CN118837542A - Antibodies and antibody conjugates and uses thereof - Google Patents

Abstract

The present invention discloses a conjugate, comprising: an antibody and at least one conjugated part, the conjugated part is connected to the antibody, the antibody comprises a CH1 fragment and a CL fragment, the CH1 fragment and the CL fragment have cysteine sites or equivalent sites selected from any of the following groups: the 67th position of the CH1 fragment and the 28th position of the CL fragment; the 9th position of the CH1 fragment and the 17th position of the CL fragment; the 9th position of the CH1 fragment and the 14th position of the CL fragment; the 54th position of the CH1 fragment and the 55th position of the CL fragment; the 53rd position of the CH1 fragment and the 55th position of the CL fragment; the 14th position of the CH1 fragment and the 12th position of the CL fragment; the 14th position of the CH1 fragment and the 102nd position of the CL fragment. The conjugate of the present invention has the advantages of high activity, high sensitivity or strong stability.

Description

Antibodies and antibody conjugates and their uses

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to Chinese patent application No. 202310453142.2 filed with the China Patent Office on April 24, 2023, entitled “Antibodies and Antibody Conjugates and Uses Thereof,” the entire contents of which are incorporated herein by reference.

Technical Field

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

Conjugates of antibodies and markers have been widely used in the field of immunoassays. The conjugation methods usually include conjugation based on lysine side chain amino groups and conjugation based on cysteine sulfhydryl groups. Conjugation based on lysine side chain amino groups is an early developed conjugation method. There are about 80-100 lysine amino groups distributed in the structure of an antibody. These lysine amines are very effective payload conjugations because they can be well exposed and the amino group is an excellent nucleophilic reagent. However, its selectivity is the worst, and conjugation at some sites will affect the stability or activity of the antibody.

The sulfur group of cysteine is reactive at neutral pH, which is different from most amines that are protonated near neutral pH and have lower nucleophilicity. Because of the relative reactivity of the sulfur group, antibodies with cysteine residues usually exist as disulfide-linked oligomers in their oxidized form or have internal bridged disulfide bonds. Extracellular antibodies generally do not have free sulfur groups. Reagents such as dithiothreitol (DTT), selenol and tris-(2-carboxyethyl)phosphine (TCEP) can be used to reduce disulfide bonds to reactive sulfur groups. In IgG1 monoclonal antibodies with four interchain disulfide bonds, this site-specific conjugation results in conjugates with up to eight ligand moieties connected. However, in the process of coupling with these different cysteine residues, the original disulfide bonds cannot always be bridged again, which may lead to structural changes and impaired antibody function.

Cysteine residues have been introduced into antibodies to form covalent connections with ligands or to form new intramolecular disulfide bonds by genetic engineering techniques. However, designing cysteine sulfhydryl groups by mutating different amino acid residues of antibodies to cysteine amino acids may be problematic, particularly when unpaired (free Cys) residues are relatively easy to react or oxidize. In concentrated solutions of antibodies, whether in the periplasm of E. coli, culture supernatant, or partially or completely purified proteins, unpaired Cys 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 modified Cys and existing Cys residues, the two Cys groups cannot be used for active site participation and interaction. In addition, by misfolding or loss of tertiary structure, the activity and specificity of the antibody can be reduced, or even inactive or non-specific, resulting in greatly reduced activity and sensitivity of the antibody conjugates obtained.

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

Summary of the invention

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.

In the first aspect of the present invention, the present invention proposes a conjugate. According to an embodiment of the present invention, the conjugate comprises: an antibody and at least one conjugated part, wherein the conjugated part is connected to the antibody; wherein the antibody comprises a CH1 fragment and a CL fragment; the CH1 fragment and the CL fragment of the antibody have a cysteine site selected from any of the following groups: 1) position 67 of the CH1 fragment and position 28 of the CL fragment, or an equivalent site thereof; 2) position 9 of the CH1 fragment and position 17 of the CL fragment, or an equivalent site thereof; 3) position 9 of the CH1 fragment and position 14 of the CL fragment, or an equivalent site thereof; 4) position 54 of the CH1 fragment and position 55 of the CL fragment, or an equivalent site thereof; 5) position 53 of the CH1 fragment and position 55 of the CL fragment, or an equivalent site thereof; 6) position 14 of the CH1 fragment and position 12 of the CL fragment, or an equivalent site thereof; 7) position 14 of the CH1 fragment and position 102 of the CL fragment, or an equivalent site thereof.

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.

In the third 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.

In a fourth 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 third aspect.

In the fifth 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 third aspect or the vector described in the fourth aspect; or expresses the antibody in the conjugate described in the first aspect or the antibody described in the second aspect.

In the sixth 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 third aspect, the vector described in the fourth aspect, or the cell or host described in the fifth aspect in the preparation of a conjugate or a kit.

In the seventh 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 first conjugation reaction with a conjugation part to obtain the conjugate.

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.

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 third aspect, the vector described in the fourth aspect, the cell or host described in the fifth aspect, or the reagent or kit described in the eighth aspect in immunoassay or preparation of immunoassay products.

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.

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 a CH1 fragment and a CL fragment in the antibody into one or more groups of cysteine sites, wherein the cysteine sites are consistent with the cysteine sites defined by the conjugate described in the first aspect.

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

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

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:

FIG1 is a SDS-Page test result of each antibody reduction in Example 1 of the present invention;

FIG2 is a non-reduced SDS-Page test result of each antibody in Example 1 of the present invention;

FIG3 is a thermal stability test result of each antibody labeled with AP in Example 1 of the present invention;

FIG4 is a CH1 and CL sequence alignment diagram of the mouse IgG1 antibody of the present invention and other subclasses of mouse antibodies (IgG2a, IgG2b, IgG2c, IgG3) and species (sheep IgG1, sheep IgG2, rabbit IgG), wherein the black boxes are cysteine sites and their equivalent sites;

FIG5 is a chromatogram of 2-(toluenesulfonyl)methacrylamide-PEG4-amide-PEG4-methyltetrazine;

FIG6 is a mass spectrum of 2-(toluenesulfonyl)methacrylamide-PEG4-amide-PEG4-methyltetrazine;

FIG7 is a mass spectrum peak analysis diagram of 2-(toluenesulfonyl)methacrylamide-PEG4-amide-PEG4-methyltetrazine;

FIG8 is a carbon NMR spectrum of 2-(toluenesulfonyl)methacrylamide-PEG4-amide-PEG4-methyltetrazine;

FIG9 is a hydrogen nuclear magnetic resonance spectrum of 2-(toluenesulfonyl)methacrylamide-PEG4-amide-PEG4-methyltetrazine.

DETAILED DESCRIPTION

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.

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.

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.

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.

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.

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.

As used herein, the term "variant" or "mutant" may refer to any naturally occurring or engineered molecule comprising one or more nucleotide or amino acid mutations.

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.

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.

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.

