID NO: 33), TGEKP (SEQ ID NO: 34), GGRR (SEQ ID NO: 35), EGKSSGSGSESK VD (SEQ ID NO: 36), KESSGSVSSEQLAQERSLD (SEQ ID NO: 37), GGRRGGGS (SEQ ID NO: 38), LRQRDGERP (SEQ ID NO: 39), LRQKDGGGSERP (SEQ ID NO: 40) andGSGSSGKPGSGEGSTKG (SEQ ID NO:23). Alternatively, if necessary, the three-dimensional structures of proteins and peptides can be simulated using computer programs, or suitable flexible linker peptides can be rationally designed using phage display methods. In a preferred embodiment, a peptide linker having the sequence shown in SEQ ID NO: 12 is used to link the Fe scaffold and its N-terminal antigen-binding domain, such as VHH or Fab. In some cases, chemically synthesized linkers can also be used to attach the binding domain to the Fe scaffold. In this case, the connection is also referred to as a "bridging". Examples of chemically synthesized connectors include: N-succinimide (INHS), disuccinimide succinate (DSS), bis(sulfosuccinimide) succinate (BS3), dithiobis(succinimide propionate JDSP), dithiobis(sulfosuccinimide propionate) (DTSSP), diethyldimethyl ...(Sulfo-DST), bis[2-(succinylaminooxy)ethyl] bis(BSOCOES), bis[2-(sulfosuccinylaminooxy)ethyl] bis(BSOCOES), etc. When multiple linkers are used in the heteropolymeric protein of the present invention, it is understood that these linkers may be the same or different from each other. In some cases, chemically synthesized linkers may also be used to combine chemically synthesized therapeutic agents (e.g., small toxin molecules) or diagnostic agents into the heteropolymeric protein of the present invention, such as bispecific antibodies, for example, on the glycan chain of the CH2 domain. Such slowed compounds are also in the TEPER SRAE of the present invention. In some embodiments, the heteropolymeric protein of the present invention is a multispecific binding protein comprising binding to different antigenic epitopes, especially a bispecific antibody. Bispecific antibodies having binding specificity against at least two different antigens or antigenic epitopes have broad clinical application potential and can be used as targeted drugs for in vitro and in vivo immunodiagnostics and therapies, as well as for diagnostic immunoassays. See, for example, WO9850431A2. Bispecific antibodies can be used in in vitro assays to detect the functional properties of cell surface molecules and determine cytotoxicity mediated by different Fc receptors (Fanger et al., CritRevImmunol.12:101-124 (1992)); for enzyme-linked and immunoassay assays (Nolan et al., Biochem. Biophys. Acta. 1040:1-11 (1990); Hammerling et al., J. Exp. Med. 128: 1461-1473 (1968)); and for the immunodiagnosis of various diseases, such as cancer, in vitro or in vivo (Songsivilai et al., Clin. Exp. Immunol.79:315 (1990)). To promote the development of BsAbs...For diagnostic applications, one arm of a BsAb can bind to antigens on the surface of diseased tissues or cells (e.g., tumor-associated antigens), while the other arm can bind to detectable markers, such as binders that tightly bind to radionuclides. For therapeutic applications, bispecific antibodies can be used to guide a patient's cellular immune defense mechanisms specifically against diseased tissues / cells, such as tumor cells, or pathogens. To this end, one arm of the BsAb can bind to antigens on the surface of immune cells (e.g., cellular or NK cells), while the other arm can bind to antigens on the surface of diseased tissues / cells. In some embodiments, the multispecific antibody according to the invention is a heterodimer consisting of or primarily of a heterodimer Fo scaffold according to the invention and at least one antigen-binding domain attached thereto, having a configuration selected from the following: -(antigen-binding domain)n-Fc / (antigen-binding domain)n-Fc, -(antigen-binding domain)n-Fc-(antigen-binding domain)ny(antigen-binding domain)n-Fc, -(antigen-binding domain)n-Fc-(antigen-binding domain)nmy(antigen-binding domain)n-Fc-(antigen-binding domain)m, wherein each of the above nm and α is independently selected from integers of 0, 1, and 2; 15 WO 2025 / 092987 PCT / CN2024 / 129416 Each of the above antigen-binding domains is independently selected from: Fy, scFv, Fab, scFab, crossFab, VHH, 8074 a K The ligand-binding domain; the N-terminal linker of α-FORPE Fe; the C-terminal linker of RES-FORPE Fe; wherein the two monomers of the heterodimer are separated by the symbol " / ; wherein, preferably one of the two Fe is a knob chain and the other is a hole chain. In some preferred embodiments, n is 1 and n is 0. In someIn a preferred embodiment, n is 0 and α is 1. In some embodiments, 0 is 1 and α is 1. In some preferred embodiments, each of the above antigen-binding domains is independently selected from: scFv, VHH, and Fab. In some specific embodiments, the multispecific heterodimeric protein according to the invention is bispecific and has an IgG conformation or an IgG-like conformation. In some specific embodiments, the multispecific heterodimeric protein according to the present invention is a bispecific antibody and has a conformation selected from the following: -SCFEV-Fc / scEv-Fc, wherein two different specific scFv domains are respectively linked to the N-terminus of the Fc scaffold according to the present invention; -Fab-FwWFab-FEc, wherein two different specific Fab domains are respectively linked to the N-terminus of the Fc scaffold according to the present invention; -VHH-Fe / VHH-Fv, wherein two different specific VHH domains are respectively linked to the N-terminus of the Fe scaffold according to the present invention; -scFv-Fe / Fab-Fc, wherein two different specific scFv domains and Fab domains are respectively linked to the N-terminus of the Fe scaffold according to the present invention; -SCFWVHH-Fc, wherein two different specific VHH domains and VHH domains are respectively linked to the N-terminus of the Fe scaffold according to the present invention; -VHHVFab-Fc, wherein two different specific ... The Fab domain and Fab domain are respectively attached to the N-terminus of the Fo scaffold according to the invention. In embodiments of the heteropolymeric protein according to the invention that contain two or more Fab domains targeting different specificities, for example when the heteropolymeric protein is a bispecific antibody with a Fab-Fc / Fab-Fc conformation, light chain misalignment can be prevented by using a common light chain.See, for example, US7951917B. Alternatively, the target heterodimer can be formed in vitro by separately generating a first Fab-Fc hapten and a second Fab-Fe hapten, followed by adding a suitable reducing agent to their mixture. In some embodiments, the heterodimer protein of the present invention is a bispecific antibody comprising a VHH domain and a Fab domain, respectively binding to different antigens. In some embodiments, the bispecific antibody binds HSA and B7-H3. In some embodiments, the VHH domain comprises the CDR1, CDR2, and CDR3 sequences of the VHH amino acid sequence shown in SEQ ID NO: 11; or comprises the amino acid sequence shown in SEQ ID NO: 11, or an amino acid sequence having at least 95% identity with it. In some embodiments, the Fab domain comprises VH and VL, wherein the VH comprises the VH shown in SEQ ID NO: 13 and the CDR1, CDR2, and CDR3 sequences of the amino acid sequence; or comprises