Fc, antibody constant region, immunoglobulin, and use thereof

Abstract

An antibody Fc, comprising a modified hinge region. The modified hinge region is obtained by modifying a parental hinge region; the parental hinge region comprises a C-O-C short peptide, and the first C and the last C in the C-O-C short peptide can mispair to form an intrachain disulfide bond in a heavy chain; and in the modified hinge region, the C-O-C short peptide in the parental hinge region is modified into a C-Z-C short peptide.

Description

Fc, antibody constant region, immunoglobulins and their uses

[0001] Priority information

[0002] This application claims priority and benefit to patent application 202510142779.9 filed with the China National Intellectual Property Administration on February 8, 2025, and patent application 202511120972.9 filed with the China National Intellectual Property Administration on August 12, 2025, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This application belongs to the field of antibody modification technology, specifically relating to an Fc, antibody constant region, immunoglobulin and its uses. Background Technology

[0004] Antibodies are produced by B lymphocytes. An antibody monomer is a Y-shaped molecule composed of four polypeptide chains. These include two identical heavy chains and two identical light chains. The Fv region of both the light and heavy chains contains the molecule's antigen-binding determinants, responsible for binding to the target antigen. The Fc region defines the antibody class (or isotype) (e.g., IgG) and is responsible for binding to many native proteins to trigger important biochemical events.

[0005] With advancements in science and technology, in vitro diagnostics has become a crucial tool in clinical medicine, acting as the "eyes" of doctors. Immunodiagnostic reagents are a subfield of in vitro diagnostic reagents. Immunodiagnostics applies the theories, techniques, and methods of immunology to diagnose various diseases and measure immune status. Methods of immunodiagnostics include radioimmunoassay, enzyme-linked immunosorbent assay (ELISA), chemiluminescence immunoassay, fluorescence chromatography, and colloidal gold chromatography. One of the main challenges in developing immunodiagnostic reagents is obtaining antibodies that strongly bind to antigens.

[0006] In in vitro immunodiagnostics, commonly used antibody sources include mice, rats, and alpacas. Compared to widely used mouse antibodies, antibodies from novel species possess unique appeal. For example, rabbit or sheep antibodies exhibit higher affinity, broader antigen recognition capabilities, or higher detection sensitivity in in vitro immunoassays compared to mouse antibodies. Therefore, research on novel species antibodies, such as rabbit antibodies or sheep monoclonal antibodies, is increasingly attracting attention in the industry. Summary of the Invention

[0007] This application provides an Fc, an antibody constant region, an antibody, and its uses.

[0008] In a first aspect of this application, an Fc is proposed. According to an embodiment of this application, the Fc includes a modified hinge region, wherein the modified hinge region is formed by modifying a COC short peptide in the parent hinge region into a CZC short peptide, wherein the O in the COC short peptide or the Z in the CZC short peptide independently represents one or more amino acids. The Fc of this application has the advantage of low half-antibody formation rate, and in particular, antibodies containing the Fc of this application can reduce the half-antibody formation rate or increase the antibody assembly rate.

[0009] In a second aspect, this application proposes an Fc. According to embodiments of this application, the Fc includes a modified hinge region, wherein the first C in the COC short peptide of the parent hinge region is replaced with any amino acid other than C, and the O in the COC short peptide represents one or more amino acids. The Fc of this application has the advantage of a low half-antibody formation rate; in particular, antibodies containing the Fc of this application can reduce the half-antibody formation rate or increase the antibody assembly rate.

[0010] In a third aspect of this application, an antibody constant region is proposed. According to embodiments of this application, the antibody constant region includes the Fc and CH1 regions described in the first or second aspect. Antibodies containing the antibody constant region of this application can reduce the rate of hapten formation or increase the antibody assembly rate.

[0011] In a fourth aspect, this application discloses an antibody. According to embodiments of this application, the antibody includes the Fc region described in the first or second aspect and the antibody constant region described in the third aspect. The antibody of this application can reduce the formation rate of haptens or increase the assembly rate of antibodies.

[0012] In a fifth aspect of this application, a nucleic acid molecule is provided. According to embodiments of this application, the nucleic acid molecule encodes the Fc described in the first or second aspect, the antibody constant region described in the third aspect, or the antibody described in the fourth aspect.

[0013] In a sixth aspect, this application provides a vector. According to an embodiment of this application, the vector carries the nucleic acid molecule described in the fifth aspect.

[0014] In a seventh aspect of this application, a host cell is proposed. According to embodiments of this application, the host cell comprises the nucleic acid molecule or vector described in the fifth aspect; or expresses the Fc described in the first or second aspect, the antibody constant region described in the third aspect, or the antibody described in the fourth aspect.

[0015] In an eighth aspect of this application, a conjugate is proposed. According to an embodiment of this application, the antibody conjugate comprises: the Fc described in the first or second aspect, the antibody constant region described in the third aspect, or the antibody described in the fourth aspect, and at least one conjugated portion connected to the Fc, the antibody constant region, or the antibody.

[0016] In a ninth aspect of this application, a kit is provided. According to embodiments of this application, the kit comprises: the Fc described in the first or second aspect, the antibody constant region described in the third aspect, or the antibody described in the fourth aspect, or the conjugate described in the eighth aspect.

[0017] In the tenth aspect of this application, the use of the Fc described in the first or second aspect, the antibody constant region described in the third aspect, the antibody described in the fourth aspect, the nucleic acid molecule described in the fifth aspect, the vector described in the sixth aspect, the host cell described in the seventh aspect, the conjugate described in the eighth aspect, or the kit described in the ninth aspect in immunoassay or in the preparation of immunoassay products is proposed.

[0018] In the eleventh aspect of this application, a method for detecting an antigen in a sample to be tested is provided. According to an embodiment of this application, the method includes: a) contacting the antibody described in the fourth aspect, the conjugate described in the eighth aspect, or the kit described in the ninth aspect with an antigen in the sample to be tested under conditions sufficient to induce an antibody / antigen binding reaction to form an immune complex; and

[0019] b) Detect the presence of the immune complex, and determine whether the sample to be tested contains an antigen based on the presence of the immune complex.

[0020] In a twelfth aspect of this application, a method for reducing antibody hemisphere formation rate or increasing antibody assembly rate is proposed. According to embodiments of this application, the method includes: modifying the Fc in the antibody to form the Fc described in the first or second aspect;

[0021] The antibody constant region in the antibody is modified to form the antibody constant region described in the third aspect; or

[0022] The antibody is modified to form the antibody described in the fourth aspect.

[0023] Additional aspects and advantages of this application will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of this application. Attached Figure Description

[0024] The above and / or additional aspects and advantages of this application will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0025] Figure 1 shows the clinical relevance of wild-type antibody, modified antibody 10, modified antibody 11, and modified antibody 12 in Example 1 of this application;

[0026] Figure 2 shows the clinical relevance of wild-type antibody, modified antibody 14, modified antibody 15, modified antibody 16, and modified antibody 19 in Example 1 of this application;

[0027] Figure 3 shows the clinical relevance of wild-type antibody, modified antibody 17, modified antibody 18, and modified antibody 21 in Example 1 of this application. Detailed Implementation

[0028] The embodiments of this application are described in detail below. The embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application.

[0029] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this application, unless otherwise stated, "multiple" means two or more.

[0030] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0031] To facilitate understanding of this application, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this application pertains. Abbreviations for amino acid residues are the standard 3-letter and / or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids.

[0032] In this document, the terms “comprising” or “including” are open-ended expressions, meaning that they include the contents specified in this application but do not exclude other contents.

[0033] In this document, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0034] In this paper, the terms “identity,” “homology,” or “similarity” are used to describe the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences relative to a reference sequence, determined by conventional methods, for example, Ausubel et al., eds. (1995), Current Protocols in Molecular Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York). and the ALIGN procedure (Dayhoff (1978), Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research Institute)). Foundation, Washington, DC). There are many algorithms for aligning sequences and determining sequence identity, including the homology alignment algorithm by Needleman et al. (1970) J. Mol. Biol. 48: 443; the local homology algorithm by Smith et al. (1981) Adv. Appl. Math. 2: 482; and the similarity search method by Pearson et al. (1988) Proc. Natl. Acad. Sci. 85: 2444. The Smith-Waterman algorithm (Meth. Mol. Biol.) .70:173-187 (1997). And the BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al. (1990) J.Mol.Biol. 215:403-410). Computer programs utilizing these algorithms are also available, and include, but are not limited to: ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (Altschul et al., Meth.Enzym., 266:460-480 (1996)). Or GAP, BESTFIT, BLAST Altschul et al., above, FASTA, and TFASTA, available in Genetics Computing Group (GCG) package, version 8, Madison, Wisconsin, USA. And the CLUSTAL PC / Gene program provided by Intelligenetics, Mountain View, California.

[0035] In this document, the term "having at least 80% sequence similarity" can mean having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence similarity. The sequence similarity described in this application can be measured using sequence analysis software, such as the computer program BLAST with default parameters, especially BLASTP or TBLASTN. All amino acid sequences mentioned in this application are shown from the N-terminus to the C-terminus.

[0036] In this document, the term "having at least 90% sequence similarity" can mean having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence similarity.

[0037] In this document, the term "variant" or "mutant" can refer to any naturally occurring or engineered molecule that contains one or more nucleotide or amino acid differences (mutations) that differ from a reference sequence. This difference can be a substitution, deletion, or insertion of one or more amino acids.

[0038] In this document, the term "vector" generally refers to a nucleic acid molecule capable of self-replication within a suitable host, transferring the inserted nucleic acid molecule into host cells and / or between host cells. The vector may include vectors primarily for inserting DNA or RNA into cells, vectors primarily for replicating DNA or RNA, and expression vectors primarily for transcription and / or translation of DNA or RNA. The vector also includes vectors having multiple of the aforementioned functions. The vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, by culturing suitable host cells containing the vector, the vector can produce the desired expression product.

[0039] In this document, the term "recombinant cell" generally refers to a cell in which the genetic material of a host cell is modified or recombined using genetic engineering or cell fusion techniques to obtain a unique trait with stable inheritance. The term "host cell" refers to a prokaryotic or eukaryotic cell into which a recombinant vector can be introduced. The terms "transformed" or "transfected" as used herein refer to the introduction of nucleic acids (e.g., vectors) into cells using various techniques known in the art. Suitable host cells can be transformed or transfected with the DNA sequence of this application and can be used for the expression and / or secretion of target proteins. Examples of suitable host cells that can be used in this application include immortalized hybridoma cells, NS / 0 myeloma cells, 293 cells, Chinese hamster ovary (CHO) cells, HeLa cells, Cap cells (cells derived from human amniotic fluid), and CoS cells.

[0040] Common antibody monomers consist of two identical light chain ("L") polypeptides and two identical heavy chain ("H") polypeptides. The four chains are linked by disulfide bonds in a "Y" configuration. At the base of the Y chain, the two H chains are covalently bonded together. The light chain, from N-terminus to C-terminus, includes a variable region and a CL region. The heavy chain, from N-terminus to C-terminus, includes a variable region and a constant region.

[0041] In this paper, the CH1 region, hinge region (optional), CH2 region, CH3 region, CH4 region (optional), and tail peptide (optional) are all components of the heavy chain constant region, which is located at the C-terminus of the heavy chain of the antibody molecule. Each heavy chain constant region, from the N-terminus to the C-terminus, includes the CH1 region, hinge region (optional), CH2 region, CH3 region, CH4 region (optional), and tail peptide (optional). Different types of antibodies (such as IgG, IgA, IgM, etc.) have different amino acid sequences and structures in their heavy chain constant regions, but they all have relatively conserved structural features. These conserved structures enable the heavy chain constant region to perform its biological function. The heavy chain constant region, CH1 region, hinge region (optional), CH2 region, CH3 region, CH4 region (optional), and tail peptide (optional) of different species and subclasses are well known in the art, and their amino acid sequences can be determined based on bioinformatics databases, such as the IMGT database (https: / / www.imgt.org / IMGTrepertoire / Proteins / ). It should be understood that different bioinformatics databases or software may not have completely consistent results in the division and sequence identification of constant regions. However, those skilled in the art have a general and unified understanding of the concept, division and sequence identification of constant regions and their segments. Therefore, the constant regions that those skilled in the art can identify and divide using common knowledge and ordinary methods are all within the scope of protection of this invention.

[0042] For example, the amino acid sequence of the corresponding segment (such as the IgM CH2 region) divided by the IMGT database can be used as the reference sequence. The start or end position of the reference sequence can be moved forward by several amino acid residues (i.e., moved to the IgM CH1 region) or backward by several amino acid residues (i.e. moved to the IgM CH3 region) to obtain a sequence of the corresponding segment that is longer or shorter than the reference sequence.

[0043] In this document, the term "CH1 region" refers to a relatively conserved region located at the N-terminus of the constant region of the heavy chain of an antibody, immediately following the variable region of the heavy chain. The specific amino acid sequence is not limited and is within the scope of protection of this application. CH1 regions of different species and alternative subclasses are well known.

[0044] In this document, the term "hinge region" refers to a polypeptide that links the CH1 and CH2 domains within the constant region of the heavy chain of an antibody. This region is rich in proline and therefore readily stretchable and flexible. Hinge regions are typically dimers, consisting of two polypeptides with the same amino acid sequence. Specific amino acid sequences are not limited and are all within the scope of protection of this application. Hinge regions of different species and subclasses are well known.

