Antibodies and antibody conjugates and uses thereof

Abstract

The present invention proposes a conjugate comprising an antibody and a conjugation moiety; the antibody comprises a heavy chain constant region, the heavy chain constant region comprises an engineered lysine residue, and 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 and the like; a conjugation moiety is linked to the antibody. The conjugate has high activity, high sensitivity or strong stability.

Description

[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 a conjugate, a method for immunoassay, and a method for improving the activity, stability or sensitivity of a conjugate. Background Art

[0004] Conjugates of antibodies and markers have been widely used in the field of immunoassays. The activity, stability or conjugation ratio of the conjugates directly affect the sensitivity or specificity of immunoassays. Currently, the commonly used conjugation methods generally include conjugation based on cysteine ​​thiol 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 bond, thereby generating free thiol side chains. Then, the thiol-reactive functional agent reacts with the free thiol groups on the antibody to generate an antibody-marker conjugate. Cysteine ​​residues have been introduced into antibodies by genetic engineering techniques, however, in a concentrated solution of the antibody, the unpaired cysteine ​​residues on the antibody surface can be paired and oxidized to form intermolecular disulfides, and thus antibody dimers or polymers are formed. In addition, if the protein is oxidized to form intramolecular disulfide bonds between the newly introduced cysteine ​​and the existing cysteine ​​residues, the two cysteine ​​residues cannot be used for active site participation and interaction, and further cause antibody misfolding or loss of tertiary structure, reducing the activity and specificity of the antibody.

[0006] Conjugation based on lysine side chain amino groups is usually based on the connection of lysine amino groups with activated carboxylate linkers through amide bonds, such as mediated by chemical reactions based on succinimide or isothiocyanate. The amino groups of lysine on antibodies are excellent nucleophiles, which can be very effective in loading markers, and the conjugation operation is simple. However, the lysine distribution on natural antibodies is dispersed, and the lysine conjugates formed based on lysine side chain amino groups will affect the structure of the antibody, reduce the activity or stability of the antibody, and thus reduce the activity and sensitivity of the obtained antibody conjugate.

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

[0008] 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 uses thereof, a method for preparing a conjugate, a method for immunoassay, and a method for improving the activity, stability or sensitivity of a conjugate.

[0009] 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 part, wherein:

[0010] a) the antibody comprises a heavy chain constant region;

[0011] The heavy chain constant region comprises an engineered lysine residue;

[0012] Wherein, the engineered lysine residue includes or is selected from at least one of the following sites or their equivalent sites:

[0013] 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;

[0014] b) The conjugate moiety is linked to the antibody.

[0015] In the 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 by the conjugate described in the first aspect of the present invention.

[0016] In the third aspect of the present invention, the present invention provides a method for preparing a conjugate. According to an embodiment of the present invention, the method comprises: using the antibody in the conjugate of the first aspect or the antibody of the second aspect to carry out a conjugation reaction with a conjugation part to obtain the conjugate.

[0017] In a fourth aspect of the present invention, 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.

[0018] In a fifth aspect of the present invention, 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.

[0019] In the sixth aspect of the present invention, 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 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.

[0020] 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 the preparation of a conjugate or a kit.

[0021] In an eighth aspect of the present invention, 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.

[0022] In the ninth aspect of the present invention, the present invention proposes the 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.

[0023] In the tenth aspect of the present invention, 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.

[0024] In the eleventh aspect of the present invention, the present invention proposes 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.

[0025] The amino acid sequence table of the present invention is as follows:

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[0041] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0043] Figure 1 The results of correlation test between each group of IgG1 antibody-AE and Roche fixed value blood in the examples of the present invention;

[0044] Figure 2 These are the correlation test results of each group of IgG2b antibody-AE and Roche fixed value blood in the examples of the present invention. DETAILED DESCRIPTION

[0045] 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 construed as limiting the present invention.

[0046] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. Further, in the description of the present invention, unless otherwise specified, the meaning of "plurality" is two or more.

[0047] The endpoints and any values ​​of the ranges disclosed in this article 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 each range, the endpoint values ​​of each range and the 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 as specifically disclosed in this article.

[0048] In order to make the present invention more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention belongs. The abbreviations for amino acid residues are standard 3-letter and / or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids.

[0049] 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.

[0050] As used herein, the terms "optionally", "optional", "optionally", "optional" or "optional" 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 occur.

[0051] 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.

