Antibody and antibody conjugate and use thereof

Abstract

Provided is a conjugate comprising an antibody and a conjugate moiety. The antibody comprises a heavy chain constant region comprising an engineered lysine residue, wherein the engineered lysine residue comprises the following sites: 1, 3, 5, 14, 31, 58, 59, 60, 61, 79, 81, 116, 141, 144, 165, 169, 190, 192, 199, 208, 209, 213, 218, 219, 220, 224, 231, 232, 233, 236, 238, 249, 255, 260, 266, 267, 271, 273, 274, 275, 279, 290, 295, 309, 313, 315, 317, 319, 320, etc.

Description

Antibodies and antibody conjugates and their uses

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese patent applications with application number 202310930054.7, entitled “Antibodies and Antibody Conjugates and Uses Thereof”, filed with the Chinese Patent Office on July 26, 2023, and application number 202410168038.3, entitled “Antibodies and Antibody Conjugates and Uses Thereof”, filed with the Chinese Patent Office on February 4, 2024, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present invention belongs to the field of biotechnology. Specifically, the present invention relates to an antibody and an antibody conjugate and uses thereof. More specifically, the present invention relates to an antibody, a conjugate, a nucleic acid molecule, a vector, a cell or a host, a reagent or a kit and uses thereof, a method for preparing the conjugate, a method for immunoassay, and a method for improving the activity, stability or sensitivity of the conjugate. Background Art

[0004] Conjugates of antibodies and markers have been widely used in immunoassays. The activity, stability, and conjugation ratio of the conjugates directly affect the sensitivity and specificity of immunoassays. Currently, commonly used conjugation methods generally include conjugation based on cysteine ​​sulfhydryl groups and conjugation based on lysine side chain amino groups.

[0005] Cysteine-based conjugation requires partial reduction of the antibody to destroy the interchain disulfide bonds, thereby generating free thiol side chains. Then, the thiol-reactive functional agent reacts with the free thiol groups on the antibody to produce an antibody-label conjugate. Cysteine ​​residues have been introduced into antibodies through genetic engineering techniques. However, in concentrated solutions of antibodies, unpaired cysteine ​​residues on the antibody surface can pair and oxidize to form intermolecular disulfides, thereby forming antibody dimers or multimers. In addition, if the protein oxidizes to form intramolecular disulfide bonds between the newly introduced cysteine ​​and the existing cysteine ​​residues, both cysteine ​​residues cannot be used for active site participation and interaction, and further lead to antibody misfolding or loss of tertiary structure, reducing the activity and specificity of the antibody.

[0006] Conjugation based on lysine side chain amino groups typically involves connecting the lysine amino group to an activated carboxylate linker via an amide bond, such as one mediated by a succinimide or isothiocyanate-based chemical reaction. Lysine amino groups on antibodies are excellent nucleophiles, making them highly effective for labeling, and the conjugation process is simple. However, lysine residues are dispersed in natural antibodies, and lysine conjugates formed based on lysine side chain amino groups can affect the antibody structure, reducing the antibody's activity or stability, thereby reducing the activity and sensitivity of the resulting antibody conjugate.

[0007] Therefore, there is an urgent need to provide antibodies and antibody conjugates with high activity, stability or sensitivity.

[0008] Summary of the Invention

[0009] The present invention aims to provide an antibody, a conjugate, a nucleic acid molecule, a vector, a cell or a host, a reagent or a kit and its use, a method for preparing the conjugate, a method for immunoassay, and a method for improving the activity, stability or sensitivity of the conjugate.

[0010] In a first aspect of the present invention, the present invention provides a conjugate. According to an embodiment of the present invention, the conjugate comprises an antibody and at least one conjugated moiety, wherein:

[0011] a) the antibody comprises a heavy chain constant region;

[0012] The heavy chain constant region comprises an engineered lysine residue;

[0013] The engineered lysine residues include or are selected from at least one of the following sites or their equivalent sites:

[0014] 1, 3, 5, 14, 31, 58, 59, 60, 61, 79, 81, 116, 141, 144, 165, 169, 190, 192, 199, 208, 209, 213, 218, 219, 220, 224, 231, 232, 233, 236, 238, 249, 255, 260, 266, 267, 271, 273, 274, 275, 279, 290, 295, 309, 313, 315, 317, 319, 320, 323, 325, 331 and 332;

[0015] b) The conjugate moiety is linked to the antibody.

[0016] In a second aspect of the present invention, the present invention provides an antibody. According to an embodiment of the present invention, the antibody is the antibody defined in the conjugate described in the first aspect of the present invention.

[0017] In a third aspect, the present invention provides a method for preparing a conjugate. According to an embodiment of the present invention, the method comprises: performing a conjugation reaction between the antibody in the conjugate of the first aspect or the antibody of the second aspect and a conjugation moiety to obtain the conjugate.

[0018] In a fourth aspect, the present invention provides a nucleic acid molecule. According to an embodiment of the present invention, the nucleic acid molecule encodes the antibody in the conjugate of the first aspect or the antibody of the second aspect.

[0019] In a fifth aspect, the present invention provides a vector. According to an embodiment of the present invention, the vector comprises the nucleic acid molecule described in the fourth aspect.

[0020] In a sixth aspect, the present invention provides a cell or host. According to an embodiment of the present invention, the cell or host comprises: the nucleic acid molecule of the fourth aspect or the vector of the fifth aspect; or expresses the antibody in the conjugate of the first aspect or the antibody of the second aspect.

[0021] In the seventh aspect of the present invention, the present invention proposes a use of the antibody described in the second aspect, the nucleic acid molecule described in the fourth aspect, the vector described in the fifth aspect, or the cell or host described in the sixth aspect in preparing a conjugate or a kit.

[0022] In an eighth aspect, the present invention provides a reagent or a kit. According to an embodiment of the present invention, the reagent or the kit comprises: the conjugate described in the first aspect or the antibody described in the second aspect.

[0023] In the ninth aspect of the present invention, the present invention proposes a use of the conjugate described in the first aspect, the antibody described in the second aspect, the nucleic acid molecule described in the fourth aspect, the vector described in the fifth aspect, the cell or host described in the sixth aspect, or the reagent or kit described in the eighth aspect in immunoassay or preparation of immunoassay products.

[0024] In a tenth aspect, the present invention provides an immunoassay method. According to an embodiment of the invention, the method comprises: contacting the conjugate described in the first aspect or the antibody described in the second aspect with a sample to be detected to form an immune complex.

[0025] In the eleventh aspect of the present invention, the present invention provides a method for improving the activity, stability or sensitivity of a conjugate, wherein the conjugate comprises an antibody and at least one conjugated portion, wherein the conjugated portion is connected to the antibody, and the method comprises: introducing an engineered lysine residue into the heavy chain constant region of the antibody, wherein the engineered lysine residue is consistent with the engineered lysine residue defined in the conjugate described in the first aspect.

[0026] The amino acid sequence table of the present invention is as follows:

[0027] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the following description of the embodiments with reference to the accompanying drawings, in which:

[0029] FIG1 is a correlation test result of each group of IgG1 antibody-AE and Roche fixed value blood in the embodiment of the present invention;

[0030] FIG2 is a correlation test result of each group of IgG2b antibody-AE and Roche fixed value blood in the embodiment of the present invention. DETAILED DESCRIPTION

[0031] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0032] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of the technical features indicated. Therefore, features defined as "first" or "second" may explicitly or implicitly include one or more of such features. Furthermore, in the description of the present invention, unless otherwise specified, "plurality" means two or more.

[0033] The endpoints of the ranges and any values ​​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 endpoints of each range, the endpoints of each range and individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.

[0034] To facilitate understanding of the present invention, certain technical and scientific terms are defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs. The abbreviations for amino acid residues are the standard three-letter and / or one-letter codes used in the art to designate one of the 20 commonly used L-amino acids.

[0035] In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present invention, but not excluding other contents.

[0036] As used herein, the terms "optionally," "optional," "optionally," "optional," or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0037] As used herein, the term "at least 80% identity" refers to at least 80% identity to the respective reference sequence, and may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% identity.

[0038] In this article, the term "vector" generally refers to a nucleic acid molecule that can be inserted into a suitable host and self-replicates, and the inserted nucleic acid molecule is transferred into and / or between cells or hosts. The vector may include a vector primarily used for inserting DNA or RNA into a cell, a vector primarily used for replicating DNA or RNA, and a vector primarily used for expression of the transcription and / or translation of DNA or RNA. The vector also includes a vector with a variety of the above-mentioned functions. The vector can be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable cell or host. Typically, the vector can produce a desired expression product by cultivating a suitable cell or host that contains the vector.