[0045] In this document, the term "CH2 region" refers to the second constant region of the antibody's heavy chain constant region, a relatively conserved region located after the CH1 region. The specific amino acid sequence is not limited and is within the scope of protection of this application. CH2 regions of different species and alternative subclasses are well known.

[0046] In this document, the term "CH3 region" refers to the third constant region of the antibody heavy chain constant region, a relatively conserved region located after the CH2 region. The CH3 region of IgM contains cysteine ​​residues involved in polymer formation; the specific amino acid sequence is not limited and is within the scope of protection of this application. The CH3 regions of different species and alternative subclasses are well known.

[0047] In this document, the term "CH4 region" refers to the fourth constant region of the heavy chain, a relatively conserved region located after the CH3 region. The specific amino acid sequence is not limited and is within the scope of protection of this application. CH4 regions of different species are well-known.

[0048] In this paper, the term "tail peptide" refers to a short peptide sequence at the end of the CH3 or CH4 region of an antibody. Tail peptides from different species are well known.

[0049] In this paper, the "CL region" refers to the constant region of the antibody's light chain, located at the C-terminus of the light chain, immediately following the variable region (VL). The CL region provides stability to the overall antibody structure by binding to the CH1 region of the heavy chain and plays a crucial role in antibody folding. The specific amino acid sequence is not limited and is within the scope of protection of this application. CL regions of different species and subtypes are well-known.

[0050] In this paper, the term "Fc" refers to the portion of the heavy chain of the antibody molecule located from the beginning of the hinge region to the end of the C-terminus. The hinge region can be further subdivided into three domains: the upper hinge domain, the middle hinge domain, and the lower hinge domain.

[0051] In this paper, the term "upper hinge region" refers to a segment of amino acid residues at the N-terminus of the middle hinge region.

[0052] In this paper, the term "intermediate hinge region" refers to a peptide segment containing all the cysteine ​​residues used to form inter-heavy chain disulfide bonds and located between the upper and lower hinge regions.

[0053] In this paper, the term "lower hinge region" refers to the segment of amino acid residues immediately adjacent to the C-terminus of the middle hinge region.

[0054] Hinge region sequences of various antibodies:

[0055] This application includes detailed descriptions of the Fc region, antibody constant region, antibody, and its uses.

[0056] The inventors of this application unexpectedly discovered a halved antibody phenomenon in the expression of rabbit IgG antibodies or sheep IgG2 antibodies during experiments. The occurrence of halved antibodies leads to poor structural uniformity and large batch-to-batch variations in antibodies. To solve this problem, the inventors analyzed rabbit IgG antibodies or sheep IgG2 antibodies and attempted to modify the antibody hinge region. The results showed that the structural uniformity of the modified antibodies was significantly improved.

[0057] Fc

[0058] In a first aspect of this application, an Fc is proposed. According to an embodiment of this application, the Fc includes a modified hinge region, wherein the modified hinge region is formed by modifying a COC short peptide in the parent hinge region into a CZC short peptide, wherein the O in the COC short peptide or the Z in the CZC short peptide each independently represents one or more amino acids.

[0059] The Fc of this application has the advantage of low half-antibody formation rate. In particular, antibodies containing the Fc of this application can reduce the half-antibody formation rate or increase the antibody assembly rate.

[0060] In this article, the term "parental hinge region" can refer to a wild-type hinge region, a variant of the wild-type hinge region, or an engineered hinge region. In this article, the antibody corresponding to this parental hinge region is a parental antibody.

[0061] In this paper, short peptide sequences, such as COC or CZC peptides, are shown in N-terminal to C-terminal order. The positions of amino acid residues on the short peptide are indicated in the order from N-terminus to C-terminus. For example, the first C in a COC peptide is the first C from the N-terminus to the C-terminus.

[0062] In this paper, all C in the short peptide sequence are cysteine ​​residues, such as COC short peptide or CZC short peptide.

[0063] In this paper, the term "half-antibody" refers to half of a Y-shaped antibody. A correctly assembled conventional antibody monomer is Y-shaped; the complete Y-shape forms inter-chain disulfide bonds through hinges, thus forming a divalent antibody, while a half-antibody is a monovalent antibody. In this paper, "antibody assembly rate" refers to the percentage of antibodies that form the correct conformation.

[0064] In this article, the term "amino acid modification" or "modification" includes amino acid substitution, insertion, and / or deletion in a polypeptide sequence. "Amino acid substitution" or "substitution" in this article means that an amino acid at a specific site in the parental polypeptide sequence is replaced by another amino acid. "Amino acid insertion" or "insertion" in this article means that an amino acid is added to a specific site in the parental polypeptide sequence. "Amino acid deletion" or "deletion" in this article means that an amino acid is removed from a specific site in the parental polypeptide sequence.

[0065] According to embodiments of this application, the Fc may further include at least one of the following technical features: In an optional embodiment of this application, the first C and the last C in the COC short peptide in the parent hinge region may mismatch to form an intra-heavy chain disulfide bond.

[0066] The first and last C in the COC short peptide in the parent hinge region mismatches to form an intra-heavy chain disulfide bond, preventing the formation of inter-heavy chain disulfide bonds and leading to the formation of a half-antibody.

[0067] In an optional embodiment of this application, the last C in the COC short peptide is a cysteine ​​residue in the parental hinge region used to form inter-heavy chain disulfide bonds. That is, when the antibody is correctly assembled, the last C in the COC short peptide is a cysteine ​​residue in the parental hinge region used to form inter-heavy chain disulfide bonds, resulting in the formation of a correct divalent Y-shaped structure.

[0068] In an optional embodiment of this application, the first C in the COC short peptide is a cysteine ​​residue in the parental hinge region used to form an intra-heavy chain disulfide bond. That is, when the antibody is correctly assembled, the first C in the COC short peptide is a cysteine ​​residue in the parental hinge region used to form an intra-heavy chain disulfide bond, resulting in a stable Y-shaped structure.

[0069] In one alternative embodiment of this application, the modification is selected from at least one of substitution, insertion, and deletion.

[0070] In one alternative embodiment of this application, the modification is selected from substitution.

[0071] In one alternative embodiment of this application, the modification is selected from substitution.

[0072] In one alternative embodiment of this application, the substitution is selected from the substitution of flexible amino acids with neutral or rigid amino acids.

[0073] In one optional embodiment of this application, the substitution is selected from the substitution of neutral amino acids with rigid amino acids.

[0074] In one alternative embodiment of this application, the substitution is selected from the interchange between different flexible amino acids.

[0075] In one alternative embodiment of this application, the substitution is selected from the interchange between different neutral amino acids.

[0076] In one alternative embodiment of this application, the substitution is selected from the interchange between different rigid amino acids.

[0077] In one alternative embodiment of this application, the substitution is selected from cysteine ​​substitution.

[0078] In this article, "cysteine ​​substitution" refers to replacing other amino acids besides cysteine ​​with cysteine.

[0079] In one optional embodiment of this application, the number of amino acids replaced is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0080] In one optional embodiment of this application, the number of cysteine ​​substitutions is 1, 2, 3, or 4 amino acids.

[0081] In an optional embodiment of this application, the modification is selected from insertion.

[0082] In one alternative embodiment of this application, the insertion is selected from neutral amino acid insertion, rigid amino acid insertion, or cysteine ​​insertion.

[0083] In one optional embodiment of this application, the number of inserted amino acids is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0084] In one optional embodiment of this application, the number of cysteine ​​insertions is 1, 2, 3, or 4 cysteines.

[0085] In an optional embodiment of this application, the modification is selected from the absence.

[0086] In one alternative embodiment of this application, the deletion is a neutral amino acid deletion or a flexible amino acid deletion.

[0087] In one optional embodiment of this application, the number of missing amino acids is 1, 2, 3, 4 or 5.

[0088] In one optional embodiment of this application, the neutral amino acid is selected from aspartic acid, lysine, arginine, or histidine.

[0089] In one optional embodiment of this application, the rigid amino acid is selected from alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, tryptophan, or proline.

[0090] In one optional embodiment of this application, the rigid amino acid is proline.

[0091] In one alternative embodiment of this application, the flexible amino acid is selected from glycine, serine, or threonine.

[0092] In one optional embodiment of this application, the flexible amino acid is threonine.

[0093] In an optional embodiment of this application, the Z in the CZC short peptide includes a C-X1-C short peptide, or includes a C-X1 short peptide in the C-X1-C short peptide, or the CZC short peptide is a C-X1-C short peptide.

[0094] In an optional embodiment of this application, X1 in the C-X1-C short peptide represents one or more amino acids.

[0095] In an optional embodiment of this application, the CZC short peptide is a C-X2-C-X1-C-X3-C short peptide.

[0096] In an optional embodiment of this application, X1, X2, and X3 in the C-X2-C-X1-C-X3-C short peptide each independently represent one or more amino acids.

[0097] In an optional embodiment of this application, C-X2 in the C-X2-C-X1-C-X3-C short peptide is a continuous peptide segment on the COC short peptide that starts with the first C at the N-terminus, and X3-C in the short peptide is a continuous peptide segment on the COC short peptide that ends with the last C at the C-terminus.

[0098] For example, see modification 21 in Table 1 of this application. The COC short peptide corresponds to the CSKPTC short peptide, the C-X2-C-X1-C-X3-C short peptide corresponds to the CSKCPPCPTC short peptide, the C-X1-C short peptide corresponds to CPCC, the C-X2 short peptide corresponds to CSK, and the X3-C short peptide corresponds to PTC.

[0099] In an optional embodiment of this application, the CZC short peptide is a C-X4-C-X1-C short peptide.

[0100] In an optional embodiment of this application, C-X4 in the C-X4-C-X1-C short peptide is a continuous peptide segment on the COC short peptide starting with the first C at the N-terminus.

[0101] For example, see Modification 15 in Table 1 of this application. The COC short peptide corresponds to the CSKPTC short peptide, the C-X4-C-X1-C short peptide corresponds to the CSKPCKHCRC short peptide, the C-X1-C short peptide corresponds to the CKHCRC, and the C-X4 short peptide corresponds to the CSKP.

[0102] In an optional embodiment of this application, C-X4 in the C-X4-C-X1-C short peptide is CO in the COC short peptide.

[0103] In an optional embodiment of this application, X1 and X4 in the C-X4-C-X1-C short peptide each independently represent one or more amino acids.

[0104] For example, see Modification 16 in Table 1 of this application. The COC short peptide corresponds to the CSKPTC short peptide, the C-X4-C-X1-C short peptide corresponds to the CSKPTCKHCRC short peptide, the C-X1-C short peptide corresponds to the CKHCRC, and the C-X4 short peptide corresponds to the CSKPT.

[0105] In an optional embodiment of this application, the C-X1-C short peptide has a higher ratio of the difference between the number of rigid amino acids and the number of flexible amino acids compared to the COC short peptide.

[0106] In this paper, the percentage difference between the number of rigid amino acids and the number of flexible amino acids is calculated as (rigid amino acids - number of flexible amino acids) / total number of amino acid residues in the short peptide.

[0107] In an optional embodiment of this application, the C-X1-C short peptide does not contain flexible amino acids.

[0108] In an optional embodiment of this application, the C-X1-C short peptide has more cysteine ​​residues than the COC short peptide.

[0109] In an optional embodiment of this application, the C-X1-C short peptide is derived from the natural hinge region of a natural antibody.

[0110] In an optional embodiment of this application, the C-X1-C short peptide is derived from a peptide segment on a natural hinge region that is different in species and subtype from the parent hinge region; hereinafter, the natural hinge region from which the C-X1-C short peptide is derived is referred to as "its source natural hinge region".

[0111] In an optional embodiment of this application, the first C and the last C in the C-X1-C short peptide are both Cs in the natural hinge region from which the peptide is derived, which are used to form inter-heavy chain disulfide bonds.

[0112] In an optional embodiment of this application, the first C in the C-X1-C short peptide is the first C in its natural hinge region used to form inter-heavy chain disulfide bonds.

[0113] In an optional embodiment of this application, the last C in the C-X1-C short peptide is the last C in its natural hinge region used to form inter-heavy chain disulfide bonds.

[0114] In an optional embodiment of this application, the C-X4-C-X1-C short peptide is a continuous fragment on its naturally occurring hinge region. That is, the C-X4-C-X1-C short peptide is a continuous fragment containing a C-X1-C short peptide on the naturally occurring hinge region of its corresponding species and subtype of C-X1-C.

[0115] In an optional embodiment of this application, the first C in the C-X4-C-X1-C short peptide is the C in the upper hinge region of its natural hinge region, and the first C is not the C in the natural hinge region used to form inter-heavy chain disulfide bonds.

[0116] In an optional embodiment of this application, the first C in the C-X4-C-X1-C short peptide is the last C in the upper hinge region of its naturally derived hinge region.

[0117] In an optional embodiment of this application, X1, X2, X3, or X4 in the short peptide each independently represent one or more amino acids.

[0118] In an optional embodiment of this application, the CZC short peptide has more amino acid residues than the COC short peptide.

[0119] In one optional embodiment of this application, the number of amino acid residues in the CZC short peptide is 1 to 10 more than the number of amino acid residues in the COC short peptide.

[0120] In this article, "number of amino acid residues" and "number of amino acids" are synonymous. "The number of amino acid residues of the CZC short peptide is 1 to 10 more than the number of amino acid residues of the COC short peptide" means that the number of amino acids of Z is 1 to 10 more than the number of amino acids of O, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0121] In one optional embodiment of this application, the CZC short peptide has 1 to 5 more amino acid residues than the COC short peptide.

[0122] In one optional embodiment of this application, the number of amino acid residues of the CZC short peptide is 1, 2, 3, 4 or 5 more than the number of amino acid residues of the COC short peptide.