[0052] In this article, the term "vector" generally refers to a nucleic acid molecule that can be inserted into a suitable host and replicates itself, and the inserted nucleic acid molecule is transferred to a cell or host and / or between cells or hosts. The vector may include a vector that is mainly used to insert DNA or RNA into a cell, a vector that is mainly used to replicate DNA or RNA, and a vector that is mainly used to express the transcription and / or translation of DNA or RNA. The vector also includes a vector with a variety of the above functions. The vector may 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.

[0053] In this article, the term "cell" generally refers to the use of genetic engineering technology or cell fusion technology to modify or reorganize the genetic material of the host cell to obtain a cell with a unique trait of stable inheritance. Among them, the term "host cell" refers to a prokaryotic cell or eukaryotic cell that can be introduced into a recombinant vector. The terms "transformed" or "transfected" used herein refer to the introduction of nucleic acids (such as vectors) into cells 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 target proteins. 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.

[0054] The present invention provides 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 a conjugate, a method for immunoassay, and a method for improving the activity, stability or sensitivity of a conjugate, which will be described in detail below.

[0055] Conjugate

[0056] 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 part, wherein:

[0057] a) the antibody comprises a heavy chain constant region;

[0058] The heavy chain constant region comprises an engineered lysine residue;

[0059] Wherein, the engineered lysine residue includes or is selected from at least one of the following sites or their equivalent sites:

[0060] 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;

[0061] b) The conjugated part is connected to the antibody. The conjugate of the present invention has the advantages of high activity, high sensitivity or strong stability.

[0062] 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.

[0063] 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. The specific structure is not limited. 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).

[0064] The heavy chain (H chain) and light chain (L chain) of an antibody can be connected, for example, by a disulfide bond. Among them, the amino acid sequence of the amino terminal (N terminal) of the peptide chain varies greatly, which is called the variable region (V region); the carboxyl terminal (C terminal) is relatively stable and varies little, which is called the constant region (C region). The V regions of the L chain and H chain are respectively called the light chain variable region (VL) and the heavy chain variable region (VH), and the C regions of the L chain and H chain are respectively called the light chain constant region (CL) and the heavy chain constant region (CH).

[0065] Herein, the heavy chain constant region includes the full length of the heavy chain constant region or a fragment thereof. The full length of the heavy chain constant region refers to the heavy chain constant region of the full-length antibody, for example, for mouse IgG, the full length of the heavy chain constant region from N-terminus to C-terminus is CH1, hinge region, CH2 and CH3. The full length fragment of the heavy chain constant region refers to a fragment of a continuous amino acid sequence taken from the full length of the heavy chain constant region, such as at least one of CH1, hinge region, CH2 or CH3. Similarly, the same is true for the light chain constant region.

[0066] 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 internationalImMunoGenetics 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. theinternational ImMunoGenetics information http: / / www.imgt.org.Created:16 / 03 / 2011.Version:17 / 01 / 2020.

[0067] 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.

[0068] Herein, the term "amino acid residue" refers to an amino acid located in an antibody, which is referred to as an "amino acid residue" because two amino acids are connected by an amide bond, resulting in the removal of -OH from the -COOH of the amino acid and / or the removal of one H from the -NH2 of the amino acid. For example, a "lysine residue", a "natural amino acid residue" or an "engineered lysine residue".

[0069] Herein, the term "natural amino acid residue" refers to the amino acid residue contained in the constant region of a wild-type antibody (e.g., a wild-type murine IgG1 antibody). For example, when the first amino acid in the heavy chain constant region of a wild-type murine IgG1 antibody is A (which can be called A1 according to its position), and it is mutated to K (or Q), A (or A1) is a natural amino acid residue, and the mutated amino acid K (or Q) is a non-natural amino acid residue.

[0070] As used herein, the term "engineered lysine residue" refers to a mutation of a natural amino acid residue at any position to lysine (K), and the lysine obtained after the mutation is an engineered lysine residue.

[0071] In this article, the term "equivalent site" refers to a site where the position and amino acid type of a certain specific site are different due to different antibody sources (e.g., antibodies derived from different sources or species), types (e.g., IgG, IgM, IgA, etc.), subclasses (e.g., IgG can be divided into subclasses such as IgG1 and IgG2), or the existence of alleles. The equivalent site of the specific site can be determined by sequence and structural homology comparison.

[0072] 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. 70:173-187 (1997); and 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, BLASTAltschul 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.

[0073] In some optional embodiments of the present invention, the above conjugate may further include at least one of the following technical features:

[0074] 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 equivalent sites:

[0075] 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.

[0076] 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 equivalent positions:

[0077] T3, P5, G14, L61, T79, T81, S116, S144, R199, P208, I209, G218, R219, Q224, D238, T255, E266, N267, Q290, E295, S319, H320;

[0078] 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 the linear position on the full length of the heavy chain constant region of mouse wild-type IgG1.