the amino acid sequence shown in SEQ ID NO: 13, or an amino acid sequence having at least 95% identity with it; and wherein the VL #4 comprises the VL shown in SEQ ID NO: 14 and the CDR1, CDR2, and CDR3 sequences of the amino acid sequence; or comprises the amino acid sequence shown in SEQ ID NO: 14, or an amino acid sequence having at least 95% identity with it. In some embodiments, the VHH domain comprises the amino acid sequence of SEQ ID NO: 11; and the Fab comprises the VH of the sequence shown in SEQ ID NO: 13 and the VL of the sequence shown in SEQ ID NO: 14. In some embodiments, the bispecific antibody is a bivalent antibody. In some embodiments, the VHH and Fab domains are respectively connected to the heterodimeric Fe scaffold according to the present invention.The N-terminus of the first and second polypeptides. In some embodiments, the binding protein of the present invention is a bispecific antibody comprising first and second Fab domains that bind to different antigens respectively. In some embodiments, the bispecific antibody binds EGFRVHER2. In some embodiments, the first Fab domain comprises VH and VL, wherein the VH comprises the VH and CDR1, CDR2 and CDR3 sequences of the amino acid sequence shown in SEQ ID NO: 15; or comprises the amino acid sequence shown in SEQ ID NO: 15, or an amino acid sequence having at least 95% identity with it; and wherein the VL comprises the VL and CDR1, CDR2 and CDR3 sequences of the amino acid sequence shown in SEQ ID NO: 16; or comprises the amino acid sequence shown in SEQ ID NO: 16, or an amino acid sequence having at least 95% identity with it. In some embodiments, the second Fab domain comprises VH and VL, wherein VH comprises the CDR1, CDR2, and CDR3 sequences of the VH amino acid sequence shown in SEQ ID NO: 17; or comprises the amino acid sequence shown in SEQ ID NO: 17, or an amino acid sequence having at least 95% identity with it; and wherein VL comprises the CDR1, CDR2, and CDR3 sequences of the VL amino acid sequence shown in SEQ ID NO: 18; or comprises the amino acid sequence shown in SEQ ID NO: 18, or an amino acid sequence having at least 95% identity with it. In some embodiments, the first Fab domain comprises VH of the sequence shown in SEQ ID NO: 15 and VL of the sequence shown in SEQ ID NO: 16; and the second Fab domain comprises VH of the sequence shown in SEQ ID NO: 17 and VL of the sequence shown in SEQ ID NO: 18.VL. In some embodiments, the bispecific antibody is a bivalent antibody. In some embodiments, the first and second Fab domains are respectively linked to the N-terminus of the first and second polypeptides of the heterodimeric Fo scaffold according to the invention. IV. SRR. BA, BE In some aspects, the present invention provides one or more polynucleotides encoding the CHB HORA, HORA RR, HRERKEGR binding protein according to the invention; one or more vectors comprising the one or more polynucleotides, wherein the one or more polynucleotides may be present in a single vector or in multiple separate vectors. In other aspects, the present invention also provides a host cell comprising the one or more nucleic acids or the one or more vectors. Depending on the circumstances, one or more vectors containing one or more nucleotides of the present invention may be introduced into the same host cell to express the desired product in one host cell; or one or more vectors containing the one or more polynucleotides may be introduced separately into different host cells to express intermediates of different chains or combinations of chains (e.g., different monomeric proteins) of the heteropolymer of the present invention in different host cells respectively, and, under suitable conditions for assembling the heteropolymer of the present invention, the heteropolymer of the present invention is produced by mixing the intermediates. In this invention, there are no particular limitations on the expression vectors that can be used, including but not limited to viruses, plasmids, granules, phages, or yeast artificial chromosomes (YAC). In this invention, there are no particular limitations on the host cells that can be used. Suitable host cells include prokaryotic microorganisms, such as *Escherichia coli*, eukaryotic microorganisms such as filamentous fungi or yeast, or various other types of microorganisms.Eukaryotic cells, such as mammalian host cells, cytokine cells, etc. Examples of useful mammalian host cell lines include monkey kidney CV1 A (COS-7) transformed with SV40, ABE}K (HEK293 or 293F 20 2), 44He (BHR), monkey bone cells (CV1), African green monkey bone cells (VERO-76), human cervical cancer cells (HELA), canine kidney cells (MDCK), Buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (HepG2), CHO cells, NSO4 40 ft, α-Ri 7g 29He Ate YO, NSO4, P3X63, and Sp2 / O, etc. In some embodiments, mammalian cell lines suitable for culture can be used. In some preferred embodiments, the host cell is CHO or HEK293 cells. In another aspect, the present invention provides a method for producing the heteropolymeric protein of the present invention. To produce the heteropolymeric protein of the present invention, the polypeptide chain of the heteropolymeric protein of the present invention can be obtained, for example, by solid-state peptide synthesis (e.g., Merrifield solid-phase synthesis) or recombinant production, and assembled under suitable conditions. As previously mentioned, in the case of recombinant production, the assembly can occur for expressing the heteropolymeric SANG EMP, or, if necessary, the assembly of the heteropolymeric protein can be performed in vitro after obtaining the intermediate polypeptide chain for expression from host cells. In one embodiment, the present invention therefore provides a method for producing the heteropolymeric protein of the present invention, the method comprising: culturing a host cell containing a polypeptide chain encoding the polypeptide chain under conditions suitable for expressing the heteropolymeric protein; and assembling the HET ARS UK chain into the heteropolymeric protein.Under the conditions of protein, the polypeptide chain is assembled to produce the heteropolymeric protein. 17 WO 2025 / 092987 PCT / CN2024 / 129416 In some embodiments, the method includes: LETRASPRARSRAE OH SU E FERS MA host cells of the polypeptide chain; and recovering the heteropolymeric protein produced by the host cells from the cell culture. Preferably, the recovered product, after protein A affinity chromatography, is determined by SEC-HPLC to have a purity greater than 80%, preferably greater than 85%, and more preferably greater than 90% for the heteropolymeric protein according to the invention. In other embodiments, the method includes: culturing host cells containing a polypeptide chain encoding the heteropolymeric protein under conditions suitable for expressing the polypeptide chain; and obtaining an intermediate of the heteropolymeric protein from the host cells in the cell culture; mixing the intermediate under conditions suitable for assembling the heteropolymeric protein, and recovering the resulting heteropolymeric protein. In the case of a heterodimeric protein, monomers containing an fnob chain and monomers containing a hole chain are expressed in different host cells, and the heteropolymeric protein is generated and recovered from the