[0123] In one optional embodiment of this application, the species of the natural hinge region is selected from humans, mice, rats, cattle, horses, pigs, sheep, goats, dogs, camels, alpacas, monkeys, orangutans, cats, rabbits, donkeys, deer, minks, chickens, ducks, geese, or humans.

[0124] In an optional embodiment of this application, the subtype of the natural hinge region is selected from IgG1, IgG2, IgG3, IgG4, IgA, and IgD.

[0125] In an optional embodiment of this application, the species and subtype of the natural hinge region are selected from bovine IgG1, sheep IgG1, human IgG1, horse IgG1, mouse IgG2a, mouse IgG1, monkey IgG1, monkey IgG3, monkey IgG4, orangutan IgG1, horse IgG3, horse IgG5, alpaca IgG1A, and alpaca IgG2B.

[0126] In an optional embodiment of this application, the C-X1-C short peptide is selected from the natural hinge region of bovine IgG1, sheep IgG1, human IgG1, horse IgG1, mouse IgG2a, and mouse IgG1.

[0127] In an optional embodiment of this application, the C-X1-C short peptide is selected from any short peptide or a portion thereof from CDCC, CKHCRC, CPPC, CPKC, CPPCKC, and CKPCIC.

[0128] In an optional embodiment of this application, the C-X4-C-X1-C short peptide is selected from sheep IgG1, human IgG1, and mouse IgG1.

[0129] In an optional embodiment of this application, the C-X4-C-X1-C short peptide is selected from any one of CGCKPCIC, CDKTHTCPPC, and CPDPCKHCRC short peptides.

[0130] In one alternative embodiment of this application, the species and subtype of the parent hinge region are selected from rabbit IgG.

[0131] In an optional embodiment of this application, the COC short peptide is selected from CSKPTC short peptide, or a polypeptide obtained by replacing at least one amino acid on CSKPTC short peptide.

[0132] In one alternative embodiment of this application, the species and subtype of the parent hinge region are selected from sheep IgG2.

[0133] In an optional embodiment of this application, the COC short peptide is selected from C-SKPP-C short peptide, or a polypeptide obtained by replacing at least one amino acid on C-SKPP-C short peptide.

[0134] In an optional embodiment of this application, the COC short peptide is selected from C-SKPM-C short peptide, C-PKPT-C short peptide, C-SPPT-C short peptide, C-CPPT-C short peptide, C-SKCPPT-C short peptide, C-CKPT-C short peptide, C-SCPT-C short peptide, and C-SKCT-C short peptide.

[0135] In an optional embodiment of this application, the parental hinge region of the rabbit IgG is selected from the short peptide APSTCSKPTCPPPELLGGPS or a portion thereof, or has at least 90% identity with it.

[0136] In an optional embodiment of this application, the parental hinge region of the sheep IgG2 is a short peptide GISSDYSKCSKPPCVSRPS or a portion thereof, or has at least 90% identity with it.

[0137] In an optional embodiment of this application, the modified hinge region has a reduced tendency for intra-heavy chain disulfide bond formation or an increased tendency for inter-heavy chain disulfide bond formation compared to the parent hinge region.

[0138] In an optional embodiment of this application, the modified hinge region further includes an N-end hinge region and / or a C-end hinge region.

[0139] In an optional embodiment of this application, the N-end hinge region in the modified hinge region includes the N-end hinge region of COC on the parent hinge region or a portion thereof.

[0140] In one optional embodiment of this application, the C-end hinge region in the modified hinge region includes the C-end hinge region of COC on the parent hinge region or a portion thereof.

[0141] In an optional embodiment of this application, the C-terminal hinge region in the modified hinge region includes the C-terminal hinge region or a portion thereof of the C-X4-C-X1-C short peptide on the natural hinge region of the corresponding species and subtype of the C-X4-C-X1-C short peptide. It should be understood that when the phrase "the natural hinge region of the corresponding species and subtype of the C-X4-C-X1-C short peptide" appears, it means that the "C-X4-C-X1-C" short peptide is a continuous fragment on its source natural hinge region; that is, the "C-X4-C-X1-C" short peptide is a continuous fragment containing the C-X1-C short peptide on the natural hinge region of its corresponding species and subtype of C-X1-C.

[0142] In an optional embodiment of this application, the C-terminal hinge region in the modified hinge region includes the C-terminal hinge region or a portion thereof of the C-X1-C short peptide on the natural hinge region corresponding to the species and subtype of C-X1-C in the C-X4-C-X1-C short peptide. It should be understood that when the phrase "the natural hinge region corresponding to the species and subtype of C-X1-C in the C-X4-C-X1-C short peptide" appears, it means that the "C-X4" short peptide and "C-X1-C" are peptides from different species or subtypes. For example, the "C-X4" short peptide is the CO in the COC short peptide of the parent hinge region, and the "C-X1-C" short peptide is derived from a peptide on a natural hinge region of a species and subtype different from the parent hinge region.

[0143] In an optional embodiment of this application, the N-terminal hinge region in the modified hinge region includes the N-terminal hinge region or a portion thereof of the natural hinge region of the C-X4-C-X1-C short peptide on the corresponding species and subtype of the C-X4-C-X1-C short peptide.

[0144] The half-life of antibody formation is lower when the N-terminal hinge region in the modified hinge region is the N-terminal hinge region of the COC on the parent hinge region than when the N-terminus is the N-terminal hinge region of the natural C-X4-C-X1-C short peptide corresponding to the species and subtype of the C-X4-C-X1-C short peptide. In other words, a higher antibody assembly rate is achieved when the N-terminal hinge region in the modified hinge region is the N-terminal hinge region of the COC on the parent hinge region.

[0145] In an optional embodiment of this application, the subtype of the parent hinge region is selected from rabbit IgG.

[0146] In one optional embodiment of this application, the COC short peptide is selected from C-SKPT-C short peptide.

[0147] Optionally, the CZC short peptide is selected from any of the following short peptides:

[0148] CCKPTC, CSCPTC, CSKCTC, CPKPTC, CSPPTC, CSKPPC, CPPPTC, CCPPTC, CSKCPPCPTC, CSKCPPTC, CSKPCDCC, CSKPCKHCRC, CSKPTCKHCR, CSKPTCPPC, CSKPTCPPC, CSKPTCPKC, CPPCKC, CKHCRC, CKPCIC, CGCKPCIC, CDKTHTCPPC, CPDPCKHCRC.

[0149] In an optional embodiment of this application, the CZC short peptide has more amino acid residues than the COC short peptide.

[0150] In an optional embodiment of this application, the CZC short peptide is selected from any of the following short peptides:

[0151] CSKCPPCPTC, CSKCPPTC, CSKPCDCC, CSKPCKHCRC, CSKPTCKHCRC, CSKPTCPPC, CSKPTCPPC, CSKPTCPKC, CGCKPCIC, CDKTHTCPPC, CPDPCKHCRC.

[0152] In an optional embodiment of this application, the C-X1-C short peptide in the short peptide does not contain flexible amino acids.

[0153] In an optional embodiment of this application, the CZC short peptide is selected from any of the following short peptides: CSKCPPCPTC, CSKPCDCC, CSKPCKHCRC, CSKPTCKHCRC, CSKPTCPPC, CSKPTCPPC, CSKPTCPKC, CGCKPCI C, CDKTHTCPPC, CPDPCKHCRC.

[0154] In an optional embodiment of this application, the N-end hinge region in the modified hinge region includes the N-end hinge region of COC on the parent hinge region or a portion thereof.

[0155] In an optional embodiment of this application, the N-terminal hinge region in the modified hinge region includes an APST short peptide or a portion thereof.

[0156] In an optional embodiment of this application, the modified hinge region is selected from any of the following short peptides, or portions of any of the following short peptides:

[0157] APSTCCKPTC,APSTCSCPTC,APSTCSKCTC,APSTCPKPTC,APSTCSPPTC,APSTCSKPPC,APSTCPPPTC,APSTCCPPTC,APSTCSKCPPCPTC,APSTCSKCPPTC,APSTCSKPCDCC,APSTCSKPCK HCRC, APSTCSKPTCKHCRC, APSTCSKPTCPPC, APSTCSKPTCPPC, APSTCSKPTCPKC, APSTCPPCKC, APSTCKHCRC, APSTCKPCIC, APSTCGCKPCIC, APSTCDKTHTCPPC, APSTCPDPCKHCRC.

[0158] In an optional embodiment of this application, the modified hinge region is selected from any of the following short peptides, or portions of any of the following short peptides:

[0159] APSTCSKCPPCPTC, APSTCSKCPPTC, APSTCSKPCDCC, APSTCSKPCKHCRC, APSTCSKPTCKHCRC, APSTCSKPTCPPC, APSTCSKPTCPPC, APSTCSKPTCPKC, APSTCGCKPCIC, APSTCDKTHTCPPC, APSTCPDPCKHCR.

[0160] In one optional embodiment of this application, the C-end hinge region in the modified hinge region includes the C-end hinge region of COC on the parent hinge region or a portion thereof.

[0161] In an optional embodiment of this application, the C-end hinge region in the modified hinge region includes PPPELLGGPS or a portion thereof.

[0162] In an optional embodiment of this application, the modified hinge region is selected from any of the following short peptides, or portions of any of the following short peptides:

[0163] APSTCCKPTCPPPELLGGPS, APSTCSCPTCPPPELLGGPS, APSTCSKCTCPPPELLGGPS, APSTCPKPTCPPPELLGGPS, APSTCSPPTCPPPELLGGPS, APSTCSKPPCPPPELLGGPS, APSTCPPPTCPPPELLGGPS, APSTCCPPTCPPPELLGGPS, APSTCSKCPPCPTCPPPELLGGPS, APSTCSK CPPTCPPPELLGGPS, APSTCSKPCDCCPPPELLGGPS, APSTCSKPCKHCRCPPPELLGGPS, APSTCSKPTCKHCRCPPPELLGGPS, APSTCSKPTCPPCPPPELLGGPS, APSTCSKPTCPKCPPPELLGGPS, APSTCPPCKCPPPELLGGPS, APSTCKHCRCPPPELLGGPS, APSTCKPCICPPPELLGGPS.

[0164] In an optional embodiment of this application, the C-terminal hinge region in the modified hinge region includes the C-terminal hinge region or a portion thereof of the natural hinge region of the C-X4-C-X1-C short peptide on the corresponding species and subtype of the C-X4-C-X1-C short peptide.

[0165] In an optional embodiment of this application, the species and subtype corresponding to the C-X4-C-X1-C short peptide is mouse IgG1, and the C-X4-C-X1-C short peptide is CGCKPCIC. Then, the C-terminal hinge region in the modified hinge region includes the C-terminal hinge region TVPEVSS or a portion thereof of the CGCKPCIC short peptide on the natural hinge region of mouse IgG1.

[0166] In an optional embodiment of this application, the C-X4-C-X1-C short peptide corresponds to the species and subtype human IgG1, and the C-X4-C-X1-C short peptide is CDKTHTCPPC. Then, the C-terminal hinge region in the modified hinge region includes the C-terminal hinge region PAPELLGGPS or a portion thereof of the CDKTHTCPPC short peptide on the natural hinge region of human IgG1.

[0167] In an optional embodiment of this application, the species and subtype corresponding to the C-X4-C-X1-C short peptide is sheep IgG1, and the C-X4-C-X1-C short peptide is CPDPCKHCRC. Then, the C-terminal hinge region in the modified hinge region includes the C-terminal hinge region PPPELLGGPS or a portion thereof of the CPDPCKHCRC short peptide on the natural hinge region of sheep IgG1.

[0168] In an optional embodiment of this application, the modified hinge region is selected from any of the following short peptides, or portions of any of the following short peptides:

[0169] APSTCGCKPCICTVPEVSS,APSTCDKTHTCPPCPAPELLGGPS,APSTCPDPCKHCRCPPPELPGGPS.

[0170] In an optional embodiment of this application, the C-terminal hinge region in the modified hinge region includes the C-terminal hinge region or a portion thereof of the natural hinge region of the C-X1-C short peptide corresponding to the species and subtype of C-X1-C in the C-X4-C-X1-C short peptide.

[0171] In an optional embodiment of this application, where C-X1-C in the C-X4-C-X1-C short peptide corresponds to the species and subtype human IgG1, and the C-X1-C short peptide is CPPC, then the C-terminal hinge region in the modified hinge region includes the C-terminal hinge region PAPELLGGPS or a portion thereof of the CPPC short peptide on the natural hinge region of human IgG1.

[0172] In an optional embodiment of this application, the modified hinge region is selected from any of the following short peptides, or portions of any of the following short peptides: APSTCSKPTCPPCPAPELLGGPS.

[0173] In an optional embodiment of this application, the subtype of the parental hinge region is selected from sheep IgG2.

[0174] In one optional embodiment of this application, the COC short peptide is selected from C-SKPP-C short peptide.

[0175] In an optional embodiment of this application, the CZC short peptide is selected from any of the following short peptides: CGCKPCIC, CDKTHTCPPC, CPDPCKHCRC.

[0176] In an optional embodiment of this application, the N-end hinge region in the modified hinge region includes the N-end hinge region of COC on the parent hinge region or a portion thereof.

[0177] In an optional embodiment of this application, the N-terminal hinge region in the modified hinge region includes a GISSDYSK short peptide or a portion thereof.