[0079] 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 equivalent positions:

[0080] 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.

[0081] Herein, "T3 / P5" means that the natural amino acid residues at the two positions of T3 and P5 are mutated into lysine residues to obtain engineered lysine residues.

[0082] Herein, engineered lysine residues may be represented by numbers (numbered positions) or by numbers + K. Exemplarily, 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 positions of other engineered lysine residues.

[0083] In this article, the site of the natural amino acid residue that mutates into an engineered lysine residue corresponds to the site of the engineered lysine residue. Exemplarily, when the natural amino acid residue that mutates into an engineered lysine residue is T3, the site of the engineered lysine residue is T3K; when the natural amino acid residue that mutates into an engineered lysine residue is P5, the site of the engineered lysine residue is P5K.

[0084] 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 equivalent positions:

[0085] 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;

[0086] 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 the linear position along the full length of the murine wild-type IgG2b heavy chain constant region.

[0087] 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 equivalent positions:

[0088] 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.

[0089] In some optional embodiments of the present invention, the mouse is selected from mice (Mus musculus).

[0090] In this article, the term "wild type" refers to an antibody type that exists naturally and is not modified by mutation. It should be understood that the engineered lysine residues in the heavy chain constant region of the antibody of the present invention are all based on the wild-type heavy chain constant region as a positioning reference, 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 site of the engineered lysine residue based on the results of homology comparison between it and the wild-type heavy chain constant region. It can be understood by those skilled in the art that the engineered lysine residues obtained after the above-mentioned homology comparison are also within the scope of protection of the present invention.

[0091] Herein, the term "IgHG1" refers to the heavy chain of IgG1 antibody; the term "IgHG2a" refers to the heavy chain of IgG2a antibody; the term "IgHG2b" refers to the heavy chain of IgG2b antibody; and the term "IgHG" refers to the heavy chain of IgG antibody.

[0092] Herein, the heavy chain constant region of murine wild-type IgG1 is shown as SEQ ID NO:1, and the heavy chain constant region of murine wild-type IgG2b is shown as SEQ ID NO:2.

[0093] Those skilled in the art can know that the various parts of the heavy chain constant region are arranged in sequence from the N-terminus to the C-terminus, and they are in a linear relationship. In this article, the term "linear position" refers to the linear position of the heavy chain constant region (including CH1-hinge region-CH2-CH3) arranged in sequence from the N-terminus to the C-terminus; that is, the heavy chain constant region (including CH1-hinge region-CH2-CH3) is numbered in sequence from the N-terminus to the C-terminus.

[0094] Those skilled in the art will understand that different numbering systems can be used to number the amino acid residue positions for an antibody sequence, and conversion between different numbering systems is well known in the art. The sites of "natural amino acid residues mutated into engineered lysine residues" or "engineered lysine" described in the present invention are numbered sequentially from the N-terminus to the C-terminus according to the amino acid sequence shown in the heavy chain constant region of a murine wild-type IgG1 antibody (such as SEQ ID NO: 1) and / or the heavy chain constant region of a murine wild-type IgG2b antibody (such as 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.

[0095] 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 compare the heavy chain constant regions of the murine wild-type IgG2a, IgG2b, IgG2c, and IgG3 antibodies with the heavy chain constant regions of the murine wild-type IgG1 antibodies for homology, so as to obtain the engineered lysine residue sites of the murine wild-type IgG2a, IgG2b, IgG2c, and IgG3 antibodies, for example, the heavy chain constant regions of the murine wild-type IgG2a, IgG2b, IgG2c, and IgG3 are compared for homology with the heavy chain constant regions of the murine wild-type IgG1. Therefore, conjugates containing murine IgG2a, IgG2b, IgG2c, and IgG3 antibodies or antibodies of other origins, and the antibodies contain the engineered lysine residue sites of the present invention, also fall within the scope of protection of the present invention.

[0096] 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.

[0097] 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.

[0098] In some optional embodiments of the present invention, the heavy chain constant region before modification includes CH1 of IgG, hinge region of IgG, CH2 of IgM, CH3 of IgM and CH4 of IgM from N-terminus to C-terminus.

[0099] In some optional embodiments of the present invention, the heavy chain constant region before modification includes CH1 of IgG, hinge region of IgG, CH2 of IgG and CH3 of IgG from N-terminus to C-terminus.

[0100] 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, 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.

[0101] 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.

[0102] 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.

[0103] 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 engineered lysine residues.

[0104] 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.

[0105] 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.

[0106] 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.