Conjugate

In the first aspect of the present invention, the present invention proposes a conjugate. According to an embodiment of the present invention, the conjugate comprises: an antibody and at least one conjugated part, wherein the conjugated part is connected to the antibody; wherein the antibody comprises a CH1 fragment and a CL fragment; the CH1 fragment and the CL fragment of the antibody have a cysteine site selected from any of the following groups: 1) position 67 of the CH1 fragment and position 28 of the CL fragment, or an equivalent site thereof; 2) position 9 of the CH1 fragment and position 17 of the CL fragment, or an equivalent site thereof; 3) position 9 of the CH1 fragment and position 14 of the CL fragment, or an equivalent site thereof; 4) position 54 of the CH1 fragment and position 55 of the CL fragment, or an equivalent site thereof; 5) position 53 of the CH1 fragment and position 55 of the CL fragment, or an equivalent site thereof; 6) position 14 of the CH1 fragment and position 12 of the CL fragment, or an equivalent site thereof; 7) position 14 of the CH1 fragment and position 102 of the CL fragment, or an equivalent site thereof. The conjugate of the present invention has the advantages of high activity, high sensitivity or strong stability.

In this article, the term "antibody" is used in the broadest sense, which can include full-length monoclonal antibodies, multispecific antibodies, and chimeric antibodies, and the specific structure is not limited as long as they show the desired biological activity. It usually includes a light chain with a lighter molecular weight and a heavy chain with a heavier molecular weight, and the heavy chain (H chain) and the light chain (L chain) are connected by a disulfide bond to form an antibody molecule. 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 changes little, which is called the constant region (C region). The V regions of the L chain and the 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 the H chain are respectively called the light chain constant region (CL or CL fragment) and the heavy chain constant region (CH or CH fragment), wherein the heavy chain constant region includes at least one of the CH1 fragment, the hinge region and the Fc fragment (CH2 and CH3 fragments), and preferably, the heavy chain constant region includes the CH1 fragment, the hinge region, the CH2 fragment and the CH3 fragment in sequence from the N-terminus to the C-terminus.

In this article, the division of variable region, constant region, CH1, CH2, and CH3 sequences refers to the IMGT division method, see Lefranc, M.-P. and Martinez-Jean C. and Bosc N. or Ehrenmann,Patrice Duroux,Chantal Ginestoux,Genetable: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.

Herein, the term "cysteine site" refers to a site where the amino acid at the site is cysteine.

In this article, the term "equivalent site" refers to a site where the position and amino acid type of certain specific sites 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), subtypes (e.g., k-type, λ-type), or alleles. The equivalent site of the specific site can be determined by sequence and structural homology comparison. For example, the serine at position 67 in CH1 of the mouse wild-type IgHG1 corresponds to the valine at position 68 in CH1 of the sheep wild-type IgHG1.

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

In some optional embodiments of the present invention, positions 9, 14, 53, 54, and 67 of the CH1 fragment are numbered sequentially from the N-terminus to the C-terminus based on the amino acid sequence shown in CH1 of mouse wild-type IgHG; positions 12, 14, 17, 28, 55, and 102 of the CL fragment are numbered sequentially from the N-terminus to the C-terminus based on the amino acid sequence shown in CL of mouse wild-type IGLK.

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

In some optional embodiments of the present invention, the IgHG is selected from IgHG1, IgHG2a, IgHG2b, IgHG2c or IgHG3.

In some optional embodiments of the present invention, the amino acid sequence of CH1 of the murine wild-type IgHG is shown as SEQ ID NO:1 or SEQ ID NO:2.

In some optional embodiments of the present invention, the amino acid sequence of CL of the murine wild-type IGLK is shown in SEQ ID NO:10.

As used herein, the term "wild type" refers to an antibody type that exists naturally and has not been modified by mutation. It should be understood that the cysteine sites of the CH1 fragment and the CL fragment of the antibody of the present invention are based on the wild-type CH1 or CL sequence as the positioning reference, which does not mean that the antibody must be wild-type. When the antibody is wild-type, the cysteine sites of the antibody are the same as the cysteine sites defined by the wild-type CH1 or CL sequence. However, for antibodies with mutations such as insertions or deletions, those skilled in the art can determine the cysteine sites based on the results of homology comparisons with the wild-type CH1 or CL sequences. It will be understood by those skilled in the art that the cysteine sites obtained after the above homology comparisons are also within the scope of protection of the present invention.

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.

In this article, the term "IGLK" refers to the light chain of a kappa-type (κ-type) antibody; the term "IGLL" refers to the light chain of a Lamda-type (λ-type) antibody. It should be noted that a person skilled in the art can compare the CL fragment sequence of a Lamda-type light chain with the CL fragment sequence of a Kappa-type light chain for homology, and determine the cysteine site of the CL fragment of the Lamda-type light chain based on the comparison result. A person skilled in the art can understand that a conjugate containing the cysteine site obtained after the above homology comparison for the CL fragment of a Lamda-type light chain or the CL fragment of a kappa-type light chain is also within the scope of protection of the present invention. Exemplarily, the homology comparison can be determined by the homology comparison 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, and the like.

In some optional embodiments of the present invention, positions 9, 14, 53, 54, and 67 of the CH1 fragment are numbered sequentially from the N-terminus to the C-terminus based on the amino acid sequence shown in CH1 of mouse wild-type IgHG1; positions 12, 14, 17, 28, 55, and 102 of the CL fragment are numbered sequentially from the N-terminus to the C-terminus based on the amino acid sequence shown in CL of mouse wild-type IGLK.

In this embodiment, the cysteine sites are numbered sequentially from N-terminus to C-terminus according to the amino acid sequences shown in CH1 of mouse wild-type IgG1 antibody (such as SEQ ID NO: 1) and CL of mouse wild-type kappa light chain (such as SEQ ID NO: 10). For example, the 1st position of the CH1 fragment is A, the 2nd position is K, the 3rd position is T, ..., and so on. The 9th position of the CH1 fragment refers to the amino acid Y located at the 9th position based on the amino acid sequence shown in SEQ ID NO: 1; the 17th position of the CL fragment refers to the amino acid Q located at the 17th position based on the amino acid sequence shown in SEQ ID NO: 10. At the same time, it should be noted that for other subtype antibodies of mouse origin (such as IgG2a, IgG2b) or IgG antibodies of other species (such as sheep, rabbit) (such as sheep IgG1 antibody, sheep IgG2 antibody, rabbit IgG antibody), those skilled in the art can determine the cysteine site based on the results of homology comparison between it and the CH1 or CL sequence of the kappa light chain of wild-type mouse IgG1. Those skilled in the art can understand that the conjugate containing the cysteine site obtained after the above-mentioned homology comparison is also within the scope of protection of the present invention.

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 cysteine sites described in this application are numbered sequentially from the N-terminus to the C-terminus based on the amino acid sequence shown in CH1 of a murine wild-type IgG1 antibody (such as SEQ ID NO: 1) and CL of a murine wild-type kappa light chain (such as SEQ ID NO: 10), but the same or similar positions numbered using other numbering systems also fall within the scope of protection of the present invention.