[0039] In this article, the term "cell" generally refers to a cell that has been modified or reorganized using genetic engineering techniques or cell fusion techniques to modify or reorganize the genetic material of a host cell to obtain a cell with a unique trait of stable inheritance. Wherein, the term "host cell" refers to a prokaryotic cell or eukaryotic cell into which a recombinant vector can be introduced. The terms "transformed" or "transfected" as used herein refer to the introduction of a nucleic acid (e.g., a vector) into a cell by various techniques known in the art. Suitable host cells can be transformed or transfected with the DNA sequence of the present invention and can be used for the expression and / or secretion of a target protein. Examples of suitable host cells that can be used in the present invention 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] The present invention provides an antibody, a conjugate, a nucleic acid molecule, a vector, a cell or host, a reagent or kit, and uses thereof, a method for preparing the conjugate, a method for immunoassay, and a method for improving the activity, stability, or sensitivity of the conjugate, each of which is described in detail below.

[0041] Conjugate

[0042] In a first aspect of the present invention, the present invention provides a conjugate. According to an embodiment of the present invention, the conjugate comprises an antibody and at least one conjugated moiety, wherein:

[0043] a) the antibody comprises a heavy chain constant region;

[0044] The heavy chain constant region comprises an engineered lysine residue;

[0045] The engineered lysine residues include or are selected from at least one of the following sites or their equivalent sites:

[0046] 1, 3, 5, 14, 31, 58, 59, 60, 61, 79, 81, 116, 141, 144, 165, 169, 190, 192, 199, 208, 209, 213, 218, 219, 220, 224, 231, 232, 233, 236, 238, 249, 255, 260, 266, 267, 271, 273, 274, 275, 279, 290, 295, 309, 313, 315, 317, 319, 320, 323, 325, 331 and 332;

[0047] b) The conjugate portion is connected to the antibody. The conjugate of the present invention has the advantages of high activity, high sensitivity or strong stability.

[0048] In some optional embodiments of the present invention, the “at least one” is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, ... or 52. In some optional embodiments of the present invention, the “at least one” is 1, 2, 3, 4 or 5.

[0049] As used herein, the term "antibody" is used in the broadest sense, including full-length antibodies or fragments thereof, such as full-length monoclonal antibodies, multispecific antibodies, and chimeric antibodies, etc., without limitation to the specific structure. A "full-length antibody" is composed of at least two identical light chains and at least two identical heavy chains connected by interchain disulfide bonds, such as immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), immunoglobulin D (IgD) or immunoglobulin E (IgE).

[0050] The heavy chain (H chain) and light chain (L chain) of an antibody can be linked, for example, by disulfide bonds. The amino acid sequence at the amino terminus (N-terminus) of the peptide chain varies greatly and is called the variable region (V region); the carboxyl terminus (C-terminus) is relatively stable and varies little, and is called the constant region (C region). The V regions of the L and H chains are called the light chain variable region (VL) and heavy chain variable region (VH), respectively, while the C regions of the L and H chains are called the light chain constant region (CL) and heavy chain constant region (CH), respectively.

[0051] As used herein, the heavy chain constant region includes the full-length heavy chain constant region or fragments thereof. The full-length heavy chain constant region refers to the heavy chain constant region of a full-length antibody. For example, for a mouse IgG, the full-length heavy chain constant region comprises, from N-terminus to C-terminus, CH1, hinge region, CH2, and CH3. A full-length heavy chain constant region fragment refers to a fragment of a contiguous amino acid sequence taken from the full-length heavy chain constant region, such as at least one of CH1, hinge region, CH2, or CH3. Similarly, the light chain constant region is also applicable.

[0052] In this article, the division of variable region, constant region, CH1, CH2, and CH3 sequences refers to the IMGT division method, see Lefranc, and Martinez-Jean C. and Bosc N. or Ehrenmann,Patrice Duroux,Chantal Ginestoux,Gene table:house mouse(Mus musculus)IGHC,IMGT Repertoire. the international ImMunoGenetics information http: / / www.imgt.org.Created:16 / 03 / 2011.Version:17 / 01 / 2020.or Ehrenmann,Patrice Duroux,Chantal Ginestoux,Gene table:house mouse(Mus musculus)IGLC,IMGT Repertoire. the international ImMunoGenetics information http: / / www.imgt.org.Created:16 / 03 / 2011.Version:17 / 01 / 2020.

[0053] It should be noted that the engineered lysine residues are obtained by sequentially numbering the full-length heavy chain constant region (including CH1-hinge region-CH2-CH3) sequence from N-terminus to C-terminus, wherein the full-length heavy chain constant region (including CH1-hinge region-CH2-CH3) sequence is obtained by referring to the above-mentioned IMGT division method.

[0054] As used herein, the term "amino acid residue" refers to an amino acid located within an antibody. Two amino acids are linked by an amide bond, resulting in the removal of an -OH group from the -COOH group of an amino acid and / or the removal of a H group from the -NH2 group of an amino acid. Examples include "lysine residue," "natural amino acid residue," or "engineered lysine residue."

[0055] As used herein, the term "natural amino acid residue" refers to an amino acid residue present in the constant region of a wild-type antibody (e.g., a wild-type murine IgG1 antibody). For example, if the first amino acid in the heavy chain constant region of a wild-type murine IgG1 antibody is A (referred to as A1 based on its position), and it is mutated to K (or Q), then A (or A1) is a natural amino acid residue, and the mutated amino acid K (or Q) is a non-natural amino acid residue.

[0056] Herein, the term "engineered lysine residue" refers to a natural amino acid residue at any position mutated to lysine (K), and the lysine obtained after the mutation is an engineered lysine residue.

[0057] As used herein, the term "equivalent site" refers to a site where the position and amino acid type of a particular site differ due to differences in antibody origin (e.g., antibodies derived from different species or genera), species (e.g., IgG, IgM, IgA, etc.), subclass (e.g., IgG can be divided into subclasses such as IgG1 and IgG2), or the presence of alleles. The equivalent site of a particular site can be determined by sequence and structural homology comparison.

[0058] As used herein, the terms "identity," "homology," or "similarity" are used to describe an amino acid sequence relative to a reference sequence, and the percentage of identical amino acids between two amino acid sequences is determined by conventional methods, for example, see Ausubel et al., eds. (1995), Current Protocols in Molecular Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York); and the ALIGN program (Dayhoff (1978), Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research Foundation, Washington, DC). There are many algorithms for aligning sequences and determining sequence identity, including the homology alignment algorithm of Needleman et al. (1970) J. Mol. Biol. 48:443; the local homology algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482; the similarity search method of Pearson et al. (1988) Proc. Natl. Acad. Sci. 85:2444; the Smith-Waterman algorithm (Meth. Mol. Biol. 10:106); the similarity search method of Pearson et al. (1988) Proc. Natl. Acad. Sci. 10:116; the similarity search method of Pearson et al. (1990) Proc. Natl. Acad. Sci. 10:117; the similarity search method of Pearson et al. (1990 ... .70:173-187 (1997); and BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al. (1990) J. Mol. Biol. 215:403-410). Computer programs that utilize 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., supra, FASTA, and TFASTA, available in the Genetics Computing Group (GCG) package, Version 8, Madison, Wisconsin, USA; and CLUSTAL in the PC / Gene program provided by Intelligenetics, Mountain View, California.

[0059] In some optional embodiments of the present invention, the conjugate may further include at least one of the following technical features:

[0060] In some optional embodiments of the present invention, the natural amino acid residue mutated into the engineered lysine residue includes or is selected from at least one of the following sites or their equivalents:

[0061] A1, T3, P5, G14, G31, Q58, S59, G60, L61, T79, T81, S116, M141, L / S144, S165, N169, R190, V192, R199, P208, I209, N213, G218, R219, P220, Q224, L231, V23 2. R233, Q236, D238, R249, T255, G260, E266, N267, T271, N273, G274, H275, N279, Q290, E295, T309, S313, N315, R317, S319, H320, N323, Y325, S331 and R332.