[0107] 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 to 53, SEQ ID NOs: 114 to 125, or SEQ ID NO: 138.

[0108] 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 to 53, SEQ ID NOs: 114 to 125, or SEQ ID NO: 138.

[0109] In some optional embodiments of the present invention, the antibody further comprises a light chain constant region.

[0110] In some optional embodiments of the present invention, the light chain constant region includes or is selected from a κ-type or λ-type light chain constant region.

[0111] In some optional embodiments of the present invention, the light chain constant region 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.

[0112] 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.

[0113] 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.

[0114] 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.

[0115] In some optional embodiments of the present invention, the antibody has a heavy chain constant region having 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 a light chain constant region having at least 80% identity thereto.

[0116] Optionally, the fragment is selected from CH1, hinge region, CH2, CH3 or a combination thereof.

[0117] In some optional embodiments of the present invention, the antibody further comprises a heavy chain variable region.

[0118] 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.

[0119] In some optional embodiments of the present invention, the antibody further comprises a light chain variable region.

[0120] 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.

[0121] In some optional embodiments of the present invention, the heavy chain variable region and the light chain variable region are connected, for example, through an interchain disulfide bond, or through 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.

[0122] Illustratively, the heavy chain variable region and the light chain variable region are linked by an interchain disulfide bond.

[0123] 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 protection scope of the present invention.

[0124] In some optional embodiments of the present invention, the target antigen is an antigen associated with infectious diseases, endocrine, tumors or drugs.

[0125] In some optional embodiments of the present invention, the antigen is an antigen associated with a viral or bacterial infectious disease.

[0126] In some optional specific 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-lymphocytic 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, syphilis treponema antigen, Borrelia treponema 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 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.

[0127] 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.

[0128] 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.

[0129] 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:

[0130]

[0131]

[0132] 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).

[0133] 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 the heavy chain connected by a disulfide bond.

[0134] 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.

[0135] As used herein, the term "Fv antibody" or "Fv fragment" generally refers to an antibody or fragment consisting only of a light chain variable region (VL) and a heavy chain variable region (VH) connected by non-covalent bonds, and is the smallest functional fragment of an antibody that retains a complete antigen binding site.

[0136] 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.

[0137] In this article, the terms "minimum recognition unit" and "MRU" both refer to antibodies or fragments consisting of only one CDR, and their molecular weight is very small, accounting for only about 1% of the complete antibody.

[0138] 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 CDR in the present invention can be adjusted according to the type of antigen, without specific limitation.

[0139] In some optional embodiments of the present invention, the conjugate portion is linked to the active group of the antibody.

[0140] 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.

[0141] 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.

[0142] In some optional embodiments of the present invention, the conjugated part 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 strands of nucleic acid) and a tag.

[0143] 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.

[0144] In this article, 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 contain or carry a liquid phase (for example, a support such as a plate, a membrane, a test tube, and a container such as a well plate, a microfluidic channel, a glass capillary, a nanocolumn, a monolithic column, etc.); it can also be a labeling carrier for labeling antibodies, such as an enzyme (for example, peroxidase, alkaline phosphatase, 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.

[0145] In some optional embodiments of the present invention, the affinity substance includes biotin / avidin.

[0146] 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.

[0147] In some optional embodiments of the present invention, the conjugated portion 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.

[0148] Antibody

[0149] In the 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 by the conjugate described in the first aspect of the present invention. The antibody of the present invention has the advantages of strong stability or high activity, and the conjugate prepared using the antibody has the advantages of strong stability, high activity or high sensitivity.

[0150] The specific implementation of the antibody described in the second aspect is described in the first aspect of the present invention and will not be repeated here.

[0151] 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 think of using genetic engineering technology or other technologies (chemical synthesis, recombinant expression) to prepare the antibody, for example, separating and purifying the antibody from the culture product of a recombinant cell that can recombinantly express the antibody as described in any of the above items, which is easy to achieve for those skilled in the art. Based on this, no matter what technology is used to prepare the antibody of the present invention, it belongs to the protection scope of the present disclosure.

[0152] Methods for preparing conjugates

[0153] In the third aspect of the present invention, the present invention provides a method for preparing a conjugate. According to an embodiment of the present invention, the method comprises: using the antibody in the conjugate described in the first aspect or the antibody described in the second aspect to carry out a conjugation reaction with a conjugation part to obtain the conjugate. Thus, the conjugate can be effectively prepared, and the preparation method is simple.

[0154] Nucleic acid molecules, vectors, cells or hosts

[0155] 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.

[0156] In a fourth aspect of the present invention, 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.

[0157] According to an embodiment of the present invention, the nucleic acid molecule includes DNA or RNA.