[0178] In an optional embodiment of this application, the modified hinge region is selected from any of the following short peptides, or portions of any of the following short peptides: GISSDYSKCGCKPCIC, GISSDYSKCDKTHTCPPC, GISSDYSKCPDPCKHCRC.

[0179] In an optional embodiment of this application, the C-terminal hinge region in the modified hinge region includes the C-terminal hinge region or a portion thereof of the natural hinge region of the C-X4-C-X1-C short peptide on the corresponding species and subtype of the C-X4-C-X1-C short peptide.

[0180] In an optional embodiment of this application, the species and subtype corresponding to the C-X4-C-X1-C short peptide is mouse IgG1, and the C-X4-C-X1-C short peptide is CGCKPCIC. Then, the C-terminal hinge region in the modified hinge region includes the C-terminal hinge region TVPEVSS or a portion thereof of the CGCKPCIC short peptide on the natural hinge region of mouse IgG1.

[0181] In an optional embodiment of this application, the C-X4-C-X1-C short peptide corresponds to the species and subtype human IgG1, and the C-X4-C-X1-C short peptide is CDKTHTCPPC. Then, the C-terminal hinge region in the modified hinge region includes the C-terminal hinge region PAPELLGGPS or a portion thereof of the CDKTHTCPPC short peptide on the natural hinge region of human IgG1.

[0182] In an optional embodiment of this application, the species and subtype corresponding to the C-X4-C-X1-C short peptide is sheep IgG1, and the C-X4-C-X1-C short peptide is CPDPCKHCRC. Then, the C-terminal hinge region in the modified hinge region includes the C-terminal hinge region PPPELPGGPS or a portion thereof of the CPDPCKHCRC short peptide on the natural hinge region of sheep IgG1.

[0183] In an optional embodiment of this application, the modified hinge region is selected from any of the following short peptides, or portions of any of the following short peptides:

[0184] GISSDYSKCGCKPCICTVPEVSS, GISSDYSKCDKTHTCPPCPAPELLGGPS, GISSDYSKCPDPCKHCRCPPPELPGGPS.

[0185] In an optional embodiment of this application, the N-terminal hinge region in the modified hinge region includes the N-terminal hinge region or a portion thereof of the natural hinge region of the C-X4-C-X1-C short peptide on the corresponding species and subtype of the C-X4-C-X1-C short peptide.

[0186] In an optional embodiment of this application, the species and subtype corresponding to the C-X4-C-X1-C short peptide is sheep IgG1, and the C-X4-C-X1-C short peptide is CPDPCKHCRC. Then, the N-terminal hinge region in the modified hinge region includes the N-terminal hinge region EPG or a portion thereof of the CPDPCKHCRC short peptide on the natural hinge region of sheep IgG1.

[0187] In an optional embodiment of this application, the modified hinge region is selected from any of the following short peptides, or a portion thereof: EPGGPPCKHCRCPPPELPGGPS. The half-antibody formation rate is lower when the N-terminal hinge region of the modified hinge region is a GISSDYSK short peptide than when the N-terminus is an EPG short peptide.

[0188] In an optional embodiment of this application, the modification further includes replacing the first C in the COC short peptide with any amino acid other than C.

[0189] In an optional embodiment of this application, the first C in the COC short peptide is replaced with S or T.

[0190] In a second aspect of this application, an Fc is proposed. According to embodiments of this application, the Fc includes a modified hinge region, wherein the first C in the COC short peptide of the parent hinge region is replaced with any amino acid other than C, and the O in the COC short peptide represents one or more amino acids. The Fc of this application has the advantage of a low half-antibody formation rate; in particular, antibodies containing the Fc of this application can reduce the half-antibody formation rate or increase the antibody assembly rate.

[0191] According to embodiments of this application, the above-mentioned Fc may further include at least one of the following technical features:

[0192] In an optional embodiment of this application, the first C in the COC short peptide is replaced with S or T.

[0193] In an optional embodiment of this application, the first C and the last C in the COC short peptide in the parent hinge region may mismatch to form an intra-heavy chain disulfide bond.

[0194] In an optional embodiment of this application, the last C in the COC short peptide is a cysteine ​​residue in the parental hinge region used to form inter-heavy chain disulfide bonds. That is, when the antibody is correctly assembled, the last C in the COC short peptide is a cysteine ​​residue in the parental hinge region used to form inter-heavy chain disulfide bonds, resulting in the formation of a correct divalent Y-shaped structure.

[0195] In an optional embodiment of this application, the first C in the COC short peptide is a cysteine ​​residue in the parental hinge region used to form an intra-heavy chain disulfide bond. That is, when the antibody is correctly assembled, the first C in the COC short peptide is a cysteine ​​residue in the parental hinge region used to form an intra-heavy chain disulfide bond, resulting in a stable Y-shaped structure.

[0196] In one alternative embodiment of this application, the species and subtype of the parent hinge region are selected from rabbit IgG.

[0197] In an optional embodiment of this application, the COC short peptide is selected from CSKPTC short peptide, or a polypeptide obtained by replacing at least one amino acid on CSKPTC short peptide.

[0198] In one alternative embodiment of this application, the species and subtype of the parent hinge region are selected from sheep IgG2.

[0199] In an optional embodiment of this application, the COC short peptide is selected from C-SKPP-C short peptide, or a polypeptide obtained by replacing at least one amino acid on C-SKPP-C short peptide.

[0200] In an optional embodiment of this application, the COC short peptide is selected from C-SKPM-C short peptide, C-PKPT-C short peptide, C-SPPT-C short peptide, C-CPPT-C short peptide, C-SKCPPT-C short peptide, C-CKPT-C short peptide, C-SCPT-C short peptide, and C-SKCT-C short peptide.

[0201] In an optional embodiment of this application, the parental hinge region of the rabbit IgG is selected from the short peptide APSTCSKPTCPPPELLGGPS or a portion thereof, or has at least 90% identity with it.

[0202] In an optional embodiment of this application, the parental hinge region of the sheep IgG2 is a short peptide GISSDYSKCSKPPCVSRPS or a portion thereof, or has at least 90% identity with it.

[0203] In an optional embodiment of this application, the modified hinge region has a reduced tendency for intra-heavy chain disulfide bond formation or an increased tendency for inter-heavy chain disulfide bond formation compared to the parent hinge region.

[0204] It should be understood that, in this document, the specific species or subtype of CH2, CH3, CH4, or the tail peptide in the Fc is not limited, and those skilled in the art should understand that the species or subtype of CH2, CH3, CH4, or the tail peptide does not affect the achievement of the purpose of this invention. The species of CH2, CH3, CH4, or the tail peptide are each independently selected from humans, mice, rats, cattle, horses, pigs, sheep, goats, dogs, camels, alpacas, monkeys, orangutans, cats, rabbits, donkeys, deer, mink, chickens, ducks, and geese. The subtypes of CH2, CH3, CH4, or the tail peptide are each independently selected from IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgM, and IgE.

[0205] In an optional embodiment of this application, the species of CH2, CH3, CH4, or tail peptide are selected from rabbits or sheep.

[0206] In one optional embodiment of this application, the species of CH2, CH3, CH4, or tail peptide are selected from cattle, humans, horses, mice, or rats.

[0207] In an optional embodiment of this application, the species of CH2, CH3, CH4, or tail peptide is the same as that of the parent antibody.

[0208] In an optional embodiment of this application, the species and subtype of the CH2, CH3, and / or tail peptide are selected from rabbit IgG or sheep IgG2.

[0209] In an optional embodiment of this application, the species and subtype of the CH2, CH3, CH4 and / or tail peptide are selected from rabbit IgM or sheep IgM.

[0210] In an optional embodiment of this application, the species of CH2, CH3, CH4, or the tail peptide is the same as the species of the natural antibody corresponding to the C-X1-C short peptide.

[0211] In an optional embodiment of this application, the species and subtypes of CH2, CH3 and tail peptide are selected from bovine IgG, human IgG, horse IgG, mouse IgG, or rat IgG.

[0212] In an optional embodiment of this application, the species and subtypes of CH2, CH3, CH4 and tail peptide are selected from bovine IgM, human IgM, horse IgM, mouse IgM, or rat IgM.

[0213] Antibody constant region

[0214] In a third aspect, this application proposes an antibody constant region. According to embodiments of this application, the antibody constant region includes the Fc described in the first or second aspect. Antibodies containing the antibody constant region of this application can reduce the rate of hapten formation or increase the antibody assembly rate.

[0215] According to embodiments of this application, the antibody constant region may further include at least one of the following technical features:

[0216] In an optional embodiment of this application, the antibody constant region includes the Fc and CH1 regions described above.

[0217] In one optional embodiment of this application, the CH1 region is selected from rabbit, sheep, cow, human, horse, mouse, or rat.

[0218] In one optional embodiment of this application, the species of the CH1 region is the same as that of the parent antibody.

[0219] In an optional embodiment of this application, the species and subtype of the CH1 region are selected from rabbit IgG or sheep IgG2.

[0220] In an optional embodiment of this application, the species and subtype of the CH1 region are selected from rabbit IgM or sheep IgM.

[0221] In an optional embodiment of this application, the species of the CH1 region is the same as the species of the natural antibody corresponding to the C-X1-C short peptide.

[0222] In one optional embodiment of this application, the species and subtype of the CH1 region are selected from bovine IgG, human IgG, horse IgG, mouse IgG, or rat IgG.

[0223] In an optional embodiment of this application, the species and subtype of the CH1 region are selected from bovine IgM, human IgM, equine IgM, mouse IgM, or rat IgM.

[0224] In an optional embodiment of this application, the antibody constant region further includes a modified CH1 region, which is obtained by modifying the parental CH1 region. The modification involves replacing the C in the parental CH1 region that forms an atypical intrachain disulfide bond with the first C in the COC short peptide in the parental hinge region.

[0225] In an optional embodiment of this application, the region selected is the modified CH1 region, which is obtained by modifying the parental CH1 region. The modification involves replacing the C in the parental CH1 region that forms an atypical intrachain disulfide bond with the first C in the COC short peptide in the parental hinge region.

[0226] In an optional embodiment of this application, the modification is to replace the C in the parent CH1 region that forms an atypical intrachain disulfide bond with the first C in the COC short peptide in the parent hinge region with S or T.

[0227] In an optional embodiment of this application, the 15th carbon in the parent CH1 region forms an atypical intrachain disulfide bond with the first carbon in the COC short peptide in the parent hinge region.

[0228] It should be noted that the 15th amino acid from the N-terminus to the C-terminus of CH1 is referred to as the "15th amino acid of the CH1 region". It should be understood that the "15th amino acid of the CH1 region" also includes positions that are equivalent to 15, and the identification of equivalent positions can be determined through sequence alignment.

[0229] Atypical intrachain disulfide bonds are defined relative to typical intrachain disulfide bonds. Typical disulfide bonds are defined by the number and position of intrachain disulfide bonds in the CH1 region of human or mouse antibodies. For example, the typical intrachain disulfide bond in the CH1 region of IgG antibodies is one pair, while atypical intrachain disulfide bonds are more than one pair. For instance, rabbit IgG antibodies and sheep IgG2 antibodies have one pair of atypical intrachain disulfide bonds.

[0230] In an optional embodiment of this application, the modification is to replace the 15th C in the parent CH1 region.

[0231] In an optional embodiment of this application, the modification is to replace the 15th C in the parent CH1 region with S or T.

[0232] In an optional embodiment of this application, the parent CH1 region has an amino acid sequence as shown in any one of SEQ ID NO:1 to 2 or an amino acid sequence having at least 90% identity with it.

[0233] According to embodiments of this application, the amino acid sequence of the parent CH1 region is shown in any one of SEQ ID NO:1 to 2.

[0234] It should be noted that in this application, "the amino acid sequence as shown in SEQ ID NO:A" includes the amino acid sequence of SEQ ID NO:A or the amino acid sequence of SEQ ID NO:A with conservative modifications, all of which are within the scope of protection of this application. For example, "the amino acid sequence of the parent CH1 region as shown in any one of SEQ ID NO:1-2" means that the parent CH1 region is the amino acid sequence of SEQ ID NO:1-2 or the amino acid sequence of SEQ ID NO:1-2 with conservative modifications, all of which are within the scope of protection of this application.

[0235] In this document, "conservatively modified amino acid sequences" refers to amino acid modifications that do not significantly affect or alter the binding properties of antibodies containing that amino acid sequence. These modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the antibodies of this application using standard techniques such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions involve replacing an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been identified in the art. These families include amino acids with basic side chains (such as lysine, arginine, and histidine), amino acids with acidic side chains (such as aspartic acid and glutamic acid), amino acids with uncharged polar side chains (such as glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), amino acids with nonpolar side chains (such as alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), amino acids with β-branched side chains (such as threonine, valine, and isoleucine), and amino acids with aromatic side chains (such as tyrosine, phenylalanine, tryptophan, and histidine). Exemplarily, the number of conserved modifications does not exceed 80% of the total number, preferably not exceeding 90%. In this document, "amino acid sequences of conserved modification form" also includes naturally occurring mutated amino acid modifications. "Naturally occurring mutated" refers to mutations caused by changes in alleles or other factors due to individual differences during the natural mutation process of antibodies.

[0236] Rabbit IgG-CH1:

[0237] Sheep IgG2-CH1:

[0238] In an optional embodiment of this application, the modified CH1 region has an amino acid sequence as shown in any one of SEQ ID NO:3 to 4 or an amino acid sequence having at least 90% identity with it.

[0239] According to embodiments of this application, the amino acid sequence of the modified CH1 region is shown in any one of SEQ ID NO:3 to 4.