For example, for the combination of cysteine sites of "position 67 of the CH1 fragment and position 28 of the CL fragment" mentioned in the present invention, a person skilled in the art can compare the homology of CH1 of mouse wild-type IgG2a, IgG2b, IgG2c, and IgG3 antibodies with CH1 of mouse wild-type IgG1 antibodies, so as to obtain the cysteine sites of mouse wild-type IgG2a, IgG2b, IgG2c, and IgG3 antibodies. For example, the homology of CH1 of mouse wild-type IgG2a, IgG2b, IgG2c, and IgG3 is compared with CH1 of mouse wild-type IgG1, that is, position 67 of the CH1 fragment of mouse wild-type IgG2a, IgG2b, IgG2c, and IgG3 and position 28 of the CL fragment. Therefore, the conjugate containing mouse IgG2a, IgG2b, IgG2c, and IgG3 antibodies, and the cysteine sites of position 67 of the CH1 fragment and position 28 of the CL fragment in the antibody, also fall within the protection scope of the present invention.

In some optional embodiments of the present invention, due to the existence of different antibody subclasses, for the above-mentioned cysteine sites of "1) position 67 of the CH1 fragment and position 28 of the CL fragment; 2) position 9 of the CH1 fragment and position 17 of the CL fragment; 3) position 9 of the CH1 fragment and position 14 of the CL fragment", their equivalent sites may be the following cysteine sites:

Example A, the CH1 fragment and CL fragment of the antibody have cysteine sites selected from any of the following groups: 1) position 67 of the CH1 fragment and position 28 of the CL fragment; 2) position 9 of the CH1 fragment and position 17 of the CL fragment; 3) position 9 of the CH1 fragment and position 14 of the CL fragment; positions 9 and 67 of the CH1 fragment are numbered sequentially from the N-terminus to the C-terminus based on the amino acid sequence shown in the CH1 of the wild-type murine IgHG2a; positions 14, 17 and 28 of the CL fragment are numbered sequentially from the N-terminus to the C-terminus based on the amino acid sequence shown in the wild-type murine IGLK light chain.

Example B: The CH1 fragment and CL fragment of the antibody have cysteine sites selected from any of the following groups: 1) position 67 of the CH1 fragment and position 28 of the CL fragment; 2) position 9 of the CH1 fragment and position 17 of the CL fragment; 3) position 9 of the CH1 fragment and position 14 of the CL fragment; positions 9 and 67 of the CH1 fragment are numbered sequentially from the N-terminus to the C-terminus based on the amino acid sequence shown in the CH1 of the wild-type murine IgHG2b; positions 14, 17 and 28 of the CL fragment are numbered sequentially from the N-terminus to the C-terminus based on the amino acid sequence shown in the wild-type murine IGLK light chain.

In some optional embodiments of the present invention, due to the existence of different antibody sources (i.e., antibodies derived from different species), for the above-mentioned cysteine sites of "1) position 67 of the CH1 fragment and position 28 of the CL fragment; 2) position 9 of the CH1 fragment and position 17 of the CL fragment; 3) position 9 of the CH1 fragment and position 14 of the CL fragment", their equivalent sites may be the following cysteine sites:

Example A, the CH1 fragment and CL fragment of the antibody have cysteine sites selected from any of the following groups: 1) position 68 of the CH1 fragment and position 29 of the CL fragment; 2) position 9 of the CH1 fragment and position 18 of the CL fragment; 3) position 9 of the CH1 fragment and position 15 of the CL fragment; positions 9 and 68 of the CH1 fragment are numbered sequentially from the N-terminus to the C-terminus based on the amino acid sequence shown in CH1 of wild-type sheep IgHG; positions 15, 18 and 29 of the CL fragment are numbered sequentially from the N-terminus to the C-terminus based on the amino acid sequence shown in CL of wild-type sheep IGLL.

In some optional embodiments of the present invention, the sheep is selected from sheep.

In some optional embodiments of the present invention, the IgHG is selected from IgHG1 or IgHG2.

In some optional embodiments of the present invention, the amino acid sequence of CH1 of the sheep wild-type IgHG is shown in SEQ ID NO:7 or SEQ ID NO:8.

In some optional embodiments of the present invention, the amino acid sequence of CL of the sheep wild-type IGLL is shown in SEQ ID NO:11.

Example B: The CH1 fragment and CL fragment of the antibody have cysteine sites selected from any of the following groups: 1) position 68 of the CH1 fragment and position 28 of the CL fragment; 2) position 9 of the CH1 fragment and position 17 of the CL fragment; 3) position 9 of the CH1 fragment and position 14 of the CL fragment; positions 9 and 68 of the CH1 fragment are numbered sequentially from the N-terminus to the C-terminus based on the amino acid sequence shown in CH1 of rabbit wild-type IgHG; positions 14, 17 and 28 of the CL fragment are numbered sequentially from the N-terminus to the C-terminus based on the amino acid sequence shown in CL of rabbit wild-type IGLK.

In some optional embodiments of the present invention, the amino acid sequence of CH1 of the rabbit wild-type IgHG is as shown in SEQ ID NO:9.

In some optional embodiments of the present invention, the amino acid sequence of CL of the rabbit wild-type IGLK is shown in SEQ ID NO:12.

In some optional embodiments of the present invention, the CH1 fragment and CL fragment of the antibody have cysteine sites selected from any of the following groups: 1) S67C in the CH1 fragment and F28C in the CL fragment; 2) Y9C in the CH1 fragment and Q17C in the CL fragment; 3) Y9C in the CH1 fragment and S14C in the CL fragment; 4) P54C in the CH1 fragment and S55C in the CL fragment; 5) F53C in the CH1 fragment and S55C in the CL fragment; 6) G14C in the CH1 fragment and P12C in the CL fragment; 7) G14C in the CH1 fragment and F102C in the CL fragment.

In some optional embodiments of the present invention, positions 9, 14, 53, 54, and 67 of the CH1 fragment are numbered sequentially from the N-terminus to the C-terminus based on the amino acid sequence shown in CH1 of mouse wild-type IgHG; positions 12, 14, 17, 28, 55, and 102 of the CL fragment are numbered sequentially from the N-terminus to the C-terminus based on the amino acid sequence shown in CL of mouse wild-type IGLK.

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

In some optional embodiments of the present invention, the IgHG is selected from IgHG1, IgHG2a, IgHG2b, IgHG2c or IgHG3.

In some optional embodiments of the present invention, the amino acid sequence of CH1 of the murine wild-type IgHG is shown as SEQ ID NO:1 or SEQ ID NO:2.

In some optional embodiments of the present invention, the amino acid sequence of CL of the murine wild-type IGLK is shown in SEQ ID NO:10.

In some optional embodiments of the present invention, due to the existence of different antibody subclasses, for the above-mentioned cysteine sites of "1) S67C in the CH1 fragment and F28C in the CL fragment; 2) Y9C in the CH1 fragment and Q17C in the CL fragment; 3) Y9C in the CH1 fragment and S14C in the CL fragment", their equivalent sites may be the following cysteine sites:

Example A, the CH1 fragment and CL fragment of the antibody have cysteine sites selected from any of the following groups: 1) S67C in the CH1 fragment and F28C in the CL fragment; 2) Y9C in the CH1 fragment and Q17C in the CL fragment; 3) Y9C in the CH1 fragment and S14C in the CL fragment; positions 9 and 67 of the CH1 fragment are numbered sequentially from the N-terminus to the C-terminus based on the amino acid sequence shown in the CH1 of the mouse wild-type IgHG2a; positions 14, 17 and 28 of the CL fragment are numbered sequentially from the N-terminus to the C-terminus based on the amino acid sequence shown in the CL of the mouse wild-type IGLK.