[0158] It should be noted that, for the nucleic acid molecules mentioned herein, those skilled in the art will understand that they actually include any one or two of the complementary double strands. For convenience, in this article, although only one strand is provided in most cases, the other strand complementary thereto is actually disclosed. In addition, the molecular sequence in the present invention includes a DNA form or an RNA form, and disclosing one of them means that the other is also disclosed.

[0159] 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 element 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, they are not from the vector itself. Of course, the nucleic acid molecule can be operably connected to the control element. Herein, "operably connected" means connecting the exogenous gene to the vector so that the control elements in the vector, such as transcription control sequences and translation control sequences, etc., can play their expected functions of regulating the transcription and translation of the exogenous gene. Commonly used vectors can be, for example, plasmids, bacteriophages, 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 a large amount of antibodies obtained in vitro.

[0160] In some specific embodiments of the present invention, the vector is a eukaryotic expression vector, a prokaryotic expression vector, a virus or a bacteriophage.

[0161] In an optional embodiment of the present invention, the expression vector is a plasmid expression vector or a lentiviral expression vector.

[0162] In the sixth aspect of the present invention, 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 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. The cell can effectively express the aforementioned antibody in the cell under suitable conditions.

[0163] According to an embodiment of the present invention, the cell is obtained by introducing the vector described in the fifth aspect into the cell.

[0164] It should be noted that the cells of the present invention are not particularly limited and can be prokaryotic cells, eukaryotic cells or bacteriophages. The prokaryotic cells can be Escherichia coli, Bacillus subtilis, Streptomyces or Proteus mirabilis, etc. The eukaryotic cells include fungi such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Trichoderma, insect cells such as armyworm, plant cells such as tobacco, and mammalian cells such as BHK cells, CHO cells, COS cells, and myeloma cells.

[0165] In an optional embodiment of the present invention, the cell is a mammalian cell, including a BHK cell, a CHO cell, a NSO cell or a COS cell, and does not include an animal germ cell, a fertilized egg or an embryonic stem cell.

[0166] 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 is easy for those skilled in the art to understand that the 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 states, 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.

[0167] Reagent or kit, use

[0168] 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 the preparation of a conjugate or a kit.

[0169] In the eighth aspect of the present invention, 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. The reagent or the kit of the present invention has the advantages of high activity and high sensitivity, and can be used for immunoassay.

[0170] In the ninth aspect of the present invention, the present invention proposes the 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.

[0171] Immunoassay method and method for improving conjugate activity, stability or sensitivity

[0172] In the tenth aspect of the present invention, 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.

[0173] In some optional embodiments of the present invention, whether the sample to be detected contains the target substance or the content of the target substance is determined based on the signal of the immune complex.

[0174] 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 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.

[0175] In some optional embodiments of the present invention, the signal comprises a fluorescent signal.

[0176] In the eleventh aspect of the present invention, the present invention proposes 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.

[0177] The scheme of the present invention will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used are not indicated by the manufacturer and are all conventional products that can be obtained commercially.

[0178] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the present disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used for the practice or testing of the preparations or unit doses herein, some methods and materials are now described. Unless otherwise stated, the techniques adopted or considered herein are standard methods. Materials, methods and examples are illustrative and non-restrictive only.

[0179] 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. This technique is fully explained in the literature, such as Molecular Cloning: A Laboratory Manual, 2nd Edition (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); PCR: The Polymerase Chain Reaction (PCR: The Polymerase Chain Reaction) (Academic Press, Inc.); Reaction" (Mullis et al., eds., 1994); and Current Protocols in Immunology (JE Coligan et al., eds., 2011), each of which is expressly incorporated herein by reference.

[0180] Embodiment 1:

[0181] 1. Selection of mutation points

[0182] By studying the sequence and structure of mouse antibodies, different natural amino acid residues were selected to mutate into lysine residues in the heavy chain constant region of mouse IgG1 and mouse IgG2b, and different engineered lysine residues were obtained. Among them, 19 mutation sites were designed for IgG1 (the mutation sites were numbered in the linear order of the heavy chain constant region SEQ ID NO: 1), and 19 mutation groups were obtained, as shown in Table 1; 32 mutation sites were designed for IgG2b (the mutation sites were numbered in the linear order of the heavy chain constant region SEQ ID NO: 2), and 32 mutation groups were obtained, as shown in Table 2.