[0240] It should be noted that "the amino acid sequence that modifies the CH1 region as shown in any one of SEQ ID NO:3 to 4" refers to the amino acid sequence that modifies the CH1 region as shown in SEQ ID NO:3 to 4 or the amino acid sequence that is conservatively modified as shown in SEQ ID NO:3 to 4, both of which are within the scope of protection of this application.

[0241] In an optional embodiment of this application, the modified CH1 region and the modified hinge region have an amino acid sequence as shown in any one of SEQ ID NO:5-6 or an amino acid sequence having at least 90% identity with it.

[0242] According to embodiments of this application, the amino acid sequences of the modified CH1 region and the modified hinge region are as shown in any one of SEQ ID NO:5 to 6.

[0243] It should be noted that "the amino acid sequences of the modified CH1 region and the modified hinge region as shown in any one of SEQ ID NO:5 to 6" refers to the amino acid sequences of the modified CH1 region and the modified hinge region as shown in SEQ ID NO:5 to 6 or the amino acid sequences of the conservative modified form of SEQ ID NO:5 to 6, all of which are within the scope of protection of this application.

[0244] In an optional embodiment of this application, the modification involves deleting the C in the parent CH1 region that forms an atypical intrachain disulfide bond with the first C in the COC short peptide in the parent hinge region.

[0245] In an optional embodiment of this application, the modification involves deleting the 15th C position in the parent CH1 region.

[0246] Antibody

[0247] In a fourth aspect, this application provides an antibody. According to embodiments of this application, the antibody includes the Fc region described in the first or second aspect, or the antibody constant region described in the third aspect. The antibody of this application can reduce the rate of hapten formation or increase the antibody assembly rate.

[0248] Unless otherwise specified herein, antibodies may be monomeric antibodies, polymeric antibodies, or mixtures thereof, and their specific types are not limited, all of which are within the scope of protection of this application. The inventors of this application have discovered that antibodies containing the modified hinge region, Fc region, or constant region of this application exhibit a reduced tendency for intra-heavy chain disulfide bond formation or an increased tendency for inter-heavy chain disulfide bond formation. Furthermore, antibodies containing the modified hinge region of this application exhibit a reduced half-antibody formation rate or an increased antibody assembly rate.

[0249] According to embodiments of this application, the antibody may further include at least one of the following technical features:

[0250] In an optional embodiment of this application, the antibody further includes a heavy chain variable region and an optional light chain variable region.

[0251] In an optional embodiment of this application, the antibody comprises a heavy chain and a light chain, wherein the heavy chain is obtained by fusing a first polypeptide and a second polypeptide; the first polypeptide comprises a heavy chain variable region located at the N-terminus, the aforementioned CH1 region, and the aforementioned modified hinge region, and the second polypeptide comprises the aforementioned IgM CH3-CH4 region located at the C-terminus.

[0252] In this paper, "the variable region of the heavy chain located at the N end" means that the variable region of the heavy chain is located at the N end of the heavy chain; "the above-mentioned IgM CH3-CH4 region located at the C end" means that the IgM CH3-CH4 region is located at the C end of the heavy chain.

[0253] In an optional embodiment of this application, the antibody comprises a heavy chain and a light chain, wherein the heavy chain is obtained by fusing a first polypeptide and a second polypeptide; the first polypeptide comprises a heavy chain variable region, the aforementioned CH1 region, and the aforementioned modified hinge region, and the second polypeptide comprises the aforementioned IgM CH3-CH4 region; wherein the heavy chain comprises, from the N-terminus to the C-terminus, the heavy chain variable region, the CH1 region, the modified hinge region, and the IgM CH3-CH4 region, respectively.

[0254] In an optional embodiment of this application, the antibody comprises a heavy chain and a light chain, wherein the heavy chain is obtained by fusing a first polypeptide and a second polypeptide; the first polypeptide consists of a heavy chain variable region located at the N-terminus, the aforementioned IgG CH1 region, and the aforementioned modified hinge region, and the second polypeptide consists of the aforementioned IgM CH2-CH3-CH4 region located at the C-terminus.

[0255] In an optional embodiment of this application, the antibody comprises a heavy chain and a light chain, wherein the heavy chain is obtained by fusing a first polypeptide and a second polypeptide; the first polypeptide comprises a heavy chain variable region, the aforementioned CH1 region, and the aforementioned modified hinge region, and the second polypeptide comprises the aforementioned IgM CH2-CH3-CH4 region; wherein the heavy chain comprises, from the N-terminus to the C-terminus, the heavy chain variable region, the CH1 region, the modified hinge region, and the IgM CH2-CH3-CH4 region, respectively.

[0256] In an optional embodiment of this application, the antibody includes a heavy chain and a light chain, wherein the heavy chain is obtained by fusing a first polypeptide and a second polypeptide; the first polypeptide includes a heavy chain variable region located at the N-terminus, the aforementioned modified CH1 region, and the aforementioned modified hinge region, and the second polypeptide includes the aforementioned IgM CH3-CH4 region located at the C-terminus.

[0257] In an optional embodiment of this application, the antibody comprises a heavy chain and a light chain, wherein the heavy chain is obtained by fusing a first polypeptide and a second polypeptide; the first polypeptide comprises a heavy chain variable region, the aforementioned modified CH1 region, and the aforementioned modified hinge region, and the second polypeptide comprises the aforementioned IgM CH3-CH4 region; wherein the heavy chain comprises, from the N-terminus to the C-terminus, the heavy chain variable region, the modified CH1 region, the modified hinge region, and the IgM CH3-CH4 region, respectively.

[0258] In an optional embodiment of this application, the antibody includes a heavy chain and a light chain, wherein the heavy chain is obtained by fusing a first polypeptide and a second polypeptide; the first polypeptide consists of the N-terminal heavy chain variable region of IgG, the aforementioned modified CH1 region, and the aforementioned modified hinge region, and the second polypeptide consists of the C-terminal IgM CH2-CH3-CH4 region.

[0259] In an optional embodiment of this application, the antibody comprises a heavy chain and a light chain, wherein the heavy chain is obtained by fusing a first polypeptide and a second polypeptide; the first polypeptide comprises a heavy chain variable region, the aforementioned modified CH1 region, and the aforementioned modified hinge region, and the second polypeptide comprises the aforementioned IgM CH2-CH3-CH4 region; wherein the heavy chain comprises, from the N-terminus to the C-terminus, the heavy chain variable region, the modified CH1 region, the modified hinge region, and the IgM CH2-CH3-CH4 region, respectively.

[0260] It should be understood that modifying IgG subtype antibodies into IgM subtype antibodies is known in the art, for example, see patent CN 114621350 B.

[0261] In one alternative embodiment of this application, the antibody has improved activity.

[0262] Nucleic acid molecules, vectors, host cells

[0263] In a fifth aspect of this application, a nucleic acid molecule is provided. According to embodiments of this application, the nucleic acid molecule encodes the Fc described in the first or second aspect, the antibody constant region described in the third aspect, or the antibody described in the fourth aspect.

[0264] According to an embodiment of this application, the isolated nucleic acid is DNA.

[0265] It should be noted that those skilled in the art will understand that the isolated nucleic acids mentioned herein actually include any one or both of the complementary double strands. For convenience, although only one strand is given in most cases, the complementary strand is also disclosed. Furthermore, the nucleic acid sequences isolated in this application include DNA or RNA forms; disclosure of one implies that the other is also disclosed.

[0266] In a sixth aspect, this application provides a vector. According to an embodiment of this application, the vector carries the nucleic acid molecule described in the fifth aspect. When the isolated nucleic acid is ligated to the vector, the isolated nucleic acid can be directly or indirectly linked to control elements on the vector, as long as these control elements can control the translation and expression of the isolated nucleic acid. Of course, these control elements can be directly derived from the vector itself or can be exogenous, i.e., not derived from the vector itself. Naturally, the operative connection between the isolated nucleic acid and the control elements is sufficient.

[0267] In this document, "operably ligated" refers to ligating a foreign gene to a vector, enabling the control elements within the vector, such as transcriptional control sequences and translational control sequences, to perform their intended functions of regulating the transcription and translation of the foreign gene. Commonly used vectors include plasmids, bacteriophages, etc. According to some specific embodiments of this application, after the vector is introduced into suitable recipient cells, the expression of the aforementioned antibody constant region or antibody can be effectively achieved under the mediation of a regulatory system, thereby enabling the large-scale in vitro production of the antibody constant region or antibody.

[0268] In some specific embodiments of this application, the vector is a eukaryotic expression vector, a prokaryotic expression vector, a virus, or a bacteriophage.

[0269] In some specific embodiments of this application, the vector is a lentiviral vector.

[0270] In one optional embodiment of this application, the expression vector is a plasmid expression vector.

[0271] In a seventh aspect of this application, a host cell is provided. According to embodiments of this application, the host cell comprises the nucleic acid molecule or vector described in the fifth or sixth aspect, or expresses the Fc described in the first or second aspect, the antibody constant region described in the third aspect, or the antibody described in the fourth aspect. Using this host cell, under suitable conditions, the aforementioned antibody constant region or antibody can be effectively expressed within the host cell.

[0272] According to an embodiment of this application, the host cell is obtained by introducing it into the host cell via the vector described in the sixth aspect.

[0273] It should be noted that the host cell in this application is not particularly limited and can be a prokaryotic cell, a eukaryotic cell, or a bacteriophage. The prokaryotic cell can be *Escherichia coli*, *Bacillus subtilis*, *Streptomyces*, or *Proteus mirabilis*, etc. The aforementioned eukaryotic cells include fungi such as *Pichia pastoris*, *Saccharomyces cerevisiae*, *Schizosaccharomyces cerevisiae*, and *Trichoderma*, insect cells such as armyworms, plant cells such as tobacco, and mammalian cells such as BHK cells, CHO cells, COS cells, and myeloma cells.

[0274] According to an embodiment of this application, the host cell is a eukaryotic cell.

[0275] According to embodiments of this application, the host cell is a mammalian cell, including but not limited to BHK cells, CHO cells, NSO cells or COS cells, and does not include animal germ cells, fertilized eggs or embryonic stem cells.

[0276] It should be noted that the "suitable conditions" mentioned in this application refer to conditions suitable for the expression of the antibody constant region or antibody described in this application. Those skilled in the art will readily understand that suitable conditions for antibody expression include, but are not limited to, suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy cell state, suitable cell density, suitable cell culture environment, and suitable cell culture time. The term "suitable conditions" is not particularly limited, and those skilled in the art can optimize the optimal conditions for antibody expression based on the specific environment of their laboratory.

[0277] Conjugate

[0278] In an eighth aspect of this application, a conjugate is proposed. According to an embodiment of this application, the antibody conjugate comprises: the Fc described in the first or second aspect, the antibody constant region described in the third aspect, or the antibody described in the fourth aspect, and at least one conjugated portion connected to the Fc, the antibody constant region, or the antibody.

[0279] According to embodiments of this application, the above-mentioned conjugate may further include at least one of the following additional technical features:

[0280] According to embodiments of this application, the antibody conjugate further includes a conjugation portion, which is conjugated to the antibody.

[0281] In one optional embodiment of this application, the coupling portion and the antibody can be coupled linearly or indirectly. The specific connection method is not limited and is within the protection scope of this application.

[0282] In one optional embodiment of this application, the coupling portion of the antibody conjugate can be directly labeled with an antibody. Compared to indirect labeling, the preparation method of antibody conjugates obtained by direct labeling is simpler and more convenient.

[0283] In an optional embodiment of this application, the indirect connection in the antibody conjugate may be that the marker is directly conjugated to a hydrophilic carrier (exemplary bovine serum albumin BSA, KLH, OVA, etc.) and indirectly connected to the antibody through a heterobifunctional linker.

[0284] According to embodiments of this application, the coupling portion is selected from biotin, biotin derivatives, markers, or solid-phase carriers.

[0285] In this paper, the term "marker" refers to a class of substances that have properties that can be directly observed by the naked eye or detected or probing by instruments, such as luminescence, color development, radioactivity, etc., which enable qualitative or quantitative detection of the corresponding target.

[0286] In one optional embodiment of this application, the markers include, but are not limited to, fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents, and nanoparticle markers.

[0287] In practical use, those skilled in the art can select appropriate markers according to the detection conditions or actual needs. Regardless of the marker used, it falls within the protection scope of this application.

[0288] In one optional embodiment of this application, the fluorescent dye includes, but is not limited to, fluorescein dyes and their derivatives (e.g., including but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc., or their analogues), rhodamine dyes and their derivatives (e.g., including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc., or their analogues), and Cy series dyes and their derivatives (e.g., including but not limited to Cy2, Cy3, Cy3B, Cy3). 5. Cy5, Cy5.5, Cy3, etc. or similar), Alexa series dyes and their derivatives (including but not limited to Alexa Fluor 350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, etc. or similar) and protein dyes and their derivatives (including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), polydiophytoxanthin-chlorophyll protein (preCP), etc.).

[0289] In one optional embodiment of this application, the enzyme includes, but is not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate dehydrogenase.

[0290] In one optional embodiment of this application, the radioactive isotope includes, but is not limited to, [other radioactive isotopes]. 212 Bi、 131 I, 111 In、 90 Y、 186 Re、 211 At、 125 I, 188 Re、 153 Sm、 213Bi、 32 P, 94 mTc, 99 mTc, 203 Pb, 67 Ga、 68 Ga、 43 Sc、 47 Sc、 110 mIn, 97 Ru、 62 Cu、 64 Cu、 67 Cu、 68 Cu、 86 Y、 88 Y、 121 Sn、 161 Tb, 166 Ho、 105 Rh、 177 Lu、 172 Lu and 18 F.