Example B: The CH1 fragment and CL fragment of the antibody have cysteine sites selected from any of the following groups: 1) S67C in the CH1 fragment and F28C in the CL fragment; 2) Y9C in the CH1 fragment and Q17C in the CL fragment; 3) Y9C in the CH1 fragment and S14C in the CL fragment; positions 9 and 67 of the CH1 fragment are numbered sequentially from the N-terminus to the C-terminus based on the amino acid sequence shown in the CH1 of the mouse wild-type IgHG2b; positions 14, 17 and 28 of the CL fragment are numbered sequentially from the N-terminus to the C-terminus based on the amino acid sequence shown in the CL of the mouse wild-type IGLK.

In some optional embodiments of the present invention, due to the existence of different antibody sources (i.e., antibodies derived from different species), for the above-mentioned cysteine sites of "1) S67C in the CH1 fragment and F28C in the CL fragment; 2) Y9C in the CH1 fragment and Q17C in the CL fragment; 3) Y9C in the CH1 fragment and S14C in the CL fragment", their equivalent sites may be the following cysteine sites:

Example A, the CH1 fragment and CL fragment of the antibody have the following cysteine sites selected from any group: 1) V68C in the CH1 fragment and L29C in the CL fragment; 2) Y9C in the CH1 fragment and E18C in the CL fragment; 3) Y9C in the CH1 fragment and S15C in the CL fragment; positions 9 and 68 of the CH1 fragment are numbered sequentially from the N-terminus to the C-terminus based on the amino acid sequence shown in the CH1 of the sheep wild-type IgHG; positions 15, 18, and 29 of the CL fragment are numbered sequentially from the N-terminus to the C-terminus based on the amino acid sequence shown in the CL of the sheep wild-type IGLL.

In some optional embodiments of the present invention, the sheep is selected from sheep.

In some optional embodiments of the present invention, the IgHG is selected from IgHG1 or IgHG2.

In some optional embodiments of the present invention, the amino acid sequence of CH1 of the sheep wild-type IgHG is shown in SEQ ID NO:7 or SEQ ID NO:8.

In some optional embodiments of the present invention, the amino acid sequence of CL of the wild-type sheep IGLL is shown in SEQ ID NO: 11. Example B, the CH1 fragment and CL fragment of the antibody have cysteine sites selected from any of the following groups: 1) V68C in the CH1 fragment and V28C in the CL fragment; 2) F9C in the CH1 fragment and Q17C in the CL fragment; 3) F9C in the CH1 fragment and A14C in the CL fragment; the 9th and 68th positions of the CH1 fragment are numbered sequentially from the N-terminus to the C-terminus based on the amino acid sequence shown in CH1 of the wild-type rabbit IgHG; the 14th, 17th and 28th positions of the CL fragment are numbered sequentially from the N-terminus to the C-terminus based on the amino acid sequence shown in CL of the wild-type rabbit IGLK.

In some optional embodiments of the present invention, the amino acid sequence of CH1 of the rabbit wild-type IgHG is as shown in SEQ ID NO:9.

In some optional embodiments of the present invention, the amino acid sequence of CL of the rabbit wild-type IGLK is shown in SEQ ID NO:12.

In some optional embodiments of the present invention, the antibody has a CH1 fragment and a CL fragment selected from any of the following groups or having more than 90% identity therewith:

CH1 fragment CL snippet 1 SEQ ID NO:13 SEQ ID NO:14 2 SEQ ID NO:15 SEQ ID NO:16 3 SEQ ID NO:15 SEQ ID NO:17 4 SEQ ID NO:18 SEQ ID NO:19 5 SEQ ID NO:20 SEQ ID NO:19 6 SEQ ID NO:21 SEQ ID NO:22 7 SEQ ID NO:21 SEQ ID NO:23 8 SEQ ID NO:46 SEQ ID NO:14 9 SEQ ID NO:47 SEQ ID NO:16 10 SEQ ID NO:47 SEQ ID NO:17 11 SEQ ID NO:52 SEQ ID NO:14 12 SEQ ID NO:53 SEQ ID NO:16 13 SEQ ID NO:53 SEQ ID NO:17 14 SEQ ID NO:58 SEQ ID NO:60 15 SEQ ID NO:59 SEQ ID NO:61 16 SEQ ID NO:59 SEQ ID NO:62 17 SEQ ID NO:67 SEQ ID NO:69 18 SEQ ID NO:68 SEQ ID NO:70 19 SEQ ID NO:68 SEQ ID NO:71

.

It should be noted that, due to the presence of alleles or other mutations, the antibodies of the present invention can be selected from CH1 fragments and CL fragments that have more than 90% identity with any group of CH1 fragments and CL fragments in the above table. For example, for a mouse wild-type IgG1 antibody, there may be multiple wild-type IgG1 antibody CH1s, and the CH1s of the multiple wild-type IgG1 antibodies have allele differences, but they are all mouse wild-type IgG1 antibodies.

Illustratively, as for the CH1 fragments of the amino acid sequences shown in SEQ ID NO:1 and SEQ ID NO:2, there are 4 allele differences in the CH1 fragments of the two sequences. The antibody in the conjugate of the present invention may include a CH1 fragment in which a cysteine mutation is performed on the basis of the CH1 fragment of the amino acid sequence shown in SEQ ID NO:2, and its cysteine site is consistent with the cysteine site on the CH1 fragment of the amino acid sequence shown in SEQ ID NO:1.

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 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.

As used herein, the term "more than 90% identity" refers to at least 90% identity to each reference sequence, and may be 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% identity.

In some optional embodiments of the present invention, the antibody further comprises an Fc fragment.

In some optional embodiments of the present invention, the C-terminus of the CH1 fragment is connected to the N-terminus of the Fc fragment.

Herein, the "Fc fragment" of the present invention includes but is not limited to the Fc fragment of the heavy chain constant region selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD or a mutant thereof. The specific sequence is not limited and is within the scope of protection of the present invention.

Illustratively, the amino acid sequence of the Fc fragment is shown in SEQ ID NO:24, SEQ ID NO:48, SEQ ID NO:54, SEQ ID NO:63 or SEQ ID NO:72.

In some optional embodiments of the present invention, the antibody further comprises a hinge region.

In some optional embodiments of the present invention, the N-terminus of the hinge region is connected to the C-terminus of the CH1 fragment, and the C-terminus of the hinge region is connected to the N-terminus of the Fc fragment. The specific sequences are not limited and are within the protection scope of the present invention.

Herein, the "hinge region" of the present invention includes but is not limited to the hinge region of the heavy chain constant region selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD or a mutant thereof. The specific sequence is not limited and is within the scope of protection of the present invention.

Illustratively, the amino acid sequence of the hinge region is shown in SEQ ID NO:25, SEQ ID NO:49, SEQ ID NO:55, SEQ ID NO:64 or SEQ ID NO:73.