[0183] Table 1: Mutation sites of IgG1

[0184]

[0185] Table 2: Mutation sites of IgG2b

[0186]

[0187] 2. Plasmid construction and expression

[0188] 2.1 Construction of recombinant antibody expression plasmid

[0189] 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 completed by a gene sequencing company. TM 3.4 Vector is a constructed recombinant antibody eukaryotic expression vector, which has been modified to introduce multiple cloning restriction sites and is hereinafter referred to as 3.4A expression vector.

[0190] 2.1. Anti-CA153 antibody gene preparation

[0191] The antibody mRNA was extracted from the hybridoma cell line secreting Anti-CA153 monoclonal antibody, and the DNA product was obtained by RT-PCR. It was inserted into the pMD-18T vector and transformed into DH5α competent cells. After the colonies grew, 4 positive clones of the heavy chain (Heavy Chain) and light chain (Light Chain) genes were taken and sent to a gene sequencing company for sequencing.

[0192] 2.2 Sequence analysis of the variable region gene of Anti-CA153 antibody

[0193] 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 determine that the genes amplified by the heavy chain and light chain primer pairs were correct.

[0194] 2.3. Construction of recombinant antibody expression plasmid

[0195] pcDNA TM 3.4 vector is a recombinant antibody eukaryotic expression vector constructed, and the expression vector is modified to introduce multiple cloning restriction sites, which are subsequently referred to as 3.4A expression vectors; according to the sequencing results of the antibody variable region genes in the above pMD-18T, the VL and VH gene-specific primers and mutant primers of the Anti-CA153 antibody are designed, with restriction endonuclease restriction sites and protective bases at both ends, and the Light Chain gene fragment and the Heavy Chain gene fragment are expanded by PCR amplification. The Heavy Chain and Light Chain gene fragments are double-digested with restriction endonucleases, and the 3.4A expression vector is double-digested with restriction endonucleases. After the fragments and vectors are purified and recovered, the Heavy Chain gene and the Light Chain gene are 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

[0196] Table 3: Amino acid sequences of heavy and light chains of each antibody

[0197]

[0198]

[0199] 2.4. Expression of recombinant antibody samples

[0200] Resuscitate HEK293 cells in advance and subculture them to 200 ml system to make the cell density reach 3-5×10 6 cells / ml, cell viability>95%; wash the cells by centrifugation, re-dissolve with culture medium, and adjust the cell density to 2.9×10 6cells / ml, as the cell diluent. Use the culture medium to prepare the plasmid DNA dilution solution and the transfection reagent dilution solution containing the recombinant expression plasmid obtained in step 2.3 of the present 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 120rmp and a CO2 content of 8%. Centrifuge and collect samples after 13 days.

[0201] 3. HPLC-SEC (High Performance Liquid Chromatography-Size Exclusion Chromatography)

[0202] 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), and the test results are shown in Tables 4 and 5. The antibodies of each mutant group were purified to obtain samples with excellent purity.

[0203] Table 4: HPLC-SEC results corresponding to IgG1 mutation to K

[0204]

[0205] Table 5: HPLC-SEC results corresponding to IgG2b mutation to K

[0206]

[0207]

[0208] 4. Determination of thermal stability Tm value

[0209] The purified multiple groups of 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 by DSF (differential scanning fluorescence). The test results are shown in Tables 6 and 7. The specific parameters are set as follows:

[0210] Sample volume: 400 μL;

[0211] Measuring range: 25℃~95℃;

[0212] Heating rate: 0.3℃ / min.

[0213] It can be seen from Tables 6 and 7 that antibodies in each mutation group have good thermal stability.

[0214] Table 6: Tm results corresponding to IgG1 mutation points

[0215] IgG1 mutation group Tm IgG1 mutation group Tm wild type 70.58 Mutation Group 10 69.49 Mutation Group 1 69.02 Mutation Group 11 69.17 Mutation Group 2 68.70 Mutation Group 12 68.67 Mutation group 3 66.53 Mutation Group 13 67.71 Mutation Group 4 71.63 Mutation Group 14 71.82 Mutation Group 5 65.79 Mutation Group 15 70.18 Mutation Group 6 73.54 Mutation Group 16 69.78 Mutation Group 7 69.02 Mutation Group 17 67.45 Mutation Group 8 69.23 Mutation Group 18 70.02 Mutation Group 9 68.65 Mutation Group 19 70.98