[0062] In some optional embodiments of the present invention, the natural amino acid residue mutated into the engineered lysine residue includes or is selected from at least one of the following positions or their equivalents:

[0063] T3, P5, G14, L61, T79, T81, S116, S144, R199, P208, I209, G218, R219, Q224, D238, T255, E266, N267, Q290, E295, S319, H320;

[0064] Among them, T3, P5, G14, L61, T79, T81, S116, S144, R199, P208, I209, G218, R219, Q224, D238, T255, E266, N267, Q290, E295, S319, and H320 are numbered according to their linear positions along the full length of the heavy chain constant region of mouse wild-type IgG1.

[0065] In some optional embodiments of the present invention, the natural amino acid residue mutated into the engineered lysine residue includes or is selected from at least one of the following positions or their equivalents:

[0066] T3 / P5, G14, L61, T79 / T81, S116, S144, R199, P208, I209, G218, R219, Q224, D238, T255, E266, N267, Q290, E295, S319, H320, T79 / G218 / D23 8 / E266 / E295, G218 / D238 / E266 / E295, I209 / G218 / D238 / E266 / E295, L61 / T79 / D238 / E266 / E295, G218 / D238 / E266, D238 / E266, G218 / D238.

[0067] Herein, "T3 / P5" refers to the mutation of the natural amino acid residues at the two sites T3 and P5 into lysine residues to obtain engineered lysine residues.

[0068] Herein, engineered lysine residues may be represented by numbers (numbered positions) or by numbers + K. For example, the position of the engineered lysine residue is selected from 3, i.e., 3K; the position of the engineered lysine residue is selected from 5, i.e., 5K; and the same applies to the positions of other engineered lysine residues.

[0069] Herein, the site of the native amino acid residue that mutates into the engineered lysine residue corresponds to the site of the engineered lysine residue. For example, when the native amino acid residue that mutates into the engineered lysine residue is T3, the site of the engineered lysine residue is T3K; when the native amino acid residue that mutates into the engineered lysine residue is P5, the site of the engineered lysine residue is P5K.

[0070] In some optional embodiments of the present invention, the natural amino acid residue mutated into the engineered lysine residue includes or is selected from at least one of the following positions or their equivalents:

[0071] A1, G31, Q58, S59, G60, M141, L144, S165, N169, R190, V192, N213, P220, L231, V232, R233, Q236, R249, G260, T271, N273, G274, H275, N279, T309, S313, N315, R317, N323, Y325, S33, and R332;

[0072] Among them, A1, G31, Q58, S59, G60, M141, L144, S165, N169, R190, V192, N213, P220, L231, V232, R233, Q236, R249, G260, T271, N273, G274, H275, N279, T309, S313, N315, R317, N323, Y325, S331 and R332 are numbered according to their linear positions along the full length of the murine wild-type IgG2b heavy chain constant region.

[0073] In some optional embodiments of the present invention, the natural amino acid residue mutated into the engineered lysine residue includes or is selected from at least one of the following positions or their equivalents:

[0074] A1, T3 / P5, G31, Q58, S59, G60, T79 / T81, M141, L144, S165, N169, R190, V192, N213, P220, L231, V232, R233, Q236, R249, G260, T271, N273, G27 4. H275, N279, T309, S313, N315, R317, N323, Y325, S331, R332, N169 / V 232 / N279, V232 / G274 / N279, N169 / V232 / G274, N169 / N279 and N169 / G274.

[0075] In some optional embodiments of the present invention, the mouse is selected from mice (Mus musculus).

[0076] As used herein, the term "wild type" refers to an antibody type that exists naturally and has not been modified by mutations. It should be understood that the engineered lysine residues in the heavy chain constant region of the antibodies of the present invention are all based on the wild-type heavy chain constant region as a positioning benchmark, which does not mean that the antibody must be wild-type. When the antibody is wild-type, the engineered lysine residues of the antibody are the same as the engineered lysine residues defined by the wild-type heavy chain constant region. However, for antibodies with, for example, insertion or deletion mutations, those skilled in the art can determine the sites of the engineered lysine residues based on the results of homology comparisons with the wild-type heavy chain constant region. It will be understood by those skilled in the art that engineered lysine residues obtained after the above-mentioned homology comparison are also within the scope of protection of the present invention.

[0077] As used herein, the term "IgHG1" refers to the heavy chain of an IgG1 antibody; the term "IgHG2a" refers to the heavy chain of an IgG2a antibody; the term "IgHG2b" refers to the heavy chain of an IgG2b antibody; and the term "IgHG" refers to the heavy chain of an IgG antibody.

[0078] Herein, the heavy chain constant region of murine wild-type IgG1 is shown in SEQ ID NO: 1, and the heavy chain constant region of murine wild-type IgG2b is shown in SEQ ID NO: 2.

[0079] As is known to those skilled in the art, the various portions of the heavy chain constant region are arranged sequentially from the N-terminus to the C-terminus, forming a linear relationship. As used herein, the term "linear position" refers to the linear position of the heavy chain constant region (including CH1, hinge region, CH2, and CH3) arranged sequentially from the N-terminus to the C-terminus; that is, the heavy chain constant region (including CH1, hinge region, CH2, and CH3) is numbered sequentially from the N-terminus to the C-terminus.

[0080] Those skilled in the art will understand that different numbering systems can be used to number amino acid residue positions in an antibody sequence, and conversion between different numbering systems is well known in the art. The positions of "natural amino acid residues mutated into engineered lysine residues" or "engineered lysine" described in the present invention are numbered sequentially from N-terminus to C-terminus based on the amino acid sequence of the heavy chain constant region of a murine wild-type IgG1 antibody (e.g., SEQ ID NO: 1) and / or the heavy chain constant region of a murine wild-type IgG2b antibody (e.g., SEQ ID NO: 2), but the same or similar positions numbered using other numbering systems also fall within the scope of protection of the present invention.

[0081] For example, for the engineered lysine residue sites of "T3 (T3K)" or "P5 (P5K)" mentioned in the present invention, those skilled in the art can perform homology comparison of the heavy chain constant region of murine wild-type IgG2a, IgG2b, IgG2c, and IgG3 antibodies with the heavy chain constant region of murine wild-type IgG1 antibodies to obtain the engineered lysine residue sites of murine wild-type IgG2a, IgG2b, IgG2c, and IgG3 antibodies, for example, by performing homology comparison of the heavy chain constant region of murine wild-type IgG2a, IgG2b, IgG2c, and IgG3 with the heavy chain constant region of murine wild-type IgG1. Therefore, conjugates containing murine IgG2a, IgG2b, IgG2c, and IgG3 antibodies or antibodies of other origins, wherein the antibodies contain the engineered lysine residue sites of the present invention, also fall within the scope of protection of the present invention.

[0082] In some optional embodiments of the present invention, the heavy chain constant region is obtained by modifying the heavy chain constant region before modification with the engineered lysine residues.

[0083] In some optional embodiments of the present invention, the heavy chain constant region before modification includes or is selected from the heavy chain constant region of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD.

[0084] In some optional embodiments of the present invention, the heavy chain constant region before modification includes, from N-terminus to C-terminus, CH1 of IgG, hinge region of IgG, CH2 of IgM, CH3 of IgM and CH4 of IgM.

[0085] In some optional embodiments of the present invention, the heavy chain constant region before modification includes, from N-terminus to C-terminus, CH1 of IgG, hinge region of IgG, CH2 of IgG and CH3 of IgG.

[0086] In some optional embodiments of the present invention, the heavy chain constant region before modification is derived from mouse antibody, human antibody, primate antibody, bovine antibody, equine antibody, bovine antibody, porcine antibody, sheep antibody, goat antibody, dog antibody, cat antibody, rabbit antibody, camel antibody, donkey antibody, deer antibody, mink antibody, chicken antibody, duck antibody, goose antibody, turkey antibody or fighting cock antibody.

[0087] In some optional embodiments of the present invention, the heavy chain constant region before modification is selected from the heavy chain constant region of IgG1, IgHG2a, IgHG2b, IgHG2c or IgHG3 derived from murine antibodies.

[0088] Herein, the term "heavy chain constant region before modification" refers to the heavy chain constant region before the lysine engineering modification described in the present invention. The heavy chain constant region before modification may be a wild-type heavy chain constant region or a non-wild-type heavy chain constant region.

[0089] It should be noted that, in this article, the “heavy chain constant region” that has not been modified “before transformation” refers to the “heavy chain constant region after transformation”, that is, the heavy chain variable region including the engineered lysine residues.