mixture of monomers under suitable assembly conditions. Preferably, the recovered product, after ion exchange chromatography, is determined by SEC-HPLC to have a purity greater than 80%, preferably greater than 85%, more preferably greater than 90% for the heteropolymeric protein according to the invention. The antibody prepared by the method described herein can be purified by known prior art techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, etc. After purification, it can be obtained through...The purity of the heteropolymeric protein of the present invention can be determined by any of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high performance liquid chromatography, etc. The physical / chemical properties and / or biological activity of the heteropolymeric protein provided herein can be identified, selected, or characterized by a variety of assays known in the art. VI. Compositions and Uses The present invention also relates to a composition comprising the heteropolymeric protein of the present invention (e.g., the binding protein according to the present invention). In embodiments where the composition is a pharmaceutical composition, the composition further comprises a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes any and all physiologically compatible solvents, dispersion media, isotonic agents, and absorption delay agents, etc. In the present invention, a pharmaceutical composition generally refers to a drug for the treatment or prevention of a disease or for the examination or diagnosis of a disease. In some preferred embodiments relating to the pharmaceutical composition of the present invention, the heteropolymeric protein of the present invention is a bispecific antibody, for example, a bispecific antibody specifically targeting a disease-associated antigen. In some embodiments, the heteropolymeric protein of the present invention is the sole active ingredient in the pharmaceutical composition. In other embodiments, the pharmaceutical composition may comprise the heteropolymeric protein described herein with one or more other therapeutic agents. The pharmaceutical compositions of the present invention can be formulated according to methods well known to those skilled in the art. Furthermore, the use of pharmaceutically acceptable carriers or media, such as sterile water or saline, vegetable oils, emulsifiers, flocculating agents, surfactants, stabilizers, flavoring agents, excipients, solvents, preservatives, binders, etc., can be considered in conjunction with the present invention.The pharmaceutical compositions of the present invention are appropriately combined to formulate unit doses suitable for drug administration. For example, a pharmaceutically acceptable carrier can be used to prepare a sterile solution or suspension for non-gastrointestinal administration in the form of an injection. The amount of active ingredient in the formulation will be set to produce an effective amount within a predetermined range after administration to an individual. The pharmaceutical compositions of the present invention are suitable for various routes of administration, including but not limited to intravenous, intramuscular, subcutaneous, parenteral, rectal, intravenous, or epidermal administration (e.g., by injection or infusion). Accordingly, the pharmaceutical compositions of the present invention can be formulated into dosage forms for administration to the intended route of administration, such as injectable, nasal, pulmonary, and transdermal dosage forms. For injectable dosage forms, the route of administration may include, but is not limited to, systemic or local administration such as intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, and intratumoral injection. Furthermore, the specific method of administration of the pharmaceutical compositions of the present invention can be appropriately selected according to the patient's age and symptoms. Furthermore, the detection or diagnostic reagent gold for heterologous multi-to-hole proteins described herein is also within the scope of this invention. The reagent gold may contain one or more other elements, such as: instructions for use; other reagents, such as markers or reagents for conjugation; a pharmaceutically acceptable carrier; and a device or other material for administration to a subject. The following examples are described to aid in understanding the invention. These examples are not intended and should not be construed as limiting the scope of the invention in any way. 18 WO 2025 / 092987 PCT / CN2024 / 129416 Examples Example 1 Design of Fe mutant candidate combination sequences Searching for Fe heterodiols in the knob-into-hole (KIHD) model from a protein database (www.pdb.org)The polymeric structures (e.g., 4NQS and SID8) were compared with the wild-type IgG1 Fe crystal structure (e.g., 1HZH) to analyze the reasons for the reduced thermal stability of the KIH model. The KIH mutation reduced the number and intensity of amino acids in contact between the two CH3 domains. In wild-type IgG1 Fe, some key amino acids at the CH3-CH3 domain interface form a water core, including L351, L368, Y407, etc., covering a large surface area (see Figure 1A). However, in the KIH model, because L368A and YY407V mutate into small side chains RAR (RARCH A BARA Bo) OLA 1B), the contact of some amino acids near the water core is also affected. For example, in wild-type IgG1 Fe, the K409 side chain of one CH3 domain is in direct contact with amino acids L368, K370, D399, F405, and Y407 of another CH3 domain; in particular, K409 forms a complex hydrogen bond network with D399, S364, T411, and a water molecule, while preventing electrostatic clash between K409 and K370. However, in the KIH structure, the K409 WHS to T366W mutation in the Knob chain results in the loss of direct contact with L368A and Y407V, as well as the loss of the stable water molecule and its mediated hydrogen bond network. To enhance CH3 interaction strength and improve thermal stability, we selected amino acids near the Fc contact interface in the KIH model as mutation targets and designed a series of Fo heterodimer mutation combinations. The specific amino acid mutation information is shown in Table 1. The KIH combination is the handle-hole mutation combination disclosed in patent UST951917B1. M1-M7 are newly designed candidate mutation combinations. Table 1. Fc heterodimer mutation combination design, where the A chain is incorporating tungsten.The knob-mutated chain, and the B chain is the chain containing the corresponding hole mutation | are ea Y-D399R a ee ee nee i See Oc a baa K Y-D399K Example 2 VHH-Fe / Fe heterodimer expression and purification In this example, to verify the effect of the Feo mutant combination on promoting heterodimer formation, an A-chain VHH-Fe fusion molecule and a B-chain Fo molecule were first designed. Based on this, the VHH-Fe and Fe genes were synthesized, and expression plasmids were constructed. After transfection of 293F cells, the culture supernatant was collected and purified by Protein A affinity. The purified product was analyzed by SDS-PAGE and SEC-HPLC methods to determine the amount of heterodimers and other impurities, so as to determine the effect of different mutant combinations on the expression yield and purity of VHH-Fe / Fe heterodimer. The expression vector