[0216] Table 7: Tm results corresponding to IgG2b mutation points

[0217] IgG2b mutation group Tm IgG2b mutation group Tm wild type 62.03 Mutation Group 17 61.97 Mutation Group 1 57.98 Mutation Group 18 61.98 Mutation Group 2 62.72 Mutation Group 19 62.04 Mutation group 3 59.72 Mutation Group 20 61.98 Mutation Group 4 61.70 Mutation Group 21 62.10 Mutation Group 5 61.79 Mutation Group 22 62.04 Mutation Group 6 61.93 Mutation Group 23 62.10 Mutation Group 7 61.95 Mutation Group 24 62.00 Mutation Group 8 62.06 Mutation Group 25 62.04 Mutation Group 9 61.97 Mutation Group 26 62.10 Mutation Group 10 61.98 Mutation Group 27 62.33 Mutation Group 11 62.01 Mutation Group 28 62.27 Mutation Group 12 61.94 Mutation Group 29 62.02 Mutation Group 13 61.93 Mutation Group 30 62.09 Mutation Group 14 61.99 Mutation group 31 61.71 Mutation Group 15 61.94 Mutation group 32 58.20 Mutation Group 16 62.02

[0218] 5. Activity identification

[0219] 5.1 Activity and specificity results after labeling AE

[0220] Marking process:

[0221] 1) Buffer exchange: The purified multiple groups of recombinant antibody samples (hereinafter referred to as antibodies or Ab) obtained in step 2.4 of this example were dialyzed in dialysis bags for 2 hours, with the buffer exchanged every 45 minutes.

[0222] 2) Reaction: The antibody was placed in a centrifuge tube, and AE mother solution activated by NHS (N-hydroxysuccinimide) was added to react for 15 min, and then Gly solution was added to terminate the reaction for 30 min to obtain a solution containing Ab-AE.

[0223] 3) Impurity removal: After the reaction, dialyze Ab-AE, and change the solution every 30 to 60 minutes on the first day (at least 4 times). After overnight, change the solution every 60 minutes on the second day (at least 4 times).

[0224] 4) Recovery and storage: Take out the Ab-AE raw material in the dialysis bag, add the same volume of high-pressure glycerol, mix well, and store it at -20°C for a long time.

[0225] Testing process:

[0226] 1) Sample addition reaction: Take 50 μL of the 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.

[0227] 2) The reaction solution was washed three times with 1*PBST.

[0228] 3) Add excitation solution A + excitation solution B to the cleaned solution and read the value immediately.

[0229] CA153 quality control products with different concentration gradients were used as test samples to detect activity.

[0230] 44 Roche certified specimens were used as test samples to test and calculate the correlation.

[0231] Among them, the activity results of each group of IgG1 antibody labeled AE (i.e., each group of IgG1 antibody-AE) obtained after mutating the natural amino acid residue 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 1The activity results of each group of IgG2b antibody labeled AE (i.e., each group of IgG2b antibody-AE) obtained after mutating the natural amino acid residue 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 .

[0232] Deviation = (activity response value of the mutant group - activity response value of the wild type) / activity response value of the wild type;

[0233] Average deviation = total deviation / number of quality control groups.

[0234] For each group of IgG1 antibodies, except for mutation groups 10 and 12, the remaining mutation groups can improve the labeled AE activity, and exemplary results are shown in Table 8-1 and Table 8-2;

[0235] 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;

[0236] from Figure 1 and Figure 2 It can be seen that antibodies in each mutation group have good test correlation.

[0237] Table 8-1: Activity results of IgG1 antibody-AE in each group

[0238]

[0239] Table 8-2: Average deviation of activity between each group of IgG1 antibody-AE and wild-type IgG1 antibody-AE

[0240]

[0241]

[0242] Table 9-1: Activity results of IgG2b antibody-AE in each group

[0243]

[0244]

[0245] Table 9-2: Average deviation of activity between each group of IgG2b antibody-AE and wild-type IgG2b antibody-AE

[0246]

[0247] 5.2 Activity and specificity results after HRP labeling

[0248] Marking process:

[0249] 1) Buffer exchange: The purified multiple groups of 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 replaced every 45 minutes.

[0250] 2) Oxidation of HRP enzyme: Weigh 2 mg of HRP dry powder (Frdbio) and dissolve it in 100 μL ddH2O in the dark. Weigh 21 mg of NaIO4 and dissolve it in 1 mL of ddH2O. Pipette 100 μL of NaIO4 solution and slowly mix with 100 μL of HRP solution. Let stand at 4°C for 30 min. At this time, the solution is green. Pipette 2 μL of ethylene glycol and slowly add it to the oxidized HRP solution. Let stand at room temperature in the dark for 30 min. At this time, the solution is brown.

[0251] 3) Antibody labeling and recovery: The oxidized HRP solution was directly added to the antibody solution treated in step 1), and the reaction was carried out at room temperature for 2 hours. 0.4 mg of NaBH4 was weighed, dissolved in 20 μL of ddH2O, and all of it was added to the reaction solution in the previous step, and allowed to stand at 4°C for 2 hours, with shaking every 30 minutes; PBS (pH 7.2) was dialyzed overnight, 30% to 50% glycerol by volume was added to the labeling solution, and the mixture was mixed and stored at -20°C to obtain Ab-HRP.