[0090] In some optional embodiments of the present invention, the heavy chain constant region has an amino acid sequence as shown in any one of SEQ ID NOs: 3 to 53, SEQ ID NOs: 114 to 125 or SEQ ID NO: 138, or a fragment thereof, or an amino acid sequence or a fragment thereof that is at least 80% identical to the amino acid sequence shown in any one of SEQ ID NOs: 3 to 53, SEQ ID NOs: 114 to 125 or SEQ ID NO: 138.

[0091] In some optional embodiments of the present invention, the heavy chain constant region has an amino acid sequence as shown in any one of SEQ ID NOs: 3 to 53, SEQ ID NOs: 114 to 125, or SEQ ID NO: 138.

[0092] In some optional embodiments of the present invention, the heavy chain constant region has a fragment of the amino acid sequence shown in any one of SEQ ID NOs: 3 to 53, SEQ ID NOs: 114 to 125, or SEQ ID NO: 138.

[0093] In some optional embodiments of the present invention, the heavy chain constant region has an amino acid sequence that is at least 80% identical to the amino acid sequence shown in any one of SEQ ID NOs: 3-53, SEQ ID NOs: 114-125, or SEQ ID NO: 138.

[0094] In some optional embodiments of the present invention, the heavy chain constant region has a fragment of an amino acid sequence that is at least 80% identical to the amino acid sequence shown in any one of SEQ ID NOs: 3-53, SEQ ID NOs: 114-125, or SEQ ID NO: 138.

[0095] In some optional embodiments of the present invention, the antibody further comprises a light chain constant region.

[0096] In some optional embodiments of the present invention, the light chain constant region includes or is selected from a kappa-type or lambda-type light chain constant region.

[0097] In some optional embodiments of the present invention, the light chain constant region is derived from a mouse antibody, a human antibody, a primate antibody, a bovine antibody, a horse antibody, a cow antibody, a porcine antibody, a sheep antibody, a goat antibody, a dog antibody, a cat antibody, a rabbit antibody, a camel antibody, a donkey antibody, a deer antibody, a mink antibody, a chicken antibody, a duck antibody, a goose antibody, a turkey antibody or a fighting cock antibody.

[0098] In some optional embodiments of the present invention, the light chain constant region has an amino acid sequence as shown in SEQ ID NO: 105 or a fragment thereof, or an amino acid sequence or a fragment thereof that is at least 80% identical to the amino acid sequence as shown in SEQ ID NO: 105.

[0099] In some optional embodiments of the present invention, the light chain constant region has a fragment of the amino acid sequence shown in SEQ ID NO:105.

[0100] In some optional embodiments of the present invention, the light chain constant region has a fragment of an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO:105.

[0101] In some optional embodiments of the present invention, the antibody has an amino acid sequence as shown in any one of SEQ ID NOs: 3 to 53, SEQ ID NOs: 114 to 125, or SEQ ID NO: 138, or a heavy chain constant region having at least 80% identity thereto, and a light chain constant region having an amino acid sequence as shown in SEQ ID NO: 105, or at least 80% identity thereto.

[0102] Optionally, the fragment is selected from CH1, hinge region, CH2, CH3 or a combination thereof.

[0103] In some optional embodiments of the present invention, the antibody further comprises a heavy chain variable region.

[0104] In some optional embodiments of the present invention, the C-terminus of the heavy chain variable region is connected to the N-terminus of the heavy chain constant region.

[0105] In some optional embodiments of the present invention, the antibody further comprises a light chain variable region.

[0106] In some optional embodiments of the present invention, the C-terminus of the light chain variable region is connected to the N-terminus of the light chain constant region.

[0107] In some optional embodiments of the present invention, the heavy chain variable region and the light chain variable region are connected, for example, by an interchain disulfide bond, or by a knob-into-hole. As long as they are connected, the specific connection method is not limited and is within the scope of protection of the present invention.

[0108] Illustratively, the heavy chain variable region and the light chain variable region are linked by an interchain disulfide bond.

[0109] Herein, the "heavy chain variable region" and "light chain variable region" of the present invention can be determined according to the target antigen. As long as they can bind to the target antigen, the specific sequence is not limited and is within the scope of protection of the present invention.

[0110] In some optional embodiments of the present invention, the target antigen is an antigen associated with infectious diseases, endocrine, tumors or drugs.

[0111] In some optional embodiments of the present invention, the antigen is an antigen related to a viral or bacterial infectious disease.

[0112] In some optional embodiments of the present invention, the antigens include but are not limited to HIV antigens, hepatitis A virus antigens, hepatitis B virus antigens, hepatitis C virus antigens, hepatitis D virus antigens, hepatitis E virus antigens, hepatitis G virus antigens, rubella virus antigens, human cytomegalovirus antigens, herpes simplex virus type 1 antigens, herpes simplex virus type 2 antigens, rabies virus antigens, human T-lymphoblastic leukemia virus antigens, dengue virus antigens, coronavirus antigens, human papillomavirus antigens, West Nile virus antigens, forest encephalitis virus Antigen, measles virus antigen, influenza virus antigen, parainfluenza virus antigen, varicella virus antigen, echovirus antigen, coxsackie virus antigen, Japanese encephalitis virus antigen, coxsackie virus antigen, Epstein-Barr virus antigen, mumps virus antigen, Treponema pallidum antigen, Borrelia burgdorferi antigen, Chlamydia trachomatis antigen, Chlamydia pneumoniae antigen, Chlamydia psittaci antigen, Ureaplasma urealyticum antigen, Mycoplasma pneumoniae antigen, Mycobacterium tuberculosis antigen, Helicobacter pylori antigen, Neisseria gonorrhoeae antigen, Plasmodium antigen, Trypanosoma cruzi antigen, Toxoplasma gondii antigen, CA153. Exemplarily, the antigen is CA153, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 108 or 109, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 110 or 111.

[0113] In some optional embodiments of the present invention, the antibody includes a heavy chain having an amino acid sequence as shown in any one of SEQ ID NOs: 54 to 104, SEQ ID NOs: 126 to 137, or SEQ ID NO: 140, or an amino acid sequence selected from an amino acid sequence having at least 80% identity with the amino acid sequence shown in any one of SEQ ID NOs: 54 to 104, SEQ ID NOs: 126 to 137, or SEQ ID NO: 140.

[0114] In some optional embodiments of the present invention, the antibody includes a light chain having an amino acid sequence as shown in any one of SEQ ID NOs: 106 to 107, or an amino acid sequence selected from an amino acid sequence having at least 80% identity with the amino acid sequence shown in any one of SEQ ID NOs: 106 to 107.

[0115] In some alternative embodiments of the present invention, the antibody has a light chain and a heavy chain selected from any of the following groups or having at least 80% identity thereto:

[0116] In some optional embodiments of the present invention, the antibody includes or is selected from at least one of a full-length antibody, a Fab antibody, a Fab' antibody, a F(ab')2 antibody, a F(ab)2 antibody, a constant region, Fc, CH1, CH2, CH3, a hinge region, an Fv antibody, a scFv fragment, a scFv-Fc fusion protein, a scFv-Fv fusion protein, a single-chain antibody, a single-domain antibody, and a minimum recognition unit; for example, it can be selected from Fc (including CH2 and CH3).

[0117] As used herein, the term "Fab antibody" or "Fab fragment" generally refers to an antibody or fragment containing only the Fab molecule, which is composed of the VH and CH1 of the heavy chain and a complete light chain, with the light chain and heavy chain connected by a disulfide bond.

[0118] As used herein, the term "F(ab')2 antibody" or "F(ab')2 fragment" has two antigen-binding F(ab') portions linked together by a disulfide bond.

[0119] As used herein, the term "Fv antibody" or "Fv fragment" generally refers to an antibody or fragment consisting solely of a light chain variable region (VL) and a heavy chain variable region (VH) linked by non-covalent bonds. It is the smallest functional fragment of an antibody that retains a complete antigen-binding site.

[0120] As used herein, the terms "single-chain antibody" and "scFv fragment" refer to antibodies or fragments formed by connecting the heavy chain variable region and the light chain variable region of an antibody via a short peptide.

[0121] In this article, the terms "minimum recognition unit" and "MRU" both refer to antibodies or fragments consisting of only one CDR, which has a very small molecular weight of only about 1% of the complete antibody.

[0122] In some optional embodiments of the present invention, the antibody is a full-length antibody or a fragment thereof having antigen-binding activity. The antibody CDRs in the present invention can be adjusted according to the type of antigen, without specific limitation.