was constructed based on the constant region amino acid sequence (POLST) of human immunoglobulin IgG from the Uniprot protein database, specifically the ABP, CER, CH2, and CH3 domains. Then, according to Table 1, a total of 10 Fc REKAKM FF (3. SEQ ID NO:1 2 SEQ ID NO:10) were designed. A VHH fragment (derived from the Ozoralizumab antibody, see SEQ ID NO:11) and a flexible linker polypeptide CL (SEQ ID NO:12) were introduced to the N-terminus of the @ A ## Fe fragment to construct the A-chain VHH-Fc fusion molecule; simultaneously, the B-chain Fc fragment remained unchanged. The molecular weight difference between VHH-Fc and Fc is beneficial for distinguishing the proportions of AB FPR-FR AK, AA / BB homodimers, and A / B EARARD TERK (HMWS) formed during recombinant expression. Codons optimized for mammalian / human expression were used to design the A-chain VHH-Fc and B-chain...The Fc DNA encoding gene was cloned into the expression vector pcDNA3.4 (Invitrogem) to obtain plasmids for expressing VHH-Fc and Fe. Gene synthesis and plasmid construction were performed by Genwiz Suzhou Co., Ltd. For 293F cell transfection, the VHH-Fc and Fe plasmids were mixed at a molar ratio of 1:1 or 0.6:1, and then 10 L / L of OPM-CD Trans293 medium (OPM, P82019) was added. FectoPRO transfection reagent (PolyPlus, PT-116-010) was added to the medium at a ratio of 2 ml / L / 1 h cells. The medium containing the plasmids and transfection reagent was mixed and incubated for 10 min. The mixture was added to Expi293F cells (Invitrogen, A14635) A94RP and cultured with shaking at 437°C and 8% CO2. 16 hours after transfection, 10 D KK-volume feed (Opmai, F081918-001) was added, and the cells were cultured for another 6 days. Protein A was purified by loading 1 ml Protein A Diamo d chromatography column (Borglon, AA0273) with 5 column volumes of equilibration buffer (20 mM sodium phosphate, pH 7.4). The cell culture medium was loaded onto the column at a flow rate of 0.5 column volumes / min. The packing material was then washed sequentially with 5 column volumes of equilibration buffer (20 mM sodium phosphate, pH 7.4) and pre-elution buffer (20 mM acetate-sodium acetate, pH 2). Finally, elute with 10 column volumes of elution buffer (20 mM acetate-sodium acetate, pH 3.2), and adjust the pH of the collected eluent to 6.0 with 1.5 SM Tris base. For SDS-PAGE analysis, the purified product from the Sug Protein A column was mixed with non-reducing loading buffer (DTT-free) and run on 10% SurePAGE Fh BK (Genscript, M00666). After electrophoresis, the gel was stained and destained with Coomassie Brilliant Blue.Images were then taken using a gel imaging system. The results are shown in Figure 2. The co-expression products of VHH-Fc plasmid and Fo plasmid mainly contained five proteins of different molecular weights: AHIR monomer, AA homodimer, BB homodimer, A monomer, and BB monomer. Under a plasmid ratio of 1:1, the heterodimer content of the KIH control, M1, M2, M5S, and M6 combinations was higher, greater than 90%, while that of M3, M4, and M7 was 80-90%. Under a plasmid ratio of 0.6:1, the heterodimer content of the M1, M3, MS, M6, and M7 combinations was higher, greater than 90%, but the heterodimer content of the KIH, M2, and M4 combinations was lower, between 80-90%. The results showed that, compared with KIH, the MI1 and M6 combinations had a higher ability to promote AB heterodimer formation and inhibit AA / BB homodimer formation. The M2 combination had a similar ability to promote AB heterodimer and AA / BB homodimer formation compared with the KIH control. Compared with the KIH combination, the M3 combination had a similar ability to promote AB heterodimer formation, but it induced more AA homodimers and fewer BB homodimers. SEC-HPLC analysis was performed on a Theromo Vanquish Core high-performance liquid chromatograph. The purified product (1 mg / ml Protein A column) was analyzed using SEC Mix and HMA A. The column was a Waters BioResolve SEC mAb (2.5µm, 7.8x300mm). Isocratic elution was performed using VA PBS as the mobile phase for 30 min at a flow rate of 0.5 ml / min. The detection wavelength was 280 nm, the column temperature was 20 °C, and the injection volume was 10 μl. The HMA A was set at 5 °C. The results are shown in Table 2. Overall, the various parameters calculated by SEC-HPLC analysis...The proportion of heterodimers produced by the mutant combinations was similar to that obtained from SDS-PAGE analysis. Under a plasmid ratio of 1:1, the heterodimer content of the KIH control, M1, M2, MS, and M6 combinations was higher, greater than 90%, while that of the M3, M4, and M7 combinations was lower, between 80% and 90%. Under the condition of plasmid ratio of 0.6:1, the heterodimer content of the M1, M3, MS, M6, and M7 combinations was relatively high, greater than 90%, while the heterodimer content of the 12% KIH, M2, and M4 combinations was relatively low, between 80-90%. The expression levels of the combined plasmids in the VHH-Fe / Fe mutant group were analyzed by SEC-HPLC. The results show that introducing pores of 1351Y RE into the KIH base does not significantly change the ratio of heterodimers to homodimers. Introducing the K409D / D399R double mutant into KIH-L351Y did not significantly alter the heterodimer ratio, but induced more AA MOR and fewer BB homodimers. Thermal stability analysis of the Kl 3 VHH-Fe / Fe heterodimer was performed. Heterodimer purification was carried out on a 1 ml Mono S ion exchange column (Cytiva, 17516801) and washed with equilibration buffer (20 mM sodium phosphate, pH 6.0) for 1 column volume or to baseline. Protein A affinity purified samples were adjusted to pH 2-6.0 with 1.5 mM Tris base or 50 mM sodium acetate (pH 3.2), loaded onto the Mono S column at a flow rate of 0.5 column volumes / min, and then washed with equilibration buffer for 10 column volumes. Finally, elution was performed using eluent (20 mM BEARGA + 1 M BL3 sodium chloride, pH 6.0) in a 0-30% gradient, and the eluent was collected in separate tubes. The heterodimer purity of each sample was determined by SEC-HPLC, and the eluents were combined.Samples with a purity greater than 98% were used. The thermal stability of the samples was analyzed using a Microcal PEAQ-DSC. The method was as follows: Protein samples were replaced with PBS (82 μmg / mL), the detection temperature range was set to 20-100°C, and the heating rate was 90°C. PBS was added to the assay cell as a blank buffer, and temperature scanning was performed. Then, the liquid in the sample cell was removed, the sample to be tested was added, and temperature scanning was performed again. The experimental data were processed using the instrument's built-in analysis software to calculate the Tm value. The Tm determination results for the VHH-Fe / Fe mutant combination are shown in Table 3 and Figure 3. The KIH control had at least two Tm values. The first, Tm{i} (Tm1) 65.68°C, reflected the thermal stability of the VHH fragment and the CH2 domain, while the second, Tm{i} (Tm2) 71.48°C, reflected the thermal stability of the CH3 domain. The Tm values ​​of the M1 and M6 mutant combinations were only 67.56°C (66.31°C, FLIRT KIH control). These results indicate that introducing electrostatic redirection design based on the KIH structure unexpectedly reduces the thermal stability of the CH3 domain. Interestingly, the