[0252] Testing process:

[0253] 1) Sample addition reaction: Take 10 μL of the 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.

[0254] 2) The reaction solution was washed three times with 1*PBST.

[0255] 3) Add excitation solution A + excitation solution B to the cleaned solution and read the value immediately.

[0256] CA153 quality control products with different concentration gradients were used as test samples to detect activity.

[0257] 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.

[0258] 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.

[0259] 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.

[0260] Table 10-1: Activity results of IgG1 antibody-HRP in each group

[0261]

[0262] Table 10-2: Average deviation of activity between IgG1 antibody-HRP and wild-type IgG1 antibody-HRP in each group

[0263]

[0264] Table 11-1: Activity results of IgG2b antibody-HRP in each group

[0265]

[0266]

[0267] Table 11-2: Average deviation of activity between each group of IgG2b antibody-HRP and wild-type IgG2b antibody-HRP

[0268]

[0269] Embodiment 2:

[0270] 2.1 The above-mentioned activity-enhancing mutations were subjected to combined mutations. Five combined mutations were designed for IgG2b, as shown in Table 12; and seven combined mutations were designed for IgG1, as shown in Table 13.

[0271] Table 12: Combined mutation sites of IgG2b

[0272]

[0273] Table 13: Combined mutation sites of IgG1

[0274]

[0275] The above-mentioned combined mutation samples were prepared with reference to the above-mentioned 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.

[0276] 2.2 Activity and specificity results after labeling AE

[0277] The activity after labeling AE was determined with reference to the above Example 1, and the activity was detected using ascites CA153 with different concentration gradients as the test sample. The activity results of the IgG2b combined mutant antibody are shown in Table 14, and the activity results of the IgG1 combined mutant antibody are shown in Table 15. The results show that the combined mutations of IgG2b or IgG1 have improved labeling activity and a correlation comparable to the wild type.

[0278] Table 14: Activity results of IgG2b combination mutant antibody-AE

[0279]

[0280] Table 15: Activity results of IgG1 combined mutant antibodies-AE

[0281]

[0282] Embodiment 3:

[0283] 3.1 Anti-CKMB wild-type antibody (mouse 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.

[0284] S319 on the heavy chain constant region of the CKMB wild-type antibody was mutated to a lysine residue to obtain a 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.

[0285] 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.

[0286] 3.2 Activity test results of labeled latex microspheres

[0287] Detection method:

[0288] 1) Take 160ul of 10% polystyrene carboxyl latex with a particle size of 188nm and dilute it to 2ml with 10mmol / L HEPES (PH7.5) buffer solution;

[0289] 2) Slowly add 50ul (10mg / ml) EDC solution and 50ul (10mg / ml) NHS solution to step 1, stir at room temperature for 20min, and then adjust the volume to 10ml with 10mmol / L HEPES (PH7.5) buffer solution;

[0290] 3) Take 70ul of denatured IgG solution, add 50ul BSA (10%) and 50mM urea;

[0291] 4) Slowly add the denatured IgG solution diluted in step 3) into step 2, and stir at room temperature for 3 hours;

[0292] 5) Centrifuge at 15000 rpm for 40 min, and resuspend by ultrasonication in 10 mmol / L PBS (containing 0.2% Tween 20, 0.1% BSA, and 10% sucrose) to obtain CKMB detection reagent 2.

[0293] 6) Use 50mmol / L glycine buffer as detection reagent 1, and CKMB detection reagent 2 to form the detection reagent of this experiment;

[0294] 7) Calibrator: Prepared by diluting the serum of myocardial CKMB patient specimens, detection instrument: Mindray BS-480, detection parameters are as follows:

[0295] Sample size (ul) 2.5 CKMB detection reagent 1(ul) 200 CKMB detection reagent 2(ul) 50 Detection wavelength (nm) 660 Reading point 1 51-51 Reading point 2 65-65

[0296] The activity data measured are as follows, and the results show that the mutant antibody has significantly improved activity compared to the wild type.

[0297] sample Concentration (ng / ml) wild type CKMB-mut Calibration 1 0 -16.97 19 Calibration 2 6 12.08 854 Calibration 3 25 109.37 1698 Calibration 4 50 239.21 4080 Calibration 5 100 545.27 6097 Calibration 6 200 1361.54 6215

[0298] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means 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 may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0299] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary 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.

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