[0123] In some optional embodiments of the present invention, the conjugate moiety is linked to the active group of the antibody.

[0124] In some optional embodiments of the present invention, the active group includes at least one of an amino group, an imino group, a thiol group, a hydroxyl group, and a carboxyl group.

[0125] In some optional embodiments of the present invention, the conjugate moiety is linked to an amino group of the antibody, preferably a primary amino group.

[0126] In some optional embodiments of the present invention, the conjugated moiety is selected from at least one of a carrier, an affinity substance (eg, one of streptavidin and biotin, one of mutually complementary sense and antisense nucleic acids), and a tag.

[0127] In some optional embodiments of the present invention, the carrier includes at least one of a solid phase carrier, a labeling carrier, a fluorescent substance, a luminescent substance, a radioactive isotope, a gold colloid and a colored substance.

[0128] Herein, the carrier can be a substance that can be suspended or dispersed in a liquid phase (for example, solid phase carriers such as particles and magnetic beads), or a solid phase that can accommodate or carry a liquid phase (for example, supports such as plates, membranes, test tubes, and containers such as well plates, microfluidics, glass capillaries, nanocolumns, and monolithic columns); it can also be a labeling carrier for labeling antibodies, such as an enzyme (for example, peroxidase, alkaline phosphatase, luciferase (luciferin), β-galactosidase), a fluorescent substance (for example, fluorescein, fluorescein isothiocyanate, rhodamine, green fluorescent protein, red fluorescent protein), a luminescent substance (for example, luciferin, aequorin, acridinium ester, tris (2,2'-bipyridine) ruthenium, luminol), a radioactive isotope (for example, 3 H. 14 C. 32 P. 35 S. 125 I) and gold colloid, etc.

[0129] In some optional embodiments of the present invention, the affinity substance includes biotin / avidin.

[0130] In some optional embodiments of the present invention, the tag includes at least one of a His tag, a Flag tag, a GST tag, an MBP tag, a SUMO tag, and a C-Myc tag.

[0131] In some optional embodiments of the present invention, the conjugated moiety is selected from one or more of acridinium ester, rhodamine and its derivatives, luciferase, luciferin, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, carbohydrate oxidase, glucose oxidase, galactose oxidase and glucose-6-phosphate dehydrogenase.

[0132] Antibody

[0133] In a second aspect, the present invention provides an antibody. According to an embodiment of the present invention, the antibody is defined as the antibody in the conjugate described in the first aspect of the present invention. The antibody of the present invention has advantages such as strong stability or high activity, and the conjugate prepared using the antibody has advantages such as strong stability, high activity, or high sensitivity.

[0134] The specific embodiments of the antibody described in the second aspect are described in the first aspect of the present invention and will not be repeated here.

[0135] It should be noted that, based on the amino acid sequence of the antibody of the present invention, those skilled in the art can easily conceive of using genetic engineering technology or other technologies (chemical synthesis, recombinant expression) to prepare the antibody. For example, the antibody can be isolated and purified from the culture product of recombinant cells that can recombinantly express the antibody as described in any of the above items. This is easy for those skilled in the art to achieve. Based on this, regardless of the technology used to prepare the antibody of the present invention, it falls within the scope of protection of the present disclosure.

[0136] Methods for preparing conjugates

[0137] In its third aspect, the present invention provides a method for preparing a conjugate. According to an embodiment of the present invention, the method comprises: performing a conjugation reaction between the antibody in the conjugate described in the first aspect or the antibody described in the second aspect and a conjugation moiety to obtain the conjugate. This method allows for efficient and simple preparation of the conjugate.

[0138] Nucleic acid molecules, vectors, cells or hosts

[0139] In the process of preparing or obtaining the antibody in the conjugate described in the first aspect or the antibody described in the second aspect, nucleic acid molecules expressing these antibodies can be connected to different vectors and then expressed in different cells to obtain the corresponding antibodies.

[0140] In a fourth aspect, the present invention provides a nucleic acid molecule. According to an embodiment of the present invention, the nucleic acid molecule encodes the antibody in the conjugate described in the first aspect or the antibody described in the second aspect. The nucleic acid molecule according to an embodiment of the present invention can encode the above-mentioned antibody.

[0141] According to an embodiment of the present invention, the nucleic acid molecule includes DNA or RNA.

[0142] It should be noted that, for nucleic acid molecules mentioned herein, those skilled in the art will understand that they actually include any one or both of the complementary double strands. For convenience, although only one strand is provided in most cases herein, the other strand complementary thereto is also disclosed. In addition, the molecular sequences in the present invention include DNA or RNA forms, and disclosure of one of them implies that the other is also disclosed.

[0143] In the fifth aspect of the present invention, the present invention proposes a vector. According to an embodiment of the present invention, the vector includes the nucleic acid molecule described in the fourth aspect. When the above-mentioned nucleic acid molecule is connected to the vector, the nucleic acid molecule can be directly or indirectly connected to the control elements on the vector, as long as these control elements can control the translation and expression of the nucleic acid molecule. Of course, these control elements can come directly from the vector itself, or they can be exogenous, that is, not from the vector itself. Of course, the nucleic acid molecule and the control elements can be operably connected. In this article, "operably connected" means that the exogenous gene is connected to the vector so that the control elements in the vector, such as transcription control sequences and translation control sequences, can play their expected function of regulating the transcription and translation of the exogenous gene. Commonly used vectors can be, for example, plasmids, phages, etc. After the vectors according to some specific embodiments of the present invention are introduced into suitable recipient cells, the expression of the aforementioned antibodies can be effectively achieved under the mediation of the regulatory system, thereby achieving large-scale in vitro acquisition of antibodies.

[0144] In some specific embodiments of the present invention, the vector is a eukaryotic expression vector, a prokaryotic expression vector, a virus or a bacteriophage.

[0145] In an optional embodiment of the present invention, the expression vector is a plasmid expression vector or a lentiviral expression vector.

[0146] In its sixth aspect, the present invention provides a cell or host. According to embodiments of the present invention, the cell or host comprises: the nucleic acid molecule described in the fourth aspect or the vector described in the fifth aspect; or expresses the antibody in the conjugate described in the first aspect or the antibody described in the second aspect. Under suitable conditions, the cell or host can effectively express the aforementioned antibodies within the cell.

[0147] According to an embodiment of the present invention, the cell is obtained by introducing the vector described in the fifth aspect into the cell.

[0148] It should be noted that the cells of the present invention are not particularly limited and may be prokaryotic cells, eukaryotic cells, or bacteriophages. The prokaryotic cells may be Escherichia coli, Bacillus subtilis, Streptomyces, or Proteus mirabilis. The eukaryotic cells include fungi such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Trichoderma, insect cells such as S. frugiperda, plant cells such as tobacco, and mammalian cells such as BHK cells, CHO cells, COS cells, and myeloma cells.

[0149] In an optional embodiment of the present invention, the cells are mammalian cells, including BHK cells, CHO cells, NSO cells or COS cells, and do not include animal germ cells, fertilized eggs or embryonic stem cells.

[0150] It should be noted that the "suitable conditions" described in the present invention refer to conditions suitable for the expression of the antibodies of the present invention. It will be readily understood by those skilled in the art that conditions suitable for the expression of the antibodies include, but are not limited to, suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy cell status, suitable cell density, suitable cell culture environment, and suitable cell culture time. "Suitable conditions" are not particularly limited, and those skilled in the art can optimize the most suitable conditions for the expression of the antibodies according to the specific environment of the laboratory.

[0151] Reagent or kit, use

[0152] In the seventh aspect of the present invention, the present invention proposes a use of the antibody described in the second aspect, the nucleic acid molecule described in the fourth aspect, the vector described in the fifth aspect, or the cell or host described in the sixth aspect in preparing a conjugate or a kit.

[0153] In an eighth aspect, the present invention provides a reagent or kit. According to an embodiment of the present invention, the reagent or kit comprises: the conjugate described in the first aspect or the antibody described in the second aspect. The reagent or kit of the present invention has advantages such as high activity and strong sensitivity, and can be used for immunoassays.

[0154] In the ninth aspect of the present invention, the present invention proposes a use of the conjugate described in the first aspect, the antibody described in the second aspect, the nucleic acid molecule described in the fourth aspect, the vector described in the fifth aspect, the cell or host described in the sixth aspect, or the reagent or kit described in the eighth aspect in immunoassay or preparation of immunoassay products.