CH3 domain Tm value of the M2 mutant combination reached approximately 74.03°C, an increase of about 25°C compared to the KIH control; while the CH3 domain Tm value of the combination with the full M3 mutant was even higher. Reaching approximately 75.84°C, the single amino acid mutation AREA, BH Fc L3S1Y enhances the stability of the CH3 domain in the KIH form; furthermore, this mutation, along with FcK409D in the A chain and FcD399R AAMR EMS in the B chain, has a synergistic effect, further improving the stability of the CH3 domain. Other mutant combinations, including M4, MS5, and M7, exhibit poor thermostability, with Tm values ​​all less than [value missing].70*C. These results indicate that introducing either the A-chain L351Y or the B-chain L351Y-D399K onto the KIH base does not improve the thermal stability of the CH3 domain. #& 3 VHH-Fe / Fe mutant combination Tm value Example 4 VHH-Fc / Fab-Fe heterodimer expression and purification To verify the universality of the mutant combination in Example 2 across different antibody sequences and molecular forms, a VHH-Fc / Fab-Fc heterodimer molecule was constructed. A VHH antibody fragment (amino acid sequence see SEQ ID NO:11) and a flexible linker polypeptide CR (SEQ ID NO:12) were introduced at the human end of the first Fc fragment to form an A-chain VHH-Fc fusion protein. A Fab antibody fragment was introduced at the N-terminus of the second Fc fragment to form a Fab-Fc half-antibody. The Fab amino acid sequence is derived from the B7-H3 DRE antibody obtained via hybridoma technology, containing the light chain domain VL-CL (RAM FIL SEQ ID NO:14) and the heavy chain domain VH-CHI (amino acid sequence see SEQ ID NO:13). The heavy chain of the Fab-Fe antibody is named the B chain, and the light chain is named the C chain. The molecular weight difference between the VHH-Fc fusion protein and the Fab-Fe half antibody is helpful in distinguishing various forms of protein components formed during recombinant expression, including ABC HORA, AA MORK, BBP dimer, BBCC homodimer, A monomer, B monomer, and C monomer. The target bispecific antibody was ABC HORA, HMA. Similar to Example 2, the genes for chains A, B, and C were synthesized, plasmids expressing chains A, B, and C were constructed, and these plasmids were transfected into 293F cells. The culture supernatant was collected, Protein A affinity purified, and the purified products were analyzed using SDS-PAGE and SEC-HPLC methods to determine the content of ABC heterodisomes and other impurities. SDS-PAGE analysis...The analytical results are shown in Figure 4. Two different plasmid transfection ratios (A-strand plasmid: B-strand plasmid: C-strand plasmid) were used, with HAL ratios of 1:1.5 or 0.6:1:13. Under both conditions, the purity of the ABC heterodimer in the KIH control was below 90%. Other mutants, gag, M1, M2, M3, M5, M6, and M7, showed ABC heterodimer purity above 90% under at least one condition. The M4 mutant combination performed poorly, with ABC heterodimer purity lower than the KIH control under both conditions. The SEC-HPLC analysis results are shown in Table 4. Under both ABC plasmid ratios of 1:1:1.5 and 470.6:1:15, the expression levels of the M1-M7 mutant combination (22 WO 2025 / 092987 PCT / CN2024 / 129416) were greater than 200 mg / L, similar to or higher than the KIH control. The proportions of ABC heterodimers produced by each mutant combination, calculated by SEC-HPLC, compared with the SDS-PAGE 34728 RS AAW KIN control under both conditions, showed that ABC HORA AMAT 90% was present in the MIL-M7 mutant combination (ABC HARA AS TF KIH control) under the ABC plasmid ratio of 1:1:1.5, with the proportions of MI, M2, M3, M5, and M7 heterodimers exceeding 90%. Under the condition of ABC plasmid ratio of 0.6:1:1.5, the proportion of heterodioxan in the M1, M6, and M7 combinations was higher than that in the KIH control, and greater than 90%. Table 4 VHH-Fe / Fab-Fe mutant combination SEC-HPLC analysis mg / l mg / l Example 5 S Fab-Fe half antibody expression and purification To verify the universality of the mutant combination in Example 2 for different antibody sequences and molecular forms, two Fab fragments binding to different antigens were also linked in the Fo region to construct two different Fab-Fc half antibody mutants. The A chain Fc designed in Table 1 of Example 1 was linked to the N-terminusLinked with the first Fab, a half antibody A is obtained, wherein the Fab is derived from the anti-EGFR monoclonal antibody Zalutumumab and contains a heavy chain domain VH-CHI (amino acid sequence shown in SEQ ID NO:15) and a light chain domain VL-CL (amino acid sequence shown in SEQ ID NO:16). The Fc fragment of the HER2-chain antibody designed in Table 1 of Example 1 was linked to a second Fab fragment at the N-terminus to obtain the HER2 hapten. This Fab fragment is derived from the anti-HER2 monoclonal antibody Pertuzumab and contains the heavy chain domain VH-CHI (α28RF5]IL SEQ ID NO:17) and the light chain domain VL-CL (amino acid sequence see SEQ ID NO:18). Similar to Example 2, the light and heavy chain genes of the A and B haptens were synthesized, plasmids expressing these genes were constructed, and 293F cells were transfected to express the A hapten and HER2 hapten, respectively. The culture supernatant was collected, purified using Protein A affinity, and the purified products were analyzed using SDS-PAGE and SEC-HPLC methods. The SDS-PAGE analysis results are shown in Figure 5S. For all tested half-antibody mutants, the expression products in 293 cells mainly formed half-antibody monomers with a molecular weight of 75 kDa and homodimers with a molecular weight of 150 kDa, as well as small amounts of light chains with a molecular weight of 25 kDa and heavy chains with a molecular weight of 50 kDa. Among them, the Fab-Fc mutants KO, K2, K7, HO, H2, and H7 had a monomer ratio greater than 80%; mutants K9 and H8 had a monomer ratio less than 50%. SEC-HPLC analysis results are shown in Table $. The expression levels of all half-antibody mutants in 293 cells were greater than 100 mg / L. The proportions of half-antibody monomers, homodimers, and polymers in the expression products of 293 cells were calculated using the built-in analysis software of SEC-HPLC. The sum of monomer and dimer contents produced by all Fab-Fc mutants is shown in Table $.All were greater than 95%, and the polymer content was less than 5%. KOPF AT produced 46.27% dimer, and K2 produced 56.17% dimer, including gold-encapsulated non-covalent dimers and disulfide-linked covalent dimers. This result suggests that the K409D mutation has little effect on the homodimerization of the CH3 domain. K9 FAT A produced 86.2% dimer. K9 produced significantly more dimers than KO, indicating that the LISLY mutation promotes the homodimerization of the CH3 domain. K7 produced 26.51% dimer and 73.24% monomer. HO FAT 2304%—RA, HP RESIERUIMG ANA RA LAREN AM IRA. H2 produced 14.38% dimer and H7 produced 15.05% RK. H2 and H7 produced significantly fewer homodimers than H0O, indicating that the D399R mutation prevents homodimerization of the CH3 domain through electrostatic repulsion. H8 produced 64.71% dimer. HS8 produced significantly more dimers than HO, indicating that the L3S1Y mutation promotes homodimerization of the CH3 domain. Table 5 SEC-HPLC Analysis