[0291] In one optional embodiment of this application, the chemiluminescent reagent includes, but is not limited to, luminol and its derivatives, luciferin, fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridine ester and its derivatives, dioxane and its derivatives, rofenine and its derivatives, and peroxazone and its derivatives.

[0292] In one optional embodiment of this application, the nanoparticle-type markers include, but are not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

[0293] In one alternative embodiment of this application, the colloid includes, but is not limited to, colloidal metals, colloidal carbon, dispersed dyes, dye-labeled microspheres, and latexes.

[0294] In one optional embodiment of this application, the colloidal metal includes, but is not limited to, colloidal gold, colloidal silver, and colloidal selenium.

[0295] In one optional embodiment of this application, the colloidal metal is colloidal gold.

[0296] In one optional embodiment of this application, the antibody conjugate further includes a solid-phase carrier conjugated to the antibody.

[0297] In this application, the term "solid support" can refer to a substance that can be suspended or dispersed in a liquid phase (e.g., solid supports such as particles or magnetic beads), or a solid phase that can contain or carry a liquid phase (e.g., supports such as plates, membranes, or test tubes, as well as containers such as perforated plates, microfluidic paths, glass capillaries, nanopillars, or monolithic columns).

[0298] In one alternative embodiment of this application, the solid support is selected from microspheres, plates, and membranes.

[0299] In one optional embodiment of this application, the solid carrier includes, but is not limited to, magnetic microspheres, plastic microspheres, plastic microparticles, microporous plates, glass, capillaries, nylon, and nitrocellulose membranes.

[0300] Reagent test kit

[0301] In a ninth aspect of this application, a kit is provided. According to embodiments of this application, the kit comprises: the Fc described in the first or second aspect, the antibody constant region described in the third aspect, the antibody described in the fourth aspect, or the conjugate described in the eighth aspect.

[0302] In this article, "kit" and "reagent" are used interchangeably. Kits or reagents do not need to have a box structure; they only require relative independence and suitable loading or containers, such as tubes, boxes, bottles, or cards. Some components are packaged in different containers, while others may be combined in one container if permissible.

[0303] According to embodiments of this application, the kit includes reagents suitable for performing immunoassays. In some embodiments, the kit may include instructions for use of the immunodiagnostic reagents (e.g., antibody conjugates) of this application in immunoassays for detecting corresponding antigens. In some embodiments, the kit may include calibrators or controls, such as standard or control antigens. In some embodiments, the antibodies or conjugates of this application are contained in containers such as test tubes, microplates, or test strips within the kit. In some embodiments, the kit may also include solid-phase supports such as magnetic beads, test tubes, microplates, cuvettes, membranes, filter paper, syringes, pipettes, buffers such as assay buffers, wash buffers, pretreatment reagents, and detectable labeled substrate solutions such as enzyme-labeled substrate solutions.

[0304] use

[0305] In the tenth aspect of this application, the use of the Fc described in the first or second aspect, the antibody constant region described in the third aspect, the antibody described in the fourth aspect, the nucleic acid molecule described in the fifth aspect, the vector described in the sixth aspect, the host cell described in the seventh aspect, the conjugate described in the eighth aspect, or the kit described in the ninth aspect in immunoassay or in the preparation of immunoassay products is proposed.

[0306] method

[0307] In its eleventh aspect, this application provides a method for detecting an antigen in a sample to be tested. According to an embodiment of this application, the method includes: a) contacting the antibody described in the fourth aspect, the conjugate described in the eighth aspect, or the kit described in the ninth aspect with an antigen in the sample to be tested under conditions sufficient to induce an antibody / antigen binding reaction, thereby forming an immune complex.

[0308] b) Detect the presence of the immune complex, and determine whether the sample to be tested contains an antigen based on the presence of the immune complex.

[0309] According to embodiments of this application, the antigen detection method described above may further include at least one of the following technical features:

[0310] According to embodiments of this application, the presence of the immune complex indicates the presence of the antigen in the sample to be tested.

[0311] According to an embodiment of this application, the immune complex further includes a second antibody, which binds to the antibody.

[0312] According to embodiments of this application, the immune complex further includes a second antibody that binds to the antigen.

[0313] The method for detecting the antigen can be any method known to those skilled in the art, for example, detecting the antigen with an additional antibody against the antigen (which is typically labeled with a signaling substance, or detected with a secondary antibody labeled with a signaling substance) after the antigen has been contacted with the solid-phase carrier (double antibody sandwich).

[0314] Alternatively, conventional antibodies can be used as antibodies coated on a solid phase and co-incubated with the antigen to be detected. The antibody is then used as a free detection antibody (which is usually labeled with a signal substance, or detected by a secondary antibody labeled with a signal substance).

[0315] Alternatively, the solid-phase carrier can be used in combination with the antibody to detect the antigen, in which case the two are paired antibodies.

[0316] The signaling substance can be any one of the following: fluorescent substance, quantum dot, digoxigenin-labeled probe, biotin, radioactive isotope, radioactive contrast agent, paramagnetic ion fluorescent microsphere, electron-dense material, chemiluminescent label, ultrasound contrast agent, photosensitizer, colloidal gold, or enzyme. In some embodiments, the signaling substance is colloidal gold, fluorescein, fluorescent microsphere, acrid ester, horseradish peroxidase, alkaline phosphatase, or β-galactosidase.

[0317] In a twelfth aspect of this application, a method for reducing antibody half-antibody formation rate or increasing antibody assembly rate is proposed. According to embodiments of this application, the method includes:

[0318] The Fc in the antibody is modified to form the Fc described in the first aspect or the second aspect;

[0319] The antibody constant region in the antibody is modified to form the antibody constant region described in the third aspect; or

[0320] The antibody is modified to form the antibody described in the fourth aspect.

[0321] The following will explain the solution of this application with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of this application. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0322] Example 1: Antibody Design and Expression

[0323] 1. Based on the wild-type rabbit antibody, the hinge region of the wild-type rabbit antibody was modified according to Table 1 (modification 13 also modified CH1). The wild-type rabbit antibody is an Anti-BNP antibody, and its heavy chain amino acid sequence is shown in SEQ ID NO:7 and its light chain amino acid sequence is shown in SEQ ID NO:8. For specific modification schemes, please refer to Table 1.

[0324] Table 1: Design Methods for Renovation

[0325] 2. Plasmid construction and expression

[0326] In this embodiment, restriction endonucleases and Prime Star DNA polymerase were purchased from Takara. The MagExtractor RNA extraction kit was purchased from TOYOBO. BD SMART TM The RACE cDNA Amplification Kit was purchased from Takara. The pMD-18T vector was purchased from Takara. The plasmid extraction kit was purchased from Tiangen Biotech. Primer synthesis and gene sequencing were performed by a gene sequencing company.

[0327] 2.1 Preparation of Anti-BNP Antibody Gene

[0328] mRNA was extracted from cell lines secreting Anti-BNP monoclonal antibodies, and DNA products were obtained by RT-PCR. The DNA products were then inserted into the pMD-18T vector and transformed into DH5α competent cells. After bacterial growth, four positive clones of the Heavy Chain and Light Chain genes were sent to a gene sequencing company for sequencing.

[0329] 2.2 Sequence analysis of the variable region gene of the Anti-BNP antibody

[0330] The gene sequences obtained from the sequencing were analyzed in the IMGT antibody database and VNTI11.5 software was used to confirm that the genes amplified by both heavy and light chain primer pairs were correct. Among the gene fragments amplified by the Light Chain primer pair, the VL gene sequence was 330 bp, with a 57 bp leader peptide sequence preceding it; among the gene fragments amplified by the Heavy Chain primer pair, the VH gene sequence was 351 bp, belonging to the VH1 gene family, with a 57 bp leader peptide sequence preceding it.

[0331] 2.3 Construction of recombinant antibody expression plasmid

[0332] pcDNA TM 3.4 TOPO The vector is a recombinant antibody eukaryotic expression vector that has been modified to include polyclonal restriction sites, and will be referred to as the 3.4A expression vector. Using wild-type Anti-BNP antibody as a template, mutant primers were designed to construct the modified heavy and light chains. The modified heavy chain gene fragments and light chain gene fragments were amplified by PCR.

[0333] The Heavy Chain and Light Chain gene fragments were double-digested with restriction endonucleases, and the 3.4A vector was also double-digested with restriction endonucleases. After purification and recovery of the fragments and vector, the Heavy Chain gene and Light Chain gene were ligated into the 3.4A expression vector to obtain recombinant expression plasmids of Heavy Chain and Light Chain, respectively.

[0334] 2.4 Expression of recombinant antibody samples

[0335] HEK293 cells were revived early and passaged to a 200ml volume to achieve a cell density of 3–5 × 10⁻⁶ cells / mL. 6 Cells / ml, cell viability >95%; centrifuge to wash cells, rehydrate with culture medium, and adjust cell density to 2.9 × 10⁶ cells / ml. 6Cells / ml were used as cell dilution buffers. Plasmid DNA and transfection reagent dilution buffers were prepared separately using culture medium. The transfection reagent dilution buffer was added to the plasmid DNA dilution buffer, mixed well, and incubated at room temperature for 15 min. This mixture was then slowly added to the cell dilution buffer over 1 min, mixed well, and samples were taken for counting. Cell viability after transfection was recorded and observed. The cells were then incubated in a 35°C incubator at 120 rpm with 8% CO2. After 13 days, the samples were centrifuged and collected.

[0336] Example 2: Determination of antibody purity and activity

[0337] 1. HPLC-SEC (High Performance Liquid Chromatography-Size Exclusion Chromatography)

[0338] Take 20 μg of purified antibody and determine the SEC purity of the sample according to the method in Table 2. The purity is expressed as the percentage of the main peak area.

[0339] Table 2: Chromatographic conditions for HPLC-SEC

[0340] 2. Non-reducing CE-SDS Page

[0341] 2.1 Desalting If the salt concentration in the final loading solution is greater than 40 mM, the sample must be desalted. Add 200 μg of sample to a 10 kDa ultrafiltration tube, bring the volume to 400 μl with ultrapure water, vortex to mix, centrifuge at 13000 rpm for 10 minutes at 4°C, collect the concentrate, and measure the concentration. If necessary, repeat the above desalting steps until the salt concentration of the resulting loading solution is less than 40 mM.

[0342] 2.2 Sample Dilution

[0343] 2.2.1 Blank control: Take 12.5 μl of ultrapure water and add 37.5 μl of 1X sample buffer to make the total volume 50 μl.

[0344] 2.2.2 Sample dilution Take an appropriate amount of 1X sample buffer and dilute the sample to a volume of 50 μl, with a concentration of 1 mg / mL.

[0345] 2.2.3 Sample Preparation: Add 2 μl of internal standard and 2.5 μl of alkylating agent (IAM) to the sample, cap the sample, mix well, and centrifuge at 6000 rpm for 1 min. Incubate the sample in a constant temperature mixer at 70℃ for 5 min, then remove and cool to room temperature. Centrifuge at 6000 rpm for 3 min. Transfer 45 μl of the prepared sample to a 96-well plate, centrifuge at 1000 × g for 10 min, and then inject for analysis.

[0346] 2.3 Hands-on Practice

[0347] The processed samples were placed into Maurice's ProteinSimple device for testing.

[0348] 3. Results of activity and specificity after biotin labeling

[0349] Labeling process: Antibody (5 mg / mL) was labeled with Zeba. TM The desalting column (10K MWCO) was replaced with PBS (100mM PB, 50mM sodium chloride, pH 8.0, 5mM EDTA). NHS-LC-BIO was dissolved in DMSO to prepare an 8mM solution. 20 eq of bio were added to the antibody solution, and the reaction was carried out at 25°C for 2 hours. Excess reagents were removed by desalting, and the biotinylated antibody was stored at 4°C for later use.

[0350] Detection procedure: 1) Sample addition reaction: Add 30Ul of sample, 50Ul of Ab-bio working solution, and 50Ul of SA-magnetic bead working solution, react at 37℃ for 10min; 2) Sample addition reaction: Add 50uL of Ab-AE working solution, react at 37℃ for 5min; 3) Wash three times with 1*TBST; 4) Add pre-activation solution A and activation solution B, and read the value immediately.

[0351] Forty-nine Roche-calibrated specimens were tested, and mean activity and clinical relevance were calculated.

[0352] 4. Test Results

[0353] 4.1 Purity Measurement Results

[0354] Table 3: Effects of different modification methods on rabbit IgG hapten antibodies

[0355] The results showed that the modified antibodies designed in Table 1 all reduced the hemitype formation rate (HL(%)) to varying degrees compared with wild-type antibodies. The order of improvement in hemitype formation rate was: Modified 10–12, Modified 14–19 or Modified 21 > Modified 22 or Modified 13 > Modified 3–9, Modified 20, Modified 24–26. Among them, Modified 10–12, Modified 14–19 and Modified 21 showed the most significant improvement in hemitype formation rate, as shown in Table 3. They reduced the hemitype formation rate from 12.54% to less than 4%, and increased the antibody assembly rate (IgG(%)) from 85% to 93%–96%.

[0356] 4.2 Activity Results

[0357] Table 4-1 Activity data for modifications 10-12

[0358] Table 4-2 Activity data of modifications 14-19 and 21

[0359] As can be seen from the tables and figures above, the average activity differences of modified types 10, 11, and 12 compared to the wild type are within ±10%, which is basically equivalent. Their clinical relevance is also comparable to that of the wild type. 2 All were 0.98. Modified 14–19 and 21 showed average activity differences within ±15% compared to wild-type, with clinical relevance similar to wild-type R. 2 Both are 0.98.