[0155] Immunoassay method and method for improving conjugate activity, stability or sensitivity

[0156] In a tenth aspect, the present invention provides an immunoassay method. According to an embodiment of the invention, the method comprises: contacting the conjugate described in the first aspect or the antibody described in the second aspect with a sample to be detected to form an immune complex.

[0157] In some optional embodiments of the present invention, whether the sample to be tested contains the target substance or the content of the target substance is determined based on the signal of the immune complex.

[0158] It should be noted that the above-mentioned "samples to be tested" may be samples to be tested from patients, such as serum samples; or they may be non-patient samples. For example, in scientific research, the above-mentioned method is only used to detect the presence or content of antigens in the sample, and does not involve disease diagnosis.

[0159] In some optional embodiments of the present invention, the signal comprises a fluorescent signal.

[0160] In the eleventh aspect of the present invention, the present invention provides a method for improving the activity, stability or sensitivity of a conjugate, wherein the conjugate comprises an antibody and at least one conjugated portion, wherein the conjugated portion is connected to the antibody, and the method comprises: introducing an engineered lysine residue into the heavy chain constant region of the antibody, wherein the engineered lysine residue is consistent with the engineered lysine residue defined in the conjugate described in the first aspect.

[0161] Below, the scheme of the present invention will be explained in conjunction with embodiment.It will be understood by those skilled in the art that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.In the embodiment, if specific technology or conditions are not indicated, the technology or conditions described in the literature in this area or the product instructions are used.The reagents or instruments used are not indicated by the manufacturer, and are all conventional products that can be obtained by commercial purchase.

[0162] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the preparations or unit doses herein, some methods and materials are now described. Unless otherwise indicated, the techniques employed or contemplated herein are standard methods. Materials, methods, and examples are illustrative and non-limiting only.

[0163] Practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of a skilled artisan. The technique is fully explained in the literature, for example, in Molecular Cloning: A Laboratory Manual, 2nd ed. (Sambrook et al., 1989); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Animal Cell Culture (RI Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FM Ausubel et al., eds., 1987); and PCR: The Polymerase Chain Reaction. Reaction" (Mullis et al., eds., 1994); and Current Protocols in Immunology (J.E. Colligan et al., eds., 2011), each of which is expressly incorporated herein by reference.

[0164] Example 1:

[0165] 1. Selection of mutation points

[0166] By studying the sequence and structure of mouse antibodies, different natural amino acid residues in the heavy chain constant region of mouse IgG1 and mouse IgG2b were mutated to lysine residues to obtain different engineered lysine residues. Among them, 19 mutation sites were designed for IgG1 (the mutation sites are numbered in the linear order of the heavy chain constant region SEQ ID NO: 1), resulting in 19 mutation groups, as shown in Table 1; 32 mutation sites were designed for IgG2b (the mutation sites are numbered in the linear order of the heavy chain constant region SEQ ID NO: 2), resulting in 32 mutation groups, as shown in Table 2.

[0167] Table 1: Mutation sites of IgG1

[0168] Table 2: Mutation sites of IgG2b

[0169] 2. Plasmid construction and expression

[0170] 2.1 Construction of recombinant antibody expression plasmid

[0171] In this example, restriction endonucleases and Prime Star DNA polymerase were purchased from Takara. 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. Primer synthesis and gene sequencing were performed by a gene sequencing company. TM 3.4 Vector is a constructed eukaryotic expression vector for recombinant antibodies. This expression vector has been modified to introduce multiple cloning restriction sites and is subsequently referred to as the 3.4A expression vector.

[0172] 2.1. Anti-CA153 Antibody Gene Preparation

[0173] Antibody mRNA was extracted from hybridoma cell lines secreting anti-CA153 monoclonal antibodies. DNA products were obtained by RT-PCR and inserted into the pMD-18T vector. The cells were transformed into DH5α competent cells. After colonies were grown, four positive clones of the heavy chain and four positive clones of the light chain genes were sent to a gene sequencing company for sequencing.

[0174] 2.2 Sequence Analysis of Anti-CA153 Antibody Variable Region Genes

[0175] The gene sequences obtained by the above sequencing were placed in the KABAT antibody database for analysis, and VNTI11.5 software was used for analysis to confirm that the genes amplified by the heavy chain and light chain primer pairs were correct.

[0176] 2.3 Construction of recombinant antibody expression plasmid

[0177] pcDNA TM 3.4 Vector is a recombinant antibody eukaryotic expression vector constructed. The expression vector has been modified to introduce multiple cloning restriction enzyme sites and is subsequently referred to as the 3.4A expression vector. Based on the sequencing results of the antibody variable region gene in the above-mentioned pMD-18T, the VL and VH gene-specific primers and mutant primers of the Anti-CA153 antibody were designed, with restriction endonuclease restriction sites and protective bases at both ends, and the Light Chain gene fragment and the Heavy Chain gene fragment were amplified by PCR amplification. The Heavy Chain and Light Chain gene fragments were double-digested with restriction endonucleases, and the 3.4A expression vector was double-digested with restriction endonucleases. After the fragments and vectors were purified and recovered, the Heavy Chain gene and the Light Chain gene were connected to the 3.4A expression vector respectively to obtain the recombinant expression plasmids of the Heavy Chain and Light Chain, respectively. Among them, the amino acid sequences corresponding to the Light Chain gene fragment and the Heavy Chain gene fragment are shown in Table 3

[0178] Table 3: Amino acid sequences of heavy and light chains of various antibodies

[0179] 2.4. Expression of recombinant antibody samples

[0180] Resuscitate HEK293 cells in advance and subculture them into 200 ml system to make the cell density reach 3-5×10 6 cells / ml, cell viability>95%; wash the cells by centrifugation, resolubilize with culture medium, and adjust the cell density to 2.9×10 6 cells / ml, as the cell dilution solution. Use culture medium to prepare plasmid DNA dilution solution and transfection reagent dilution solution containing the recombinant expression plasmid obtained in step 2.3 of this application respectively. Add the transfection reagent dilution solution to the plasmid DNA dilution solution, mix well and let it stand at room temperature for 15 minutes; slowly add the mixture to the cell dilution solution within 1 minute, mix well and take samples to count, record and observe the viability of the cells after transfection, and place them in a 35°C constant temperature incubator for culture. The culture conditions are a speed of 120 rpm and a CO2 content of 8%. Centrifuge and collect samples after 13 days.

[0181] 3. HPLC-SEC (High Performance Liquid Chromatography-Size Exclusion Chromatography)

[0182] 20 μg of the purified recombinant antibody samples obtained in step 2.4 of this example were taken and the SEC purity of the recombinant antibody samples was determined by high performance liquid chromatography-size exclusion chromatography (HPLC-SEC). The test results are shown in Tables 4 and 5. All mutant antibody groups were purified to obtain samples of excellent purity.

[0183] Table 4: HPLC-SEC results corresponding to IgG1 mutation to K

[0184] Table 5: HPLC-SEC results corresponding to IgG2b mutation to K

[0185] 4. Determination of thermal stability Tm value

[0186] The purified recombinant antibody samples obtained in step 2.4 of this example were diluted to 1 mg / ml, and the thermal stability Tm of the samples was measured using DSF (differential scanning fluorimetry). The test results are shown in Tables 6 and 7. The specific parameters were set as follows:

[0187] Sample volume: 400 μL;

[0188] Measuring range: 25℃~95℃;

[0189] Heating rate: 0.3°C / min.

[0190] It can be seen from Tables 6 and 7 that antibodies in each mutation group have good thermal stability.

[0191] Table 6: Tm results corresponding to IgG1 mutation points

[0192] Table 7: Tm results corresponding to IgG2b mutation points

[0193] 5. Activity identification

[0194] 5.1 Activity and specificity results after labeling AE

[0195] Marking process:

[0196] 1) Buffer exchange: The purified recombinant antibody samples (hereinafter referred to as antibodies or Ab) obtained in step 2.4 of this example were dialyzed using dialysis bags for 2 h, with the buffer exchanged every 45 min.

[0197] 2) Reaction: The antibody was placed in a centrifuge tube, and AE stock solution activated with NHS (N-hydroxysuccinimide) was added to react for 15 min. Then, Gly solution was added to terminate the reaction for 30 min to obtain a solution containing Ab-AE.

[0198] 3) Impurity Removal: After the reaction, dialyze the Ab-AE. Change the solution every 30 to 60 minutes on the first day (at least 4 times). Change the solution every 60 minutes on the second day after overnight (at least 4 times).