In some optional embodiments of the present invention, the antibody includes a heavy chain constant region as shown in any one of SEQ ID NO:26 to SEQ ID NO:30, SEQ ID NO:50 to SEQ ID NO:51, SEQ ID NO:56 to SEQ ID NO:57, SEQ ID NO:65 to SEQ ID NO:66 and SEQ ID NO:74 to SEQ ID NO:75, or an amino acid sequence having more than 90% identity therewith.

In some optional embodiments of the present invention, the CH1 fragment and the CL fragment are connected via an interchain disulfide bond.

In some optional embodiments of the present invention, a disulfide bond is formed between the sulfhydryl groups of two cysteine residues in each group of cysteine sites.

In some optional embodiments of the present invention, the antibody further comprises a heavy chain variable region and/or a light chain variable region.

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 CH1 fragment, and/or the C-terminus of the light chain variable region is connected to the N-terminus of the CL fragment.

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

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

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

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:31, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:32.

In some optional embodiments of the present invention, the antibody has a heavy chain and a light chain selected from any of the following groups:

Heavy chain Light chain 1 SEQ ID NO:33 SEQ ID NO:34 2 SEQ ID NO:35 SEQ ID NO:36 3 SEQ ID NO:35 SEQ ID NO:37 4 SEQ ID NO:38 SEQ ID NO:39 5 SEQ ID NO:40 SEQ ID NO:39 6 SEQ ID NO:41 SEQ ID NO:42 7 SEQ ID NO:41 SEQ ID NO:43 8 SEQ ID NO:78 SEQ ID NO:79 9 SEQ ID NO:80 SEQ ID NO:81

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

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

In some optional embodiments of the present invention, the conjugated moiety is linked to the sulfhydryl group of the cysteine site.

In some optional embodiments of the present invention, the conjugated part is selected from at least one of a carrier and a tag.

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

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

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

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.

In some specific 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.

In some optional embodiments of the present invention, a linker molecule is further included, and the conjugated part is connected to the active group through the linker molecule.

In some optional embodiments of the present invention, the linker molecule is linked to the sulfhydryl groups of two paired cysteine residues in the antibody.

In some optional embodiments of the present invention, the linker molecule is linked to the sulfhydryl groups of the two paired cysteine residues in the antibody, and the linker molecule is linked to the conjugated moiety.

In some optional embodiments of the present invention, the linker molecule forms a sulfur-carbon bridge connection with the antibody chain.

In some optional embodiments of the present invention, the linker molecule has a structure selected from the following:

In some optional embodiments of the present invention, the antibody conjugate comprises the following structure:

wherein n and m are each independently selected from any integer between 0 and 24 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24); A is the conjugated portion; and B is the antibody.

In some optional embodiments of the present invention, A is a vector.

In some optional embodiments of the present invention, the antibody conjugate comprises the following structure:

wherein n and m are each independently selected from any integer between 0 and 24 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24), and the S atom in the sulfur-carbon bridge is the S atom of cysteine in the antibody.

As used herein, the term "sulfur-carbon bridge" refers to a -S-C- structure.

In some optional embodiments of the present invention, n=4, m=4.

Antibody

In the second aspect of the present invention, the present invention proposes an antibody. According to an embodiment of the present invention, the antibody comprises a heavy chain constant region and a light chain constant region; wherein the heavy chain constant region comprises a CH1 fragment, and the CH1 fragment is consistent with the CH1 fragment defined by the conjugate described in the first aspect; the light chain constant region comprises a CL fragment, and the CL fragment is consistent with the CL fragment defined by the conjugate described in the first aspect. 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.

In some optional embodiments of the present invention, the above antibody may further include at least one of the following additional technical features:

In some optional embodiments of the present invention, the antibody further comprises an Fc fragment.

In some optional embodiments of the present invention, the C-terminus of the CH1 fragment is connected to the N-terminus of the Fc fragment.

Herein, the "Fc fragment" of the present invention includes but is not limited to the Fc fragment of the heavy chain constant region selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD or a mutant thereof. The specific sequence is not limited and is within the scope of protection of the present invention.

Illustratively, the amino acid sequence of the Fc fragment is shown in SEQ ID NO:24, SEQ ID NO:48, SEQ ID NO:54, SEQ ID NO:63 or SEQ ID NO:72.

In some optional embodiments of the present invention, the antibody further comprises a hinge region.

In some optional embodiments of the present invention, the N-terminus of the hinge region is connected to the C-terminus of the CH1 fragment, and the C-terminus of the hinge region is connected to the N-terminus of the Fc fragment. The specific sequences are not limited and are within the protection scope of the present invention.

Herein, the "hinge region" of the present invention includes but is not limited to the hinge region of the heavy chain constant region selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD or a mutant thereof. The specific sequence is not limited and is within the scope of protection of the present invention.

Illustratively, the amino acid sequence of the hinge region is shown in SEQ ID NO:25, SEQ ID NO:49, SEQ ID NO:55, SEQ ID NO:64 or SEQ ID NO:73.

In some optional embodiments of the present invention, the antibody includes a heavy chain constant region as shown in any one of SEQ ID NO:26 to SEQ ID NO:30, SEQ ID NO:50 to SEQ ID NO:51, SEQ ID NO:56 to SEQ ID NO:57, SEQ ID NO:65 to SEQ ID NO:66 and SEQ ID NO:74 to SEQ ID NO:75, or an amino acid sequence having more than 90% identity therewith.

In some optional embodiments of the present invention, the CH1 fragment and the CL fragment are connected via an interchain disulfide bond.

In some optional embodiments of the present invention, a disulfide bond is formed between the sulfhydryl groups of two cysteine residues in each group of cysteine sites.

In some optional embodiments of the present invention, the antibody further comprises a heavy chain variable region and/or a light chain variable region.

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 CH1 fragment, and/or the C-terminus of the light chain variable region is connected to the N-terminus of the CL fragment.

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. Please refer to the target antigen in the conjugate described in the first aspect.

Illustratively, the target antigen is CA153, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:31, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:32.

In some optional embodiments of the present invention, the antibody comprises at least one selected from polyclonal antibody, full-length monoclonal antibody, Fab antibody, Fab' antibody, F(ab') ₂ antibody, F(ab) ₂ antibody, Fv antibody, single-chain antibody, single-domain antibody and minimum recognition unit.

As used herein, the terms "full-length antibody", "full-length monoclonal antibody" or "full-length monoclonal antibody" are 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).

In this article, the terms "polyantibody" and "multispecific antibody" are synonymous and both refer to antibodies that can recognize multiple antigenic epitopes, such as antibodies that can recognize two antigenic epitopes (bispecific antibodies, abbreviated as bispecific antibodies), antibodies that can recognize three antigenic epitopes, or antibodies that can recognize four antigenic epitopes. This is understood in a broad sense and the specific structure is not limited as long as it can recognize multiple antigenic epitopes.