[0360] Example 3: Validation results of antibodies from different species

[0361] 1. Based on wild-type sheep antibodies, the hinge region of the wild-type sheep antibodies was modified according to Table 5. The amino acid sequence of the heavy chain of the wild-type sheep antibody is shown in SEQ ID NO:32, and the amino acid sequence of the light chain is shown in SEQ ID NO:33. For specific modification schemes, please refer to Table 1.

[0362] Table 5: Design Methods for Renovation

[0363] 2. Antibodies were prepared according to the antibody preparation process described in Example 1, and the purity of the antibodies (reduced CE-SDS + non-reduced CE-SDS) was determined according to Example 2. The results showed that the modification methods verified as effective in rabbits were also verified as effective in sheep. As exemplified in Table 7, the modification methods of mutations 1-2 showed a reduced half-antibody formation rate (HL (%)) and an increased antibody assembly rate (IgG (%)) in both rabbits and sheep compared to the wild type.

[0364] Table 6: Reduction of CE-SDS

[0365] Table 7: Non-reducing CE-SDS

[0366] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0367] Although embodiments of this application have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting this application. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of this application.

Claims

1. An Fc, characterized in that, It includes a modified hinge region, wherein the modified hinge region is formed by modifying the COC short peptide in the parent hinge region into a CZC short peptide, wherein the O in the COC short peptide or the Z in the CZC short peptide independently represents one or more amino acids.

2. The Fc according to claim 1, characterized in that, The first C and the last C in the COC short peptide in the parent hinge region mismatch to form an intra-heavy chain disulfide bond. Optionally, the last C in the COC short peptide is a cysteine ​​residue in the parent hinge region used to form inter-heavy chain disulfide bonds.

3. The Fc according to any one of claims 1-2, characterized in that, The modification is selected from at least one of substitution, insertion, and deletion; Optionally, the modification is selected from substitution; Optionally, the substitution is selected from the substitution of flexible amino acids with neutral or rigid amino acids; Optionally, the substitution is selected from the substitution of neutral amino acids with rigid amino acids; Optionally, the substitution is selected from the interchange between different flexible amino acids; Optionally, the substitution is selected from the interchange between different neutral amino acids; Optionally, the substitution is selected from the interchange between different rigid amino acids; Optionally, the substitution is selected from cysteine ​​substitution; Optionally, the number of amino acids replaced is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. Optionally, the number of cysteine ​​substitutions is 1, 2, 3, or 4 amino acids; Optionally, the modification is selected from insertion; Optionally, the insertion is selected from neutral amino acid insertion, rigid amino acid insertion, or cysteine ​​insertion; Optionally, the number of amino acids inserted is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. Optionally, the number of cysteine ​​insertions is 1, 2, 3, or 4 cysteines; Optionally, the modification is selected from missing values; Optionally, the deletion is a deletion of neutral amino acids or a deletion of flexible amino acids; Optionally, the number of missing amino acids is 1, 2, 3, 4 or 5; Optionally, the neutral amino acid is selected from aspartic acid, lysine, arginine, or histidine; Optionally, the rigid amino acid is selected from alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, tryptophan, or proline; Optionally, the rigid amino acid is proline; Optionally, the flexible amino acid is selected from glycine, serine, or threonine; Optionally, the flexible amino acid is threonine.

4. The Fc according to any one of claims 1-3, characterized in that, The Z in the CZC short peptide includes a C-X1-C short peptide, or includes a C-X1 short peptide in the C-X1-C short peptide, or the CZC short peptide is a C-X1-C short peptide. Optionally, the CZC short peptide is a C-X2-C-X1-C-X3-C short peptide; Optionally, C-X2 in the C-X2-C-X1-C-X3-C short peptide is a continuous peptide segment on the COC short peptide starting with the first C at the N-terminus, and X3-C in the short peptide is a continuous peptide segment on the COC short peptide ending with the last C at the C-terminus. Optionally, the CZC short peptide is a C-X4-C-X1-C short peptide; Optionally, C-X4 in the C-X4-C-X1-C short peptide is a continuous peptide segment on the COC short peptide starting with the first C at the N-terminus; Optionally, the C-X4 in the C-X4-C-X1-C short peptide is the CO in the COC short peptide; Optionally, the C-X1-C short peptide has a higher ratio of the difference between the number of rigid amino acids and the number of flexible amino acids compared to the COC short peptide; Optionally, the C-X1-C short peptide does not contain flexible amino acids; Optionally, the C-X1-C short peptide has more cysteine ​​residues compared to the COC short peptide; Optionally, the C-X1-C short peptide is derived from the natural hinge region of a natural antibody; Optionally, the first C and the last C in the C-X1-C short peptide are both Cs in the natural hinge region from which it is derived, which are used to form inter-heavy chain disulfide bonds; Optionally, the first C in the C-X1-C short peptide is the first C in its native hinge region used to form inter-heavy chain disulfide bonds; Optionally, the last C in the C-X1-C short peptide is the last C in its natural hinge region used to form inter-heavy chain disulfide bonds; Optionally, the C-X4-C-X1-C short peptide is a continuous fragment on its natural hinge region; Optionally, the first C in the C-X4-C-X1-C short peptide is the C in the upper hinge region of its natural hinge region, and the first C is not the C in the natural hinge region used to form inter-heavy chain disulfide bonds; Optionally, the first C in the C-X4-C-X1-C short peptide is the last C in the upper hinge region of its natural hinge region; Optionally, X1, X2, X3, or X4 in the short peptide each independently represent one or more amino acids; Optionally, the CZC short peptide has more amino acid residues than the COC short peptide; Optionally, the CZC short peptide has 1 to 10 more amino acid residues than the COC short peptide; Optionally, the CZC short peptide has 1, 2, 3, 4 or 5 more amino acid residues than the COC short peptide; Optionally, the species of the natural hinge region is selected from humans, mice, rats, cattle, horses, pigs, sheep, goats, dogs, camels, alpacas, monkeys, orangutans, cats, rabbits, donkeys, deer, minks, chickens, ducks, geese, or humans; Optionally, the subtype of the natural hinge region is selected from IgG1, IgG2, IgG3, IgG4, IgA, and IgD; Optionally, the species and subtype of the natural hinge region are selected from bovine IgG1, sheep IgG1, human IgG1, horse IgG1, mouse IgG2a, mouse IgG1, monkey IgG1, monkey IgG3, monkey IgG4, orangutan IgG1, horse IgG3, horse IgG5, alpaca IgG1A, and alpaca IgG2B. Optionally, the C-X1-C short peptide is selected from the natural hinge region of bovine IgG1, sheep IgG1, human IgG1, horse IgG1, mouse IgG2a, and mouse IgG1. Optionally, the C-X1-C short peptide is selected from any short peptide or a portion thereof from CDCC, CKHCRC, CPPC, CPKC, CPPCKC, and CKPCIC; Optionally, the C-X4-C-X1-C short peptide is selected from sheep IgG1, human IgG1, and mouse IgG1; Optionally, the C-X4-C-X1-C short peptide is selected from any one of CGCKPCIC, CDKTHTCPPC, and CPDPCKHCRC short peptides; Optionally, the species and subtype of the parent hinge region are selected from rabbit IgG; Optionally, the COC short peptide is selected from CSKPTC short peptide, or a polypeptide obtained by replacing at least one amino acid on CSKPTC short peptide; Optionally, the species and subtype of the parent hinge region are selected from sheep IgG2; Optionally, the COC short peptide is selected from C-SKPP-C short peptide, or a polypeptide obtained by replacing at least one amino acid on C-SKPP-C short peptide; Optionally, the COC short peptide is selected from C-SKPM-C short peptide, C-PKPT-C short peptide, C-SPPT-C short peptide, C-CPPT-C short peptide, C-SKCPPT-C short peptide, C-CKPT-C short peptide, C-SCPT-C short peptide, and C-SKCT-C short peptide; Optionally, the parental hinge region of the rabbit IgG is selected from the short peptide APSTCSKPTCPPPELLGGPS or a portion thereof, or has at least 90% identity with it; Optionally, the parental hinge region of the sheep IgG2 is a short peptide GISSDYSKCSKPPCVSRPS or a portion thereof, or has at least 90% identity with it; Optionally, the modified hinge region has a reduced tendency for intra-heavy chain disulfide bond formation or an increased tendency for inter-heavy chain disulfide bond formation compared to the parent hinge region.

5. The Fc according to any one of claims 1-4, characterized in that, The modified hinge area further includes an N-end hinge area; and / or a C-end hinge area; Optionally, the N-end hinge region in the modified hinge region includes the N-end hinge region of COC on the parent hinge region or a portion thereof; Optionally, the C-end hinge area in the modified hinge area includes the C-end hinge area of ​​COC on the parent hinge area or a portion thereof; Optionally, the C-terminal hinge region in the modified hinge region includes the C-terminal hinge region or a portion thereof of the natural hinge region of the C-X4-C-X1-C short peptide on the corresponding species and subtype of the C-X4-C-X1-C short peptide. Optionally, the C-terminal hinge region in the modified hinge region includes the C-terminal hinge region or a portion thereof of the natural hinge region of the C-X1-C short peptide corresponding to the species and subtype of C-X1-C in the C-X4-C-X1-C short peptide.

6. The Fc according to any one of claims 1-5, characterized in that, The subtype of the parental hinge region was selected from rabbit IgG; Optionally, the COC short peptide is selected from C-SKPT-C short peptide; Optionally, the CZC short peptide is selected from any of the following short peptides: CCKPTC, CSCPTC, CSKCTC, CPKPTC, CSPPTC, CSKPPC, CPPPTC, CCPPTC, CSKCPPCPTC, CSKCPPTC, CSKPCDCC, CSKPCKHCRC, CSKPTCKHCRC, CSKPTCPPC, CSKPTCPPC, CSKPTCPKC, CPPCKC, CKHCRC, CKPCIC, CGCKPCIC, CDKTHTCPPC, CPDPCKHCRC; Optionally, the CZC short peptide is selected from any of the following short peptides: CSKCPPCPTC, CSKCPPTC, CSKPCDCC, CSKPCKHCRC, CSKPTCKHCRC, CSKPTCPPC, CSKPTCPPC, CSKPTCPKC, CGCKPCIC, CDKTHTCPPC, CPDPCKHCRC; Optionally, the CZC short peptide is selected from any of the following short peptides: CSKCPPCPTC, CSKPCDCC, CSKPCKHCRC, CSKPTCKHCRC, CSKPTCPPC, CSKPTCPPC, CSKPTCPKC, CGCKPCI C, CDKTHTCPPC, CPDPCKHCRC; Optionally, the N-end hinge region in the modified hinge region includes the N-end hinge region of COC on the parent hinge region or a portion thereof; Optionally, the N-terminal hinge region in the modified hinge region includes an APST short peptide or a portion thereof; Optionally, the modified hinge region is selected from any of the following short peptides, or portions of any of the following short peptides: APSTCCKPTC,APSTCSCPTC,APSTCSKCTC,APSTCPKPTC,APSTCSPPTC,APSTCSKPPC,APSTCPPPTC,APSTCCPPTC,APSTCSKCPPCPTC,APSTCSKCPPTC,APSTCSKPCDCC,APSTCSKPCK HCRC, APSTCSKPTCKHCRC, APSTCSKPTCPPC, APSTCSKPTCPPC, APSTCSKPTCPKC, APSTCPPCKC, APSTCKHCRC, APSTCKPCIC, APSTCGCKPCIC, APSTCDKTHTCPPC, APSTCPDPCKHCRC; Optionally, the modified hinge region is selected from any of the following short peptides, or a portion thereof: APSTCSKCPPCPTC, APSTCSKCPPTC, APSTCSKPCDCC, APSTCSKPCKHCRC, APSTCSKPTCKHCRC, APSTCSKPTCPPC, APSTCSKPTCPPC, APSTCSKPTCPKC, APSTCCKPCIC, APSTCKTHTCPPC, APSTCPDPCKHCRC; Optionally, the C-end hinge area in the modified hinge area includes the C-end hinge area of ​​COC on the parent hinge area or a portion thereof; Optionally, the C-end hinge area in the modified hinge area includes PPPELLGGPS or a portion thereof, or has at least 80% identity with it; Optionally, the modified hinge region is selected from any of the following short peptides, or portions of any of the following short peptides: APSTCCKPTCPPPELLGGPS, APSTCSCPTCPPPELLGGPS, APSTCSKCTCPPPELLGGPS, APSTCPKPTCPPPELLGGPS, APSTCSPPTCPPPELLGGPS, APSTCSKPPCPPPELLGGPS, APSTCPPPTCPPPELLGGPS, APSTCCPPTCPPPELLGGPS, APSTCSKCPPCPTCPPPELLGGPS, APSTCSK CPPTCPPPELLGGPS, APSTCSKPCDCCPPPELLGGPS, APSTCSKPCKHCRCPPPELLGGPS, APSTCSKPTCKHCRCPPPELLGGPS, APSTCSKPTCPPCPPPELLGGPS, APSTCSKPTCPKCPPPELLGGPS, APSTCPPCKCPPPELLGGPS, APSTCKHCRCPPPELLGGPS, APSTCKPCICPPPELLGGPS; Optionally, the C-terminal hinge region in the modified hinge region includes the C-terminal hinge region or a portion thereof of the natural hinge region of the C-X4-C-X1-C short peptide on the corresponding species and subtype of the C-X4-C-X1-C short peptide. Optionally, if the species and subtype corresponding to the C-X4-C-X1-C short peptide is mouse IgG1, and the C-X4-C-X1-C short peptide is CGCKPCIC, then the C-terminal hinge region in the modified hinge region includes the C-terminal hinge region TVPEVSS or a portion thereof of the CGCKPCIC short peptide on the natural hinge region of mouse IgG1. Optionally, if the species and subtype corresponding to the C-X4-C-X1-C short peptide is human IgG1, and the C-X4-C-X1-C short peptide is CDKTHTCPPC, then the C-terminal hinge region in the modified hinge region includes the C-terminal hinge region PAPELLGGPS or a portion thereof of the CDKTHTCPPC short peptide on the natural hinge region of human IgG1. Optionally, if the species and subtype corresponding to the C-X4-C-X1-C short peptide is sheep IgG1, and the C-X4-C-X1-C short peptide is CPDPCKHCRC, then the C-terminal hinge region in the modified hinge region includes the C-terminal hinge region PPPELLGGPS or a portion thereof of the CPDPCKHCRC short peptide on the natural hinge region of sheep IgG1. Optionally, the modified hinge region is selected from any of the following short peptides, or portions of any of the following short peptides: APSTCGCKPCICTVPEVSS, APSTCDKTHTCPPCPAPELLGGPS, APSTCPDPCKHCRCPPPELPGGPS; Optionally, the C-terminal hinge region in the modified hinge region includes the C-terminal hinge region or a portion thereof of the natural hinge region of the C-X1-C short peptide corresponding to the species and subtype of C-X1-C in the C-X4-C-X1-C short peptide. Optionally, if the species and subtype of C-X1-C in the C-X4-C-X1-C short peptide is human IgG1, and the C-X1-C short peptide is CPPC, then the C-terminal hinge region in the modified hinge region includes the C-terminal hinge region PAPELLGGPS or a portion thereof of the CPPC short peptide on the natural hinge region of human IgG1. Optionally, the modified hinge region is selected from any of the following short peptides, or a portion of any of the following short peptides: APSTCSKPTCPPCPAPELLGGPS.