Results of Fab-Fe Hapmids we | im | sso] ass | 0 mw | | sao] isos | 0 _—* Example 6 Preparation and Purification of Fab-Fe / Fab-Fe Heterodimers In Vitro Assembly The hapten solution obtained by affinity chromatography in Example $ was mixed with the corresponding hapten combinations in an equimolar ratio (1:1) as shown in Table 6. Then, 1.5M Tris base was added to neutralize to pH 8-8.2. The total protein content of the reaction system was 2 mg. After standing at room temperature for 30 DAG, 200x molar ratio GSH %i& (Sigma, SO073) was added. The reaction sample was incubated at 37"C for 4 hours, and the results were analyzed separately.Samples were taken at 0.5, 1, 2, and 4 hours and immediately tested for in vitro assembly efficiency. The reaction samples were transferred to a 42°C refrigerator and left standing overnight. HIC-HPLC (hydrophobic interaction high performance liquid chromatography) was used to analyze the purity. Only HIC tests were performed using a Theromo Vanquish Core high performance liquid chromatograph. The chromatographic column was a TOSOH TSKgel Bulty-NPR (specification: 2.5μm, 4.6x100mm). 1.3SM eRe Fe 20MM BRA SIR (pH 7.0) was used as mobile phase A, and 20 mM phosphoric acid 3h YER (pH 7.0) was used as mobile phase B for gradient elution (0 - 15min 0%B - 100%B, 15.5 - 22min 0%B - 0%B). The flow rate was 0.6ml / min, the detection wavelength was 280nm, the column temperature was 23 °C, the injection volume was 10 μl, and the temperature of the temperature control plate was 5 °C. The HIC-HPLC spectra of the half-antibody and the bispecific antibody reacted for 4 hours are shown in Figure 6. This method can sensitively detect the assembly efficiency of bispecific antibody heterodimer molecules. Because there are hydrophobic differences in the monomers or homodimers formed by the half-antibodies, the peak elution times in the HIC spectra are different. The peak elution times of the bispecific antibody molecules generated by the in vitro assembly of two Fab-Fc are also different from those of the half-antibody. The HIC-HPLC analysis results are shown in Table 6. Under the conditions of 37°C and 0.5 hours of CH FA, the proportion of AB heterodimers produced by the combination of KIH, M1, M2, and M3 mutants was already greater than 90%. Under the conditions of 37°C, 2 hours, and 4 hours of CH FA, the proportion of AB heterodimers produced by the combination of KIH, M1, M2, and M3 mutants increased to approximately 93%. Table 6 HIC-HPLC analysis of bispecific antibody assembly efficiency 24 WO 2025 / 092987 PCT / CN2024 / 129416 FORM lml Capto S ImpAct ion exchange column (Cytiva, 17371751) was rinsed with the equilibration solution (20mM sodium phosphate, pH 6.0) for 5 column volumes or until the baseline.Externally assembled samples were adjusted to pH 0.8-6.2 with 1M acetic acid, then diluted 1-2 times with ultrapure water and filtered through a 0.22µm PES membrane. Samples were loaded onto a Capto S column at a flow rate of 0.5 column volumes / min. The column was then washed with equilibration buffer for 10 CV. Finally, elution was performed with eluent (20mM sodium phosphate + 1M sodium hydroxide, pH 6.0) at a 0-30 volt gradient, and the eluent was collected in separate tubes. The purity of the dimer peaks in each tube was determined by SEC-HPLC, and samples with a purity greater than 98% were combined. The SEC-HPLC analysis results are shown in Table 7. Under 37°C CHA conditions for 4 hours, the KIH, M1, M2, and M3 mutant combinations produced 96% RAMP AF, with the main component being AB heterodimers and a small amount of AA or BB homodimers. Other impurities include monomers A or B, or high molecular weight aggregates, all in proportions less than 2%. Table 7 shows the purity of the bispecific antibody produced by SEC-HPLC #49 37°C CH#FA 4h. Example 7: Test of bispecific antibody binding activity to antigen. Example 6: Binding activity of purified bispecific antibody to antigen. Recombinant human EGFR protein (ACRO Biosystems, EGR-HS222) and HER2 protein (ACRO Biosystems, HE2-H5225) were diluted to 1 μg / mL with coating buffer (Solarbio, C1055), and 100 μL of 4hte was added to each well, incubated at 4°C CHEA for 16 hours. Preparation of blocking agent: Add 3% BSA (AL, 9048-46-8) and 0.05% Tween 20 (4 I, A600560-0500) to PBS solution. After washing the plate 3 times with 0.05% PBST, add 300 μL of blocking agent to each well and incubate at room temperature for 2 hours. For stable release of the bispecific antibody, use OS% BSA - 0.05% PBST blocking agent, with a maximum concentration of 1 Sug / mL and a 3-fold continuous gradient. After washing the plate 3 times with 0.05% PBST, add 100 μL of bispecific antibody solution to each well and incubate at room temperature for 1 hour.After washing the plate three times, add 100 μL of HRP-labeled secondary antibody (Bethyl, A80-304P, 1:5000 dilution) to each well and incubate at room temperature for 1 hour. After washing the plate three times with 0.0% PBST, add 100 μL of chromogenic buffer (Solarbio, PR1210) to each well and incubate at room temperature in the dark for 10 minutes. Add 100 μL of stop buffer (Solarbio, C1058) to each well and read the OD450 absorbance using a multi-mode microplate reader (Tecan, Spark). The ELISA results are shown in Figure 7. The bispecific antibodies generated by the combinations of KIH, M1, M2, and M3 mutants showed very similar antigen-binding curves. The IC50 values ​​for binding to EGFR with the bispecific antibody ranged from 17.0 to 24.9 ng / ml, and for binding to HER2 from 12.8 to 14.6 ng / ml. Mutations in the CH3 domain of the Fe region of the RGA do not affect the antigen-binding ability of the bispecific antibody. Example 8. The binding activity of the bispecific antibody to the Fe receptor was determined using thin-layer interferometry (BLD). The affinity of the purified bispecific antibody from Example 6 for binding to human FoyRI receptor and human FcRn was measured. The equilibrium dissociation constant (KD) was calculated using a Fortebio Octet Red 96 instrument. A column of HISIK (Fortebio, Cat 18-S120) sensors was immersed in a standard buffer (1xPBS, pH 7.4 with 0.1% BSA, 0.02% Tween-20). After the sensor was equilibrated in the standard buffer, Sug / ml recombinant human FeyRI protein (ACRO Biosystems, Cat FCA-H52H1) was immobilized, and the antibody bound and dissociated in the standard buffer. Instrument operation steps: Baseline 1 (60 s), Loading(~15 s, 0.Snm), Baseline2(60 s), Association(60 s) and Dissociation(120s), 4324 1000rpm, i A 30°C. TakeOne SA (Fortebio, Cat 18-5019) & Siz iz F pH6.0 buffer (1xPBS, pH 6.0, with 0.1% BSA, 0.02% Tween-20). After equilibration in pH6.0 buffer, 200 μL of pH6.0 buffer, 100 nM antibody, and human FeRn were added to a 96 JLB ERR CT bi HK ILA (Greiner, 655209)? . 