[0199] 4) Recovery and storage: Take out the Ab-AE raw material from the dialysis bag, add the same volume of high-pressure glycerol, mix well, and store at -20°C for a long term.

[0200] Testing process:

[0201] 1) Sample addition reaction: Take 50 μL of test sample, add 50 μL of Ab-magnetic bead working solution, add 50 μL of Ab-AE working solution, and react at 37°C for 15 min.

[0202] 2) The reaction solution was washed three times with 1*PBST.

[0203] 3) Add excitation solution A + excitation solution B to the cleaned solution and read the value immediately.

[0204] CA153 quality control products with different concentration gradients were used as test samples to detect activity.

[0205] 44 Roche certified specimens were used as test samples, and the correlation was tested and calculated.

[0206] Among them, the activity results of each group of IgG1 antibody-labeled AE (i.e., each group of IgG1 antibody-AE) obtained after the natural amino acid residue was mutated to K are shown in Table 8-1, the average deviation of the activity of each group of IgG1 antibody-AE is shown in Table 8-2, and the correlation results of each group of IgG1 antibody-AE are shown in Figure 1; the activity results of each group of IgG2b antibody-labeled AE (i.e., each group of IgG2b antibody-AE) obtained after the natural amino acid residue was mutated to K are shown in Table 9-1, the average deviation of the activity of each group of IgG2b antibody-AE is shown in Table 9-2, and the correlation results of each group of IgG2b antibody-AE are shown in Figure 2.

[0207] Deviation = (activity response value of the mutant group - activity response value of the wild type) / activity response value of the wild type;

[0208] Average deviation = total deviation / number of quality control groups.

[0209] For each group of IgG1 antibodies, except for mutation groups 10 and 12, the remaining mutation groups can improve the labeled AE activity. Exemplary results are shown in Table 8-1 and Table 8-2;

[0210] For each group of IgG2b antibodies, mutation groups 1 to 32 can improve the labeled AE activity, and exemplary results are shown in Table 9;

[0211] As can be seen from Figures 1 and 2, antibodies in each mutation group have good test correlation.

[0212] Table 8-1: Activity results of IgG1 antibody-AE in each group

[0213] Table 8-2: Average deviation of activity between IgG1 antibody-AE and wild-type IgG1 antibody-AE in each group

[0214] Table 9-1: Activity results of IgG2b antibody-AE in each group

[0215] Table 9-2: Average deviation of activity between IgG2b antibody-AE and wild-type IgG2b antibody-AE in each group

[0216] 5.2 Activity and specificity results after HRP labeling

[0217] Marking process:

[0218] 1) Buffer exchange: The purified recombinant antibody samples (hereinafter referred to as antibodies or Ab) obtained in step 2.4 of this example were dialyzed using dialysis bags for 4 hours, with the buffer exchanged every 45 minutes.

[0219] 2) HRP enzyme oxidation: 2 mg of HRP dry powder (Frdbio) was weighed and dissolved in 100 μL of ddH2O in the dark. 21 mg of NaIO4 was weighed and dissolved in 1 mL of ddH2O. 100 μL of NaIO4 solution was slowly mixed with 100 μL of HRP solution and allowed to stand at 4°C for 30 min. The solution turned green. 2 μL of ethylene glycol was slowly added to the oxidized HRP solution and allowed to stand at room temperature in the dark for 30 min. The solution turned brown.

[0220] 3) Antibody Labeling and Recovery: The oxidized HRP solution was added directly to the treated antibody solution from step 1) and allowed to react at room temperature for 2 hours. 0.4 mg of NaBH₄ was weighed and dissolved in 20 μL of ddH₂O. The mixture was added to the reaction solution from the previous step and allowed to stand at 4°C for 2 hours, shaking every 30 minutes. The solution was dialyzed overnight against PBS (pH 7.2). 30% to 50% by volume of glycerol was added to the labeling solution, mixed thoroughly, and stored at -20°C to obtain Ab-HRP.

[0221] Testing process:

[0222] 1) Sample addition reaction: Take 10 μL of test sample, add 50 μL of Ab-magnetic bead working solution, add 150 μL of Ab-HRP working solution, and react at 37°C for 15 min.

[0223] 2) The reaction solution was washed three times with 1*PBST.

[0224] 3) Add excitation solution A + excitation solution B to the cleaned solution and read the value immediately.

[0225] CA153 quality control products with different concentration gradients were used as test samples to detect activity.

[0226] Among them, the activity results of each group of IgG1 antibodies labeled with HRP (i.e., each group of IgG1 antibody-HRP) obtained after mutating the natural amino acid residue to K are shown in Table 10-1, and the average deviation of the activity of each group of IgG1 antibody-HRP is shown in Table 10-2; the activity results of each group of IgG2b antibodies labeled with HRP (i.e., each group of IgG2b antibody-HRP) obtained after mutating the natural amino acid residue to K are shown in Table 11-1, and the average deviation of the activity of each group of IgG2b antibody-HRP is shown in Table 11-2.

[0227] For each group of IgG1 antibodies, except for mutation groups 10 and 12, the remaining mutation groups can improve the activity of labeled HRP. Exemplary results are shown in Table 10.

[0228] For each group of IgG2b antibodies, mutation groups 1 to 32 can all increase the activity of labeled HRP. Exemplary results are shown in Table 11.

[0229] Table 10-1: Activity results of IgG1 antibody-HRP in each group

[0230] Table 10-2: Average deviation of activity between IgG1 antibody-HRP and wild-type IgG1 antibody-HRP in each group

[0231] Table 11-1: Activity results of IgG2b antibody-HRP in each group

[0232] Table 11-2: Average deviation of activity between IgG2b antibody-HRP and wild-type IgG2b antibody-HRP in each group

[0233] Example 2:

[0234] 2.1 Combination mutations were performed on the above-mentioned activity-enhancing mutations. Five combination mutations were designed for IgG2b, as shown in Table 12; and seven combination mutations were designed for IgG1, as shown in Table 13.

[0235] Table 12: Combined mutation sites of IgG2b

[0236] Table 13: Combined mutation sites of IgG1

[0237] The above-mentioned combined mutation samples were prepared with reference to Example 1, and the expression level, purity and thermal stability of each combined mutation were determined with reference to Example 1. The results showed that the expression level, purity and thermal stability of each combined mutation were basically consistent with those of the wild-type antibody.

[0238] 2.2 Activity and specificity results after labeling AE

[0239] The activity of labeled AE was determined as described in Example 1 above, using ascites CA153 at varying concentrations as the test sample. Activity results for the IgG2b combination mutant antibodies are shown in Table 14, and for the IgG1 combination mutant antibodies are shown in Table 15. The results showed that both IgG2b and IgG1 combination mutants exhibited enhanced labeling activity and comparable correlation to wild-type activity.

[0240] Table 14: Activity results of IgG2b combination mutant antibody-AE

[0241] Table 15: Activity results of IgG1 combination mutant antibody-AE

[0242] Example 3:

[0243] 3.1 Anti-CKMB wild-type antibody (murine IgG1) was prepared according to the method of Example 1. The heavy chain and light chain sequences of the wild-type antibody are shown in SEQ ID NO: 139 and SEQ ID NO: 141, respectively.

[0244] S319 in the heavy chain constant region of the wild-type CKMB antibody was mutated to a lysine residue to obtain the mutant antibody CKMB-mut. The heavy chain and light chain sequences of CKMB-mut are shown in SEQ ID NO: 140 and SEQ ID NO: 141, respectively.

[0245] The expression level, purity and thermal stability of CKMB-mut were determined with reference to Example 1. The results showed that the expression level, purity and thermal stability of CKMB-mut were basically consistent with those of the wild-type antibody.

[0246] 3.2 Activity test results of labeled latex microspheres

[0247] Detection method:

[0248] 1) Take 160 μl of 10% polystyrene carboxyl latex with a particle size of 188 nm and dilute to 2 ml with 10 mmol / L HEPES (pH 7.5) buffer solution;

[0249] 2) Slowly add 50 μl (10 mg / ml) EDC solution and 50 μl (10 mg / ml) NHS solution to the mixture in step 1. Stir at room temperature for 20 min, then adjust the volume to 10 ml with 10 mmol / L HEPES (pH 7.5) buffer solution.