7. The Fc according to any one of claims 1-5, characterized in that, The subtype of the parental hinge region was selected from sheep IgG2; Optionally, the COC short peptide is selected from C-SKPP-C short peptide; Optionally, the CZC short peptide is selected from any of the following short peptides: CGCKPCIC, CDKTHTCPPC, CPDPCKHCRC; Optionally, the N-end hinge region in the modified hinge region includes the N-end hinge region of COC on the parent hinge region or a portion thereof; Optionally, the N-terminal hinge region in the modified hinge region includes a GISSDYSK short peptide or a portion thereof; Optionally, the modified hinge region is selected from any of the following short peptides, or a portion of any of the following short peptides: GISSDYSKCGCKPCIC, GISSDYSKCDKTHTCPPC, GISSDYSKCPDPCKHCRC; Optionally, the C-terminal hinge region in the modified hinge region includes the C-terminal hinge region or a portion thereof of the natural hinge region of the C-X4-C-X1-C short peptide on the corresponding species and subtype of the C-X4-C-X1-C short peptide. Optionally, if the species and subtype corresponding to the C-X4-C-X1-C short peptide is mouse IgG1, and the C-X4-C-X1-C short peptide is CGCKPCIC, then the C-terminal hinge region in the modified hinge region includes the C-terminal hinge region TVPEVSS or a portion thereof of the CGCKPCIC short peptide on the natural hinge region of mouse IgG1. Optionally, if the species and subtype corresponding to the C-X4-C-X1-C short peptide is human IgG1, and the C-X4-C-X1-C short peptide is CDKTHTCPPC, then the C-terminal hinge region in the modified hinge region includes the C-terminal hinge region PAPELLGGPS or a portion thereof of the CDKTHTCPPC short peptide on the natural hinge region of human IgG1. Optionally, if the species and subtype corresponding to the C-X4-C-X1-C short peptide is sheep IgG1, and the C-X4-C-X1-C short peptide is CPDPCKHCRC, then the C-terminal hinge region in the modified hinge region includes the C-terminal hinge region PPPELPGGPS or a portion thereof of the CPDPCKHCRC short peptide on the natural hinge region of sheep IgG1. Optionally, the modified hinge region is selected from any of the following short peptides, or portions of any of the following short peptides: GISSDYSKCGCKPCICTVPEVSS, GISSDYSKCDKTHTCPPCPAPELLGGPS, GISSDYSKCPDPCKHCRCPPPELPGGPS; Optionally, the N-terminal hinge region in the modified hinge region includes the N-terminal hinge region or a portion thereof of the natural hinge region of the C-X4-C-X1-C short peptide on the corresponding species and subtype of the C-X4-C-X1-C short peptide. Optionally, if the species and subtype corresponding to the C-X4-C-X1-C short peptide is sheep IgG1, and the C-X4-C-X1-C short peptide is CPDPCKHCRC, then the N-terminal hinge region in the modified hinge region includes the N-terminal hinge region EPG or a portion thereof of the CPDPCKHCRC short peptide on the natural hinge region of sheep IgG1. Optionally, the modified hinge region is selected from any of the following short peptides, or a portion of any of the following short peptides: EPGCPDPCKHCRCPPPELPGGPS.

8. The Fc according to any one of claims 1-7, characterized in that, The modification also includes replacing the first C in the COC short peptide with any amino acid other than C; Optionally, the first C in the COC short peptide is replaced with S or T.

9. An Fc, characterized in that, It includes a modified hinge region, wherein the first C in the COC short peptide in the parent hinge region is replaced with any amino acid other than C, and the O in the COC short peptide represents one or more amino acids; Optionally, the first C in the COC short peptide is replaced with S or T; Optionally, the first C and the last C in the COC short peptide in the parent hinge region may mismatch to form an intra-heavy chain disulfide bond; Optionally, the last C in the COC short peptide is a cysteine ​​residue in the parent hinge region used to form inter-heavy chain disulfide bonds; Optionally, the species and subtype of the parent hinge region are selected from rabbit IgG; Optionally, the COC short peptide is selected from CSKPTC short peptide, or a polypeptide obtained by replacing at least one amino acid on CSKPTC short peptide; Optionally, the species and subtype of the parent hinge region are selected from sheep IgG2; Optionally, the COC short peptide is selected from C-SKPP-C short peptide, or a polypeptide obtained by replacing at least one amino acid on C-SKPP-C short peptide; Optionally, the COC short peptide is selected from C-SKPM-C short peptide, C-PKPT-C short peptide, C-SPPT-C short peptide, C-CPPT-C short peptide, C-SKCPPT-C short peptide, C-CKPT-C short peptide, C-SCPT-C short peptide, and C-SKCT-C short peptide; Optionally, the parental hinge region of the rabbit IgG is selected from the short peptide APSTCSKPTCPPPELLGGPS or a portion thereof, or has at least 90% identity with it; Optionally, the parental hinge region of the sheep IgG2 is a short peptide GISSDYSKCSKPPCVSRPS or a portion thereof, or has at least 90% identity with it; Optionally, the modified hinge region has a reduced tendency to form intra-heavy chain disulfide bonds or an increased tendency to form inter-heavy chain disulfide bonds compared to the parent hinge region. Optionally, CH2, CH3, CH4, or tail peptide in Fc are each independently selected from rabbit, sheep, cow, human, horse, mouse, or rat; Optionally, the species of CH2, CH3, CH4, or tail peptide is the same as that of the parent antibody; Optionally, the species and subtype of the CH2, CH3, and / or tail peptide are selected from rabbit IgG or sheep IgG2; Optionally, the species and subtype of CH2, CH3, CH4, and / or tail peptide are selected from rabbit IgM or sheep IgM; Optionally, the species of CH2, CH3, CH4, or the tail peptide is the same as the species of the natural antibody corresponding to the C-X1-C short peptide; Optionally, the species and subtypes of CH2, CH3 and tail peptide are selected from bovine IgG, human IgG, horse IgG, mouse IgG, or rat IgG; Optionally, the species and subtypes of CH2, CH3, CH4 and tail peptide are selected from bovine IgM, human IgM, horse IgM, mouse IgM, or rat IgM.

10. An antibody constant region comprising the Fc and CH1 regions as described in any one of claims 1 to 9; Optionally, the CH1 region is selected from rabbits, sheep, cattle, humans, horses, mice, or rats; Optionally, the species of the CH1 region is the same as that of the parent antibody; Optionally, the species and subtype of the CH1 region are selected from rabbit IgG or sheep IgG2; Optionally, the species and subtype of the CH1 region are selected from rabbit IgM or sheep IgM; Optionally, the species of the CH1 region is the same as the species of the natural antibody corresponding to the C-X1-C short peptide; Optionally, the species and subtype of the CH1 region are selected from bovine IgG, human IgG, horse IgG, mouse IgG, or rat IgG; Optionally, the species and subtype of the CH1 region are selected from bovine IgM, human IgM, horse IgM, mouse IgM, or rat IgM; Optionally, the antibody constant region further includes a modified CH1 region, which is obtained by modifying the parental CH1 region; The modification involves replacing the C in the parent CH1 region that forms an atypical intrachain disulfide bond with the first C in the COC short peptide in the parent hinge region; Optionally, the CH1 region is selected from the modified CH1 region, which is obtained by modifying the parental CH1 region; The modification involves replacing the C in the parent CH1 region that forms an atypical intrachain disulfide bond with the first C in the COC short peptide in the parent hinge region; Optionally, the modification involves replacing the C in the parental CH1 region that forms an atypical intrachain disulfide bond with the first C in the COC short peptide in the parental hinge region with S or T. Optionally, the 15th carbon in the parent CH1 region forms an atypical intrachain disulfide bond with the first carbon in the COC short peptide in the parent hinge region; Optionally, the modification involves replacing the 15th C position in the CH1 region of the parent. Optionally, the modification involves replacing the 15th C in the parent CH1 region with S or T; Optionally, the amino acid sequence of the CH1 region of the parent is as shown in any one of SEQ ID NO: 1 to 2; Optionally, the amino acid sequence of the modified CH1 region is as shown in any one of SEQ ID NO:3 to 4; Optionally, the amino acid sequences of the modified CH1 region and the modified hinge region are as shown in any one of SEQ ID NO: 5 to 6; Optionally, the modification involves deleting the C in the parent CH1 region that forms an atypical intrachain disulfide bond with the first C in the COC short peptide in the parent hinge region; Optionally, the modification involves deleting the 15th C position in the parent CH1 region.

11. An antibody, characterized in that, The antibody includes the Fc as described in any one of claims 1 to 9, or the antibody constant region as described in claim 10; Optionally, the antibody further includes a heavy chain variable region and an optional light chain variable region; Optionally, the antibody comprises a heavy chain and a light chain, wherein the heavy chain is obtained by fusing a first polypeptide and a second polypeptide: the first polypeptide comprises a heavy chain variable region located at the N-terminus, a CH1 region in the constant region of the antibody according to claim 10, and a modified hinge region in the Fc according to any one of claims 1-9, and the second polypeptide comprises an IgM CH3-CH4 region in the Fc according to claim 9 located at the C-terminus; Optionally, the antibody comprises a heavy chain and a light chain, wherein the heavy chain is obtained by fusing a first polypeptide and a second polypeptide: the first polypeptide comprises a heavy chain variable region located at the N-terminus, a modified CH1 region in the constant region of the antibody of claim 10, and a modified hinge region in the Fc of any one of claims 1-9, and the second polypeptide comprises an IgM CH3-CH4 region in the Fc of claim 9 located at the C-terminus.

12. A nucleic acid molecule, a vector, and a host cell, characterized in that, The nucleic acid molecule encodes the Fc as described in any one of claims 1 to 9, the antibody constant region as described in claim 10, or the antibody as described in claim 11; The vector carries the aforementioned nucleic acid molecules; The host cell includes the above-described nucleic acid molecules or vectors; or expresses the Fc as described in any one of claims 1 to 9, the antibody constant region as described in claim 10, or the antibody as described in claim 11.

13. A conjugate, characterized in that, include: The Fc according to any one of claims 1 to 9, the antibody constant region according to claim 10, or the antibody according to claim 11, and At least one conjugation portion, said conjugation portion being linked to the Fc, antibody constant region, or antibody.

14. A reagent kit, characterized in that, include: The Fc according to any one of claims 1 to 9, the antibody constant region according to claim 10, the antibody according to claim 11, or the conjugate according to claim 13.

15. The use of the Fc according to any one of claims 1 to 9, the antibody constant region according to claim 10, the antibody according to claim 11, the nucleic acid molecule, vector, or host cell according to claim 12, the conjugate according to claim 13, or the kit according to claim 14 in immunoassay or the preparation of immunoassay products.

16. A method for detecting antigens in a sample to be tested, characterized in that, include: a) Under conditions sufficient to allow an antibody / antigen binding reaction to occur, the antibody of claim 11, the conjugate of claim 13, or the kit of claim 14 is brought into contact with the antigen in the sample to be tested to form an immune complex; and b) Detect the presence of the immune complex, and determine whether the sample to be tested contains an antigen based on the presence of the immune complex.

17. A method for reducing antibody half-antibody formation rate or increasing antibody assembly rate, characterized in that, The method includes: The Fc in the antibody is modified to form the Fc according to any one of claims 1 to 9; The antibody constant region in the antibody is modified to form the antibody constant region of claim 10; or The antibody is modified to form the antibody of claim 11.

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