4% Jf] Fortebio Octet Red96 for detection. The sensor position was selected based on the sample location. After equilibration in pH6.0 buffer, 2 μg / ml of recombinant human FcRn protein (ACROBiosystems Cat FCM-H8286) was immobilized, then bound to 100 nM antibody and dissociated in pH6.0 buffer. Instrument operation steps: Baseline 1 (60 s), Loading (~100 s, 3.0 nm), Baseline 2 (60 s), Association (60 s), and Dissociation (60 s), with a rotation speed of 1000 rpm and a temperature of 30°C. Fortebio test results are shown in Figure 8. The affinity of the control antibody IgG1 45A for FoyRI was 6.33 x 10⁻⁶ M; the affinity of the KIH, M1, M2, and M3 mutant combinations for human FecyRI ranged from 1.01 x 10⁻⁶ M to 1.03 x 10³⁵ M. The affinity A for human FcRn was 141 x 10⁻⁶ M for the control antibody IgG1; the affinity of the KIH, M1, M2, and M3 mutant combinations for FcRn was slightly lower, ranging from 1.44 x 10⁻⁶ M to 1.85 x 10⁻⁶ M. These affinity values ​​were within the range of instrument detection error. Therefore, the Fe mutant combinations obtained in this study essentially maintained their binding to both human FcRn and human FoyRI. Furthermore, the affinity values ​​of the M1, M2, and M3 mutant combinations were very similar to the KIH control. Example 9: Accelerated Stability Test of Bispecific Antibodies. The antibody purified in Example 6...The double antibody replacement buffer was 20 mM ketamine, diluted to 1 mg / ml, and filtered through a 0.22 μm PES filter. Samples were placed in a 425°C oven, and taken on days 7 and 14 for purity analysis by SEC-HPLC and SDS-PAGE. SEC-HPLC results are shown in Table 9. After storage at 42°C for 7 days and 14 days, the purity of each mutant combination (KIH, M1, M2, and M3) only decreased slightly. Specifically, the purity of the KIH, M1, M2, and M3 mutant combinations decreased by 0.21%, 0.73%, 0.47%, and 0.57% on day 7, respectively; and by 1.43%, 1.50%, 1.46%, and 0.84% ​​on day 14, respectively. These results indicate that the newly designed Fe mutant combinations have good stability under high-temperature storage conditions. SDS-PAGE analysis also showed that the proportions of protein bands of different molecular weights remained basically unchanged after the KIH, M1, M2 and M3 mutant combinations were stored at 42°C for 14 days compared with storage for 0 days. Table 9. Discussion of Storage Stability of Bispecific Antibodies in Accelerated Experiments by SEC-HPLC Analysis. By analyzing the reasons for the decreased thermal stability of the handle-hole model, we selected amino acids near the CH3 contact interface of the Fe fragment as mutation targets to investigate the effects of different mutation combinations on the production performance, physicochemical properties, and biological functions of Fo-based IgG-like single / multispecific antibodies. We do not wish to be bound by theory, believing that in wild-type IgG1 Fe, amino acid 1351 in one chain directly contacts 1351, P352, P353, S354, and T366 in the opposite side chain; while in the KIH structure, 351 in the hole chain directly contacts L351, P352, P353, S354, and T366 in the handle chain, but with increased contact distance, the interaction force is expected to weaken. Regarding the hole...Mutations of 13S1, such as those resulting in a ketone acid with a larger side chain (L35S1Y), are presumably designed to strengthen the contact between the L351 residues in the pore chain and the handle chain amino acids P352, P353, S354, and TIOOW, thereby generating a stronger hydration effect (see Figure 1C). This also allows for new contacts with other handle chain amino acids; for example, #E&(OH) in L351Y forms a new amino bond network with T366W and E357. Building upon the L351 mutation in the pore chain, further mutations combining handle chain K409D and pore chain D399R are introduced. To avoid being bound by theory, it is speculated that this mutation combination could lead to a pair of salt bridge connections similar to those in IgG1 Fo; and that the porous chain D399R will form new contacts with the handle chains K392, T411, T366W amino acids, while the handle chain 409D mutation will retain direct contact with the porous chain F405 (COLA 1D). The experimental results of the above embodiments show that combining the above mutations in the KIH structure, namely porous chain 1L351Y or porous chain L3S1Y + handle chain K409D-D399R RE, WHR Fe scaffold and the production and physicochemical properties of mono / multispecific proteins (especially bispecific antibodies) containing it, can impart a good promoting effect. After expression in cells and purification by affinity chromatography, the Fe heterodimer scaffold protein with the mutant combination described above produced a Fo heterodimer ratio of over 90%, which is higher than the control Fc EO with only the knob-into-hole mutation; and after further purification by ion exchange column, a purity of over 98% can be achieved. Furthermore, compared to the control Fc protein, the purified Fe heterodimer scaffold protein exhibits better thermal stability, with the CH3 domain Tm value measured by DSC reaching 74°C. Further, the above embodiments...Experimental results also show that combining the above mutations in the KIH structure, namely the L351Y pore chain or the L351Y+ handle chain K409D-D399R RE, WF HORA Fe scaffold, and the antigen-binding function of the mono / multispecific proteins (especially bispecific antibodies) containing it, as well as the FcRn and FoyR receptor binding functions, has virtually no impact. These results suggest that introducing the L351Y pore chain mutation or the combination of L351YD399R and the K409D handle mutation into the KIM structure will benefit the production and stability of heterodimeric target antibodies. 27 WO 2025 / 092987 PCT / CN2024 / 129416 Sequence Listing SEQ ID | Sequence Description / Name BIRR ARF RR hoe 1 Fe KO ® & (knob) | DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS (T366W) HEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ (EU 221-447) DWLNGKEYKCK VSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG SFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 2 Fe K2 MutationDKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVV VDVS (K409D) HEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCK VSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG SFFLY SDLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPGK 3 Fe K9 MutationDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS (L351Y)HEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTY PPSREE MTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG SFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 4 Fe K7 mutation DKTHTCPPCPAPELLGGPSVELFPPKPKDTLMISRTPEVTCVVVDVS (L351Y-K409D) HEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTY PPSREE MTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG SFFLY SDLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPGK 5 Fe HO mutation (hole) | DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS (T366S-L368A-Y40_ | HEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ TV) DWLNGKEYKCK VSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG SFFLVSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPGK Fe H2 mutation DKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVV VDVS (D399R) HEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCK VSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTIPPVLRSDG SFFLVSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPGK 7 Fe H8 mutationDKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVV VDVS (L351Y) HEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTY PPSREE MTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG SFFLVSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPGK Fe H7 mutation DKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVS (L351 