[0250] 3) Take 70ul of denatured IgG solution, add 50ul of BSA (10%) and 50mM urea;

[0251] 4) Slowly add the denatured IgG solution diluted in step 3) to step 2, and stir at room temperature for 3 hours;

[0252] 5) Centrifuge at 15,000 rpm for 40 min, and resuspend in 10 mmol / L PBS (containing 0.2% Tween 20, 0.1% BSA, and 10% sucrose) by ultrasonication to obtain CKMB detection reagent 2.

[0253] 6) Use 50 mmol / L glycine buffer as detection reagent 1, and CKMB detection reagent 2 to form the detection reagent for this experiment;

[0254] 7) Calibrator: Prepared by diluting serum from myocardial CKMB patient specimens. Detection instrument: Mindray BS-480. Test parameters are as follows:

[0255] The activity data measured are as follows, and the results show that the mutant antibody has significantly improved activity compared to the wild type.

[0256] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0257] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A conjugate, characterized in that comprising an antibody and at least one conjugated moiety, wherein: a) the antibody comprises a heavy chain constant region; The heavy chain constant region comprises an engineered lysine residue; Wherein, the engineered lysine residue includes or is selected from at least one of the following sites or their equivalent sites: 1, 3, 5, 14, 31, 58, 59, 60, 61, 79, 81, 116, 141, 144, 165, 169, 190, 192, 199, 208, 209, 213, 218, 219, 220, 224, 231, 232, 233, 236, 238, 249, 255, 260, 266, 267, 271, 273, 274, 275, 279, 290, 295, 309, 313, 315, 317, 319, 320, 323, 325, 331, and 332; b) The conjugate moiety is linked to the antibody.

2. The conjugate according to claim 1, characterized in that The natural amino acid residue mutated into the engineered lysine residue includes or is selected from at least one of the following positions or their equivalent positions: A1, T3, P5, G14, G31, Q58, S59, G60, L61, T79, T81, S116, M141, L / S144, S165, N169, R190, V192, R199, P208, I209, N213, G218, R219, P220, Q224, L231, V23 2. R233, Q236, D238, R249, T255, G260, E266, N267, T271, N273, G274, H275, N279, Q290, E295, T309, S313, N315, R317, S319, H320, N323, Y325, S331 and R332; Optionally, the heavy chain constant region is obtained by modifying the heavy chain constant region before modification with the engineered lysine residues; Optionally, the heavy chain constant region before transformation is selected from the heavy chain constant region of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; Optionally, the heavy chain constant region before transformation is derived from mouse antibody, human antibody, primate antibody, bovine antibody, horse antibody, dairy cow antibody, porcine antibody, sheep antibody, goat antibody, dog antibody, cat antibody, rabbit antibody, camel antibody, donkey antibody, deer antibody, mink antibody, chicken antibody, duck antibody, goose antibody, turkey antibody or fighting cock antibody; Optionally, the heavy chain constant region before transformation is selected from IgG1, IgHG2a, IgHG2b, IgHG2c or IgG3 derived from mouse antibodies; Optionally, the heavy chain constant region has an amino acid sequence as shown in any one of SEQ ID NOs: 3 to 53, SEQ ID NOs: 114 to 125 or SEQ ID NO: 138, or a fragment thereof, or an amino acid sequence having at least 80% identity to an amino acid sequence as shown in any one of SEQ ID NOs: 3 to 53, SEQ ID NOs: 114 to 125 or SEQ ID NO: 138, or a fragment thereof; Optionally, the antibody further comprises a light chain constant region; Optionally, the light chain constant region includes or is selected from a κ-type or λ-type light chain constant region; Optionally, the light chain constant region is derived from a mouse antibody, a human antibody, a primate antibody, a bovine antibody, a horse antibody, a dairy cow antibody, a porcine antibody, a sheep antibody, a goat antibody, a dog antibody, a cat antibody, a rabbit antibody, a camel antibody, a donkey antibody, a deer antibody, a mink antibody, a chicken antibody, a duck antibody, a goose antibody, a turkey antibody or a fighting cock antibody; Optionally, the light chain constant region has an amino acid sequence as shown in SEQ ID NO: 105 or a fragment thereof, or an amino acid sequence having at least 80% identity with the amino acid sequence as shown in SEQ ID NO: 105 or a fragment thereof; Optionally, the fragment is selected from CH1, hinge region, CH2 or CH3; Optionally, the antibody further comprises a heavy chain variable region; Optionally, the C-terminus of the heavy chain variable region is connected to the N-terminus of the heavy chain constant region; Optionally, the antibody further comprises a light chain variable region; Optionally, the C-terminus of the light chain variable region is connected to the N-terminus of the light chain constant region; Optionally, the heavy chain variable region and the light chain variable region are linked; Optionally, the heavy chain variable region and the light chain variable region are linked by an interchain disulfide bond; Optionally, the antibody comprises a heavy chain having an amino acid sequence as shown in any one of SEQ ID NOs: 54 to 104, SEQ ID NOs: 126 to 137 or SEQ ID NO: 140, or an amino acid sequence selected from an amino acid sequence having at least 80% identity with an amino acid sequence as shown in any one of SEQ ID NOs: 54 to 104, SEQ ID NOs: 126 to 137 or SEQ ID NO: 140; Optionally, the antibody comprises a light chain having an amino acid sequence as shown in any one of SEQ ID NOs: 106, 107, 141, or an amino acid sequence selected from an amino acid sequence having at least 80% identity with the amino acid sequence as shown in any one of SEQ ID NOs: 106, 107, 141; Optionally, the antibody has a light chain and a heavy chain selected from any of the following groups or having at least 80% identity thereto: Optionally, the antibody includes or is selected from at least one of a full-length antibody, a Fab antibody, a Fab' antibody, a F(ab')2 antibody, a F(ab)2 antibody, a constant region, an Fc, a CH1, a CH2, a CH3, a hinge region, an Fv antibody, a scFv fragment, a scFv-Fc fusion protein, a scFv-Fv fusion protein, a single-chain antibody, a single-domain antibody, and a minimum recognition unit; Optionally, the conjugated moiety is attached to an active group of the antibody; Optionally, the active group includes at least one of an amino group, an imino group, a thiol group, a hydroxyl group and a carboxyl group; Optionally, the conjugate moiety is attached to an amino group of the antibody, preferably a primary amino group; Optionally, the conjugated moiety is selected from at least one of a carrier, an affinity substance and a tag; Optionally, the carrier includes at least one of a solid phase carrier, a labeling carrier, a fluorescent substance, a luminescent substance, a radioactive isotope, a gold colloid and a colored substance; Optionally, the affinity substance comprises biotin / avidin; Optionally, the tag includes at least one of a His tag, a Flag tag, a GST tag, a MBP tag, a SUMO tag and a C-Myc tag.

3. An antibody, characterized in that The antibody is the antibody defined in any one of claims 1 to 2.

4. A method for preparing a conjugate, characterized in that: include: The antibody according to claim 3 is subjected to a conjugation reaction with a conjugation moiety to obtain the conjugate.

5. A nucleic acid molecule, vector, cell or host, characterized in that: The nucleic acid molecule encodes the antibody of claim 3; The vector comprises the above-mentioned nucleic acid molecule; the cell or host comprises: the above-mentioned nucleic acid molecule or vector; or expresses the antibody described in claim 3.

6. Use of the antibody according to claim 3, the nucleic acid molecule, vector, cell or host according to claim 5 in preparing a conjugate or a kit.

7. A reagent or a kit, characterized in that: include: The conjugate according to any one of claims 1 to 2 or the antibody according to claim 3.

8. Use of the conjugate according to any one of claims 1 to 2, the antibody according to claim 3, the nucleic acid molecule, vector, cell or host according to claim 5, or the reagent or kit according to claim 7 in immunoassay or in the preparation of immunoassay products.

9. A method of immunoassay, characterized in that: include: The conjugate according to any one of claims 1 to 2 or the antibody according to claim 3 is contacted with a sample to be detected to form an immune complex; Optionally, based on the signal of the immune complex, it is determined whether the sample to be detected contains the target substance or the content of the target substance.

10. A method for improving the activity, stability or sensitivity of a conjugate, wherein the conjugate comprises an antibody and at least one conjugated portion, wherein the conjugated portion is linked to the antibody, characterized in that: include: An engineered lysine residue is introduced into the heavy chain constant region of the antibody, wherein the engineered lysine residue is consistent with the engineered lysine residue defined in the conjugate according to any one of claims 1 to 2.

Images