In this article, the terms "single domain antibody", "nanoantibody" and "VHH antibody" are used interchangeably, which were originally described as antigen-binding immunoglobulin (variable) domains of "heavy chain antibodies" (i.e., "antibodies lacking light chains") (Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R.: "Naturally occurring antibodies devoid of light chains"; Nature 363, 446-448 (1993)), comprising a heavy chain variable region (VH) and conventional CH2 and CH3 regions, which specifically bind to antigen proteins through the heavy chain variable region.

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.

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

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.

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.

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.

In some optional embodiments of the present invention, the antibody comprises or is a heavy chain and a light chain as shown below:

Heavy chain Light chain 1 SEQ ID NO:33 SEQ ID NO:34 2 SEQ ID NO:35 SEQ ID NO:36 3 SEQ ID NO:35 SEQ ID NO:37 4 SEQ ID NO:38 SEQ ID NO:39 5 SEQ ID NO:40 SEQ ID NO:39 6 SEQ ID NO:41 SEQ ID NO:42 7 SEQ ID NO:41 SEQ ID NO:43 8 SEQ ID NO:78 SEQ ID NO:79 9 SEQ ID NO:80 SEQ ID NO:81

.

It should be noted that, based on the amino acid sequence of the antibody disclosed herein, 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, 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 disclosed herein, it belongs to the protection scope of the present disclosure.

Nucleic acid molecules, vectors, cells or hosts

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.

In the third 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.

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

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.

In the fourth 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 third 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.

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

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

In the fifth 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 third aspect or the vector described in the fourth 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.

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

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.

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.

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.

Use and method of preparing conjugates

In the sixth 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 third aspect, the vector described in the fourth aspect, or the cell or host described in the fifth aspect in the preparation of a conjugate or a kit.

In the seventh 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 and the conjugation part to carry out a first conjugation reaction to obtain the conjugate. Thus, the conjugate can be effectively prepared, and the preparation method is simple.

In some optional embodiments of the present invention, before the conjugation reaction, a second conjugation reaction is performed on the cross-linking agent and the antibody.

In some optional embodiments of the present invention, the cross-linking agent is selected from at least one of bissulfone compounds, maleimide compounds, pyridazine compounds, bisvinylsulfonamide compounds, allyl sulfone compounds, brominated pyridinedione compounds, divinylpyridine compounds, N-substituted-3-bromo-5-methylenepyrrole-2-one compounds, succinimide compounds, imidoester compounds and azidebenzene compounds.

In some optional embodiments of the present invention, the cross-linking agent is selected from at least one of bissulfone compounds, maleimide compounds, pyridazine compounds, bisvinylsulfonamide compounds, allyl sulfone compounds, brominated pyridinedione compounds, divinylpyridine compounds and N-substituted-3-bromo-5-methylenepyrrole-2-one compounds.

In some optional embodiments of the present invention, the cross-linking agent is selected from at least one of 2-(tosyl)methacrylamide-PEG4-amide-PEG4-methyltetrazine, maleimide, pyridine disulfide, vinyl sulfone, iodoacetyl, bromoacetyl and cysteine derivatives.

In some optional embodiments of the present invention, the cross-linking agent is selected from 2-(toluenesulfonyl)methacrylamide-PEG4-amide-PEG4-methyltetrazine.

In some optional embodiments of the present invention, the conjugation reaction is carried out by at least one of the following methods: mixed anhydride method, carbodiimide method, glutaraldehyde method, glutaric anhydride method, diazotization method, succinic anhydride method, carbonyldiimidazole method, click chemistry method and sodium periodate method.

Reagents or kits and their uses

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.

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 third aspect, the vector described in the fourth aspect, the cell or host described in the fifth aspect, or the reagent or kit described in the eighth aspect in immunoassay or preparation of immunoassay products.

Immunoassay method and method for improving conjugate activity, stability or sensitivity

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.

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.

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.

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

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 a CH1 fragment and a CL fragment in the antibody into one or more groups of cysteine sites, wherein the cysteine sites are consistent with the cysteine sites defined by the conjugate described in the first aspect.

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.

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.

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 (M. J. Gait, ed., 1984); Animal Cell Culture (R. I. Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos, eds., 1987); Current Protocols in Molecular Biology (Current Protocols in Molecular Biology, 1996). "PCR: The Polymerase Chain Reaction" (Mullis et al., 1994); and "Current Protocols in Immunology" (J.E. Coligan et al., 2011), each of which is expressly incorporated herein by reference.

Preparation Example 1: Preparation of 2-(Toluenesulfonyl)methacrylamide-PEG4-amide-PEG4-methyltetrazine

The preparation steps are as follows:

(1) Preparation of 2-(Toluenesulfonyl)methacrylamide-(PEG)4-amide (TSMA)

①

a. Dissolve tert-butyloxycarbonyl-imino-tetraethylene glycol-amine (compound A) in 10 mL DMF (N,N-dimethylformamide), add methacrylic acid (compound B), DCC (dicyclohexylcarbodiimide) and NHS (N-hydroxysuccinimide), and stir magnetically at 25°C for 12 hours.

b. The solvent was removed by rotary evaporation, and the crude product was dissolved in 10 mL of deionized water. The precipitate was removed by filtration. After the solvent was removed by rotary evaporation, column chromatography was used for purification to obtain tert-butyloxycarbonyl-imino-tetraethylene glycol-methacrylamide (Compound C) with a yield of about 90%.

In the above steps, methacrylic acid can be replaced by, but not limited to, methacrylic halide (such as methacrylic chloride, methacrylic bromide, methacrylic fluoride, etc.) or methacrylic anhydride. When methacrylic halide or methacrylic anhydride is used, the synthesis steps are as follows:

a. The tert-butyloxycarbonyl-imino-tetraethylene glycol-amine (compound A) was dissolved in 10 mL of dichloromethane, triethylamine, methacrylic halide or methacrylic anhydride were added at 0°C, and magnetic stirring was performed for 12 hours.

b. The solvent was removed by rotary evaporation, and the crude product was dissolved in 30 mL of dichloromethane, washed with 1 M HCl and saturated sodium chloride solution, and the organic layer was dried with anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. Column chromatography was used for purification to obtain tert-butyloxycarbonyl-imino-tetraethylene glycol-methacrylamide (Compound C), with a yield of about 90%.

②

a. Dissolve compound C in 5 mL of dichloromethane, add 4-toluenesulfonyl chloride (compound D), and stir magnetically at 25°C for 24 hours.

b. Then triethylamine was added and stirred for 12 hours. The crude product was washed with 1M HCl, saturated sodium bicarbonate solution, and saturated sodium chloride solution, dried over anhydrous magnesium sulfate, and the solvent was removed by rotary evaporation.

c. The crude product was dissolved in 10 mL of ethyl acetate, and triethylamine was added and refluxed at 0°C for 12 hours. After the solvent was removed by rotary evaporation, column chromatography was used for purification to obtain tert-butyloxycarbonyl-2-(tosyl)methacrylate (Compound E) with a yield of about 70%.