Y-D399R) HEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTY PPSREE MTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTIPPVLRSDG SFFLVSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPGK [9 | Fe2-D399K _| DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDYS | 28 WO Serial number 2025 / 092987 PCT / CN2024 / 129416 HEDPEVKFNW Y VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPOQVY TLPPSREE MTKNQVSLSCAVKGFY PSDIAVEWESNGQPENNYKTTPPVLKSDG SFFLVSKLTVDKSRW QOGNVEFSCS VMHEALHNHY TOKSLSLSPGK 10 Fe H7-D399K mutation DKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVS (L351 Y-D399K) HEDPEVKFNW Y VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY TY PPSREE MTKNQVSLSCAVKGFY It should be noted that this text seems to be a very specific and technical biological or genetic sequence description, and the translation is mainly for the purpose of maintaining the original content in English while keeping the specific tags intact. If there are further requirements for understanding or processing this text, additional biological or technical knowledge may be needed.PSDIAVEWESNGQPENNYKTTPPVLKSDG SFFLVSKLTVDKSRW QOGNVEFSCS VMHEALHNHY TOKSLSLSPGK 11 Ozoralizuamab VHH | EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMS W VRQAP KGL EWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTA VY YCTIGGSLSRSSQGTLVTVSS 13 hz32F8.9- VH+CH1 | QVOLVQSGAEVKKPGSSVKVSCKTSGYTFTDY VITW VRQAPGQG LEWMGETYPRSDRTY YNENFKGRVTITADKSTNTAY MELSSLRSED TAVY YCARWDNYYFDYWGQGTLVTVSSASTKGPS VFPLAPSSKST SGGTAALGCLVKD YFPEPVTVSWNSGALTSGVHTFPAVLOSSGLYS LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC 14 hz32F8.9-VL+CL DVVMTQSPLSLPVTLGQPASISCRSSQSLVHSNAITY LWY QQRPG QSPRLLIY KVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVY CSQTTHVPWTFGGGTK VEIKRTVAAPS VFIFPPSDEQLKSGTAS VVC LLNNFYPREAK VOQWKVDNALOSGNSQESVTEQDSKDSTYSLSSTL TLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 15 Zalutumumab-VH+ | QVQLVESGGGVVQPGRSLRLSCAASGFTFSTY GMHW VRQAPGKG CH1 LEW VAVIWDDGSYKY YGDSVKGRFTISRDNSKNTLYLQMNSLRAE DTAVY YCARDGITMVRGVMKDYFDYWGQGTLVTVSSASTKGPSV FPLAPSSKSTSGGTAALGCLVKD YFPEPVTVSWNSGALTSGVHTFP AVLQOSSGLYSLSSVVTVPSSSLGTQTYICN VNHKPSNTKVDKKVEP KSC 16Zalutumumab - AIQLTQSPSSLSASVGDRVTITCRASQDISSALVWYQQKPGKAPKLLVL+CLTYDASSLESGVPSRFSGSESGTDFTLTISSLQPEDFATYYCQQFNSYPLTFGGGTKVEIKRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALOSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC17Pertuzumab - VH+CEVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKG H1LEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLOSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC18Pertuzumab - DIQMTQSPSSLSASVGDRVTITCKAQDVSIGVAWYQQKPGKAPLVL+CL LIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYTPYTFGQGTKVEIKRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALOQOSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC29WO2025 / 092987PCT / CN2024 / 129416(EU341 - 447)PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQOQGNVFSCSVMHEA Se [Enwoeuuroe20Wild - typeCH3domain|GQPREPOVY TLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQWithoutK447residuePENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQOQGNVFSCSVMHEA [Gerke [eames21 Sh 4 CH2 domain | APELLGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFEN (EU 231-340) WY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY a 30 WO 2025 / 092987 PCT / CN2024 / 129416 Claim 1. A heterodimeric Fe scaffold comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises a first Fec region, the second polypeptide comprises a second Fc region, wherein the first Fc region comprises a first CH3 domain, the second Fe region comprises a second CH3 domain, wherein, relative to the wild-type CH3 domain, the first Fc region and the second Fe region respectively contain a KIH-mutated knob mutation and a hole mutation in their CH3 domains, and wherein, (a) the Fe region contains a hole-mutated Fo The region also contains an L351Y mutation; or (b) a hole-mutated Fe region also contains an L351Y and D399R FB, HS knob-mutated Fe Ri K409D, wherein the first and second polypeptides are paired and heterodimerized through the first and second Fo regions to form the Fo XE, LP ARR RARE Kabat numbered by EU index. 2. The heterodimeric Fe scaffold of claim 1, wherein the first and second Fe regions contain CH2 and CH3 AHI, ROH chain regions, CH2 and CH3 structural domains. 3. A heterodimeric Fo scaffold according to any one of claims 1-2, wherein the first and second Fe RA IgG types, such as IgG1, IgG2, IgG3 or IgG4 subtypes, preferably, the first and second Fo regions are human IgG1 LH. 4. A heterodimeric Fo scaffold according to any one of claims 1-3, wherein the knob mutation is T366W; and the hole mutation is Y407V or...T366S-L368A-Y407V, preferably, the first Fc region includes T366W, and the second Fc region includes T366S-L368A-Y407V-L351Y; or the first Fe region includes T366W-K409D, and the second Fo region includes T366S-L368A-Y407V-L351Y-D399R. 5. The heterodimeric Fo scaffold of claims 1-4, wherein: (a) relative to the wild-type CH3 domain, the first and second CHB domains respectively further comprise 0-3 amino acid residue changes; preferably, the wild-type CH3 domain is the CH3 domain of natural human IgG1 immunoglobulin, more preferably comprising the amino acid sequence of SEQ ID NO: 19 or 20; (b) the first and second Fo regions comprise the CH2 domain of natural human IgG1 immunoglobulin, or comprise the SEQ ID NO: 21 PR RH amino acid sequence or CH2 BHA having 1-5 amino acid residue changes therewith; (c) the Fe region with the &4 knob mutation comprises the amino acid sequence of SEQ ID NO: 1 or 2 or an amino acid sequence having at least 95%, 96%, 97%, 989% or 99% identity therewith; (d) the Fe region containing the hole mutation comprises SEQ ID NO: 7 or 8. The amino acid sequence of the scaffold or an amino acid sequence having at least 93%, 96%, 979%, 989%, or 99% identity with it, and / or (e) the Fo scaffold does not form non-natural disulfide bonds between the first Fc region and the second Fo region. 6. A heterodimeric Fo scaffold of any one of claims 1-5, wherein the Fo scaffold, compared to the corresponding control Fe scaffold containing only tungsten containing the KIH mutation, has increased thermal stability, and preferably, by DSC determination, the CH3 domain of the Fe scaffold has a Tm value of approximately 70°C or higher. 7. A CH3 HARA comprising a heterodimeric first immunoglobulin.The first CH3 domain and the second immunoglobulin CH3 domain, wherein: relative to the wild-type CH3 domain, the first CH3 domain and the second CH3 domain respectively contain the knob mutation and the hole mutation in the KIH mutation, and wherein, (a) the CH3 domain containing the hole mutation also contains the L351Y mutation; or (b) the CH3 domain containing the hole mutation also contains the L351Y and D399R mutations, and the CH3 domain containing the knob mutation 31 Correction page (Detailed Rules Article 91) 1ISA / CN WO 2025 / 092987 PCT / CN2024 / 129416 ELS K409D mutation, wherein the amino acid residues are numbered according to the EU index in Kabat, preferably, the first and second CH3 domains are of the IgG type, such as IgG1, IgG2, IgG3 or IgG4 subtype, Preferably, the wild-type CH3 domain is a natural human IgG1 RARE A CH3 domain; more preferably, relative to the wild-type CH3 domain, the first a...

Images