In the above steps, 4-toluenesulfonyl chloride can be replaced by, but not limited to, sodium 4-toluenesulfinate. When sodium 4-toluenesulfinate is used, steps a and b in the above synthesis steps are as follows:

a. Dissolve compound C in 5 mL of dichloromethane, add sodium 4-toluenesulfinate and iodine, and stir magnetically at 25°C for 72 hours.

b. Then triethylamine was added and stirred for 12 hours. The crude product was washed with 1M HCl, saturated sodium bicarbonate solution, saturated sodium thiosulfate solution, and saturated sodium chloride solution, dried over anhydrous magnesium sulfate, and the solvent was removed by rotary evaporation.

cThe steps are the same.

③ Compound E was dissolved in 5 mL of dichloromethane, trifluoroacetic acid (278 mg, 2.50 mmol) was added, and magnetic stirring was performed at 25° C. for 12 hours. The solvent was removed by rotary evaporation to obtain TSMA with a yield of about 98%.

(2) Preparation of 2-(Toluenesulfonyl)methacrylamide-PEG4-amide-PEG4-methyltetrazine (TSMA-MTz)

① Dissolve TSMA (13 mg, 0.03 mmol) in 2 mL of dichloromethane, add DIEA (N, N-diisopropylethylamine, 7.5 mg, 0.06 mmol) or triethylamine (6.1 mg, 8.4 μL, 0.06 mmol), and stir to dissolve. Dissolve methyl tetrazine-PEG4-NHS ester (compound F, purchased from Xi'an Kangfuno Biotechnology Co., Ltd., 16.7 mg, 0.03 mmol) in 1 mL of dichloromethane, dissolve evenly, and add to the above TSMA solution.

② The mixture was magnetically stirred at 25° C. for 12 hours, and the solvent was removed by rotary evaporation. The crude product was dissolved in 20 mL of dichloromethane, washed with saturated sodium chloride solution, and dried over anhydrous magnesium sulfate. The solvent was removed by rotary evaporation, and then purified by column chromatography (dichloromethane: methanol = 20:1) to obtain 2-(toluenesulfonyl)methacrylamide-PEG4-amide-PEG4-methyltetrazine (Compound G, TSMA-MTz) with a yield of about 50%.

The product was subjected to liquid chromatography-mass spectrometry and nuclear magnetic resonance detection. The detection results are shown in Figures 5-9, and the results prove that the method successfully prepared 2-(toluenesulfonyl)methacrylamide-PEG4-amide-PEG4-methyltetrazine.

Embodiment 1:

1. Determination of mutation point

The heavy chain and light chain amino acid sequences of mouse IgG1 (the amino acid sequences of the heavy chain and light chain are shown in SEQ ID NO: 44 and SEQ ID NO: 45, respectively, which are wild type) were input into Molecular Operating Environment (MOE), and 7 groups of amino acids were selected on CH1 and CL1 to be mutated to C. Among them, the mutation sites (mutation sites are numbered sequentially from the N-terminus of CH1 or CL to the first amino acid at the C-terminus) and amino acid sequences of the 7 groups (mutation group 1 to mutation group 7) of mouse IgG1 antibodies after mutation are shown in Table 1:

Table 1: Mutation sites and amino acid sequences of mutation groups 1 to 7

2. Plasmid construction and expression

2.1 Construction of recombinant antibody expression plasmid

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 RACE cDNA Amplification Kit was purchased from Takara. pMD-18T vector was purchased from Takara. Plasmid extraction kit was purchased from Tiangen. Primer synthesis and gene sequencing were completed by Gene Sequencing Company. pcDNA ^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.

According to the sequencing results of the antibody variable region genes in the above pMD-18T, the VL and VH gene-specific primers of the Anti-CA153 antibody were designed, with restriction endonuclease cutting sites and protective bases at both ends, respectively. The 0.72KB light chain (Light Chain) gene fragment and the 1.40kb heavy chain (Heavy Chain) gene fragment were amplified by PCR amplification method.

The Heavy Chain and Light Chain gene fragments were double-digested with restriction endonucleases, and the 3.4A 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 respectively connected to the 3.4A expression vector to obtain the recombinant expression plasmids of the Heavy Chain and Light Chain, respectively.

2.2 Expression of recombinant antibody samples

Resuscitate CHOK1 cells in advance, subculture to a 500ml system, so that the cell density reaches (3-5)×10 ⁶ cells/ml and the cell viability is >95%; wash the cells by centrifugation, re-dissolve with culture medium, and adjust the cell density to 2.9×10 ⁶ cells/ml as a cell diluent. Use culture medium to prepare transfection reagent diluent and plasmid DNA diluent of the recombinant expression plasmid obtained in step 2.1 of this application. Add the transfection reagent diluent to the plasmid DNA diluent, mix well and let stand at room temperature for 15 minutes; slowly add the mixture to the cell diluent 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, with a rotation speed of 120rmp and a CO ₂ content of 8%. Centrifuge and collect samples after 13 days to obtain each cell culture.

3. Purification of recombinant antibody samples and SDS-PAGE identification

Each cell culture obtained in Example 2.2 was centrifuged at 1000 rpm for 5 minutes, and the culture supernatant was recovered. The culture supernatant contained the secreted antibodies from the transfected cells. The obtained culture supernatant was centrifuged again at 10000×g for 10 minutes, and the supernatant was recovered and filtered through a 0.22 μm membrane. The clarified supernatant was affinity purified using a protein A (Mab select SuRe LX, Cytiva) affinity chromatography column.

6 μg of purified antibody was subjected to reducing SDS-PAGE (4-12% GenScript precast gel) and non-reducing SDS-PAGE (4-12% GenScript precast gel). The electrophoretic patterns were shown in Figures 1 and 2, wherein, in Figures 1 and 2, lane 1: wild type, lane 2: mutant group 1, lane 3: mutant group 2, lane 4: mutant group 3, lane 5: mutant group 4, lane 6: mutant group 5, lane 7: mutant group 6, lane 8: mutant group 7.

In Figure 1, two bands were shown after reducing SDS-PAGE, one with Mr of 50 KD (heavy chain) and the other with Mr of 28 KD (light chain).

In Figure 2, the newly added disulfide bonds of mutation groups 1 to 7 were fully assembled, and the expression stability and assembly of mutation groups 2, 3, 4, 5, 6 and 7 were comparable to those of WT.

4. HPLC-SEC (High Performance Liquid Chromatography-Size Exclusion Chromatography) Detection of Recombinant Antibody Samples

20 μg of each of the purified recombinant antibody samples obtained in step 3 of this example were taken, and the SEC purity of each recombinant antibody (mutation group 1 to mutation group 7) was determined by high performance liquid chromatography-size exclusion chromatography (HPLC-SEC). The peak area normalization method was used to analyze the purification, and the SEC results are shown in Table 2.

Table 2: HPLC-SEC results corresponding to mutation groups 1 to 7

name Polymer % Main peak SEC% Wild type 1.5 98.5 Mutation Group 1 0.5 99.5 Mutation Group 2 0.92 99.18 Mutation group 3 2.66 98.34 Mutation Group 4 0.46 99.54 Mutation Group 5 0.21 99.79 Mutation Group 6 0.29 99.71 Mutation Group 7 1.06 98.94

As shown in Table 2, mutation groups 1 to 7 all formed complete IgG1 antibodies.

5. Determination of thermal stability Tm value

Take multiple groups of recombinant antibody samples obtained in step 3 of this example after purification, dilute each recombinant antibody (mutation group 1 to mutation group 7) to 1 mg/ml, and measure the thermal stability Tm of the samples by DSF (differential scanning fluorescence). The test results are shown in Table 3.

Table 3: Test results of thermal stability Tm of mutation group 1 to mutation group 7

From the △Tm in Table 3, it can be seen that the thermal stability of mutation groups 1 to 6 is significantly improved, and mutation groups 2 and 6 have the best thermal stability, with the highest improvement reaching 9°C.

6. Stability test of mutation groups 1 to 7 after labeling with AE (acridinium ester)

Steps for labeling AE (acridinium ester):

1) Buffer exchange: The purified multiple groups of recombinant antibody samples (hereinafter referred to as antibodies) obtained in step 3 of this example were dialyzed using dialysis bags for 2 hours, with the buffer exchanged every 45 minutes.

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 antibody-AE.

3) Removal of impurities: After the reaction is completed, the antibody-AE is dialyzed, and the solution is changed every 30 to 60 minutes on the first day (at least 4 times), and after overnight, the solution is changed every 60 minutes on the second day (at least 4 times).

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

Steps for testing the stability of antibody-AE using the double antibody sandwich method:

Take 50 μL of the above-prepared antibody-AE, add 50 μL of Ab-magnetic bead working solution, add 50 μL of Ab-AE working solution, and react at 37°C for 15 minutes. Wash the reacted solution three times with 1*PBST. Then add excitation solution A + excitation solution B, read the value immediately, mark mutation group 1 to mutation group 7 with AE, and test at 37°C for 7 days, with 4°C as the control, and compare the stability (within 10%, it is considered qualified for 7 days), and the test results are shown in Figure 3.

As can be seen from FIG3 , compared with the wild-type antibody, the stability of mutation groups 1 to 7 after AE labeling is improved.

7. Site-directed labeling activity identification of mutation groups 1 to 7

Select mutation group 2, mutation group 3, mutation group 5, mutation group 6 and mutation group 7 to arrange for site-specific labeling to detect activity. The specific detection method is as follows:

1) Antibody reduction: Take the purified multiple groups of recombinant antibody samples obtained in step 3 of this example (hereinafter referred to as antibodies), and replace the antibodies (5 mg/mL) with PB (50 mM, pH 7.4, 1 mM EDTA) buffer using a Zeba ^TM desalting column (10K MWCO). TCEP (Sigma) was prepared into a 10 mM solution using PB (50 mM, pH 7.4, 1 mM EDTA). 2 eq (2 equal amounts) of TCEP was added to the antibody solution, and the reaction was carried out at 37°C with gentle shaking (400 rpm) for 2 hours. The reduced antibodies were stored at 4°C for future use.

2) Reconstruction of antibody disulfide bonds 2-(Toluenesulfonyl)methacrylamide-PEG4-amide-PEG4-methyltetrazine (TSMA-MTz, for its preparation method, see Preparation Example 1) Introduction of linker molecules: TSMA-MTz was prepared into a 10mM solution with DMSO under dry conditions. 20eq of TSMA-MTz was added to the reduced antibody solution and reacted at 4°C for 16 hours. The excess chemical reagents in the antibody solution were replaced with a PB (50mM, pH7.4) buffer using a Zeba ^TM desalting column (10K MWCO). Antibody-methyltetrazine (antibody-MTz) was prepared, diluted to 4mg/mL with PB (50mM, pH7.4), and stored at 4°C for later use.

3) Preparation of antibody-AE (acridinium ester) conjugate: antibody-methyl tetrazine (4 mg/mL) and acridinium ester-trans-cyclooctene (4 mg/mL, dissolved in DMSO) were cross-linked at a molar ratio of 1:10 and reacted at 25°C for one hour to obtain antibody-AE (acridinium ester) conjugate. After the reaction, it was stored at 4°C for future use.

4) Activity and stability test of antibody-AE (acridinium ester) conjugate: Take 100μl of bio-labeled paired antibody and 100μl of step 3) antibody-AE conjugate, mix with different concentrations of CA153 quality control (0U/mL, 1U/mL, 10.3U/mL, 292.9U/mL), incubate at 37℃ for 10min, then add 23μl magnetic bead working solution, incubate for another 10min, wash 6 times with 280μL of washing solution PBST, add 100μL of excitation solution A. On-machine detection: add 100μL of excitation solution B, gain 100000.

The antibody activity during directional labeling is calculated as shown in Table 4, and the signal-to-noise ratio P/N (P is the reading value of the experimental group at different sample concentrations, and N is the control reading value) is shown in Table 5.

The improvement in low-value activity and low-value sensitivity of antibodies during directional labeling are calculated as shown in Table 6.

Table 4: Activity results of each antibody after site-specific labeling

Table 5: Signal-to-noise ratio P/N after site-specific labeling of each antibody

Table 6: Improvement in low-value activity and low-value sensitivity after site-specific labeling of each antibody

As can be seen from Table 6, the antibodies of mutation group 3, mutation group 5, mutation group 6 and mutation group 7 are superior to the wild-type antibody, the low-value activity is improved by more than 10%, and the low-value sensitivity P/N is improved by more than 4%.

Example 2: Mutation design of other species and subtype antibodies and verification of their effects

1. The above mouse IgG1 antibody was sequence aligned and spatially simulated with other subclasses of mouse antibodies (IgG2a, IgG2b) and antibodies of other species (sheep, rabbit). The sequence alignment results are shown in Figure 4 (Figure 4 is a CH1 and CL sequence alignment diagram of different subclasses and species, and the black boxes are mutation sites and their equivalent sites).

The mutation sites of other subclasses of mouse antibodies (IgG2a, IgG2b) and antibodies of other species (sheep, rabbit) as well as the amino acid sequences of CH1 and CL are shown in Table 7.

Table 7: Mutation sites of mouse antibodies of other subclasses and antibodies of other species and the amino acid sequences of CH1 and CL

2. Verification of stability effects of other species and subtypes of antibodies

The purified target antibody was prepared by the method of step 2 and step 3 of Example 1, and the stability test was performed. The results showed that the stability of the antibodies described in Table 7 was improved compared with the wild type. The stability verification process and results of the mouse antibody IgG2a are shown in detail as follows. 2.1 Stability Verification of Mouse Antibody IgG2a

The mutation effects of mutation group 3 and mutation group 5 were verified on mouse IgG2a antibody (anti-CRP antibody). The amino acid sequences of the mutant antibodies are shown in Table 8:

Table 8: Mutant antibody sequences of mouse anti-IgG2a

The purified target antibody was prepared by the method of step 2 and step 3 of Example 1, and then 2 mg of each antibody sample was placed at 4°C, 37°C, 45°C for 7 days, -20°C for 21 days, or frozen and thawed 5 times at -20°C. The sample after the test was taken to detect the activity of the antibody through the fluorescent POCT platform. The specific method is as follows:

1) Antibody labeling: Take 100ul of 1% fluorescent microspheres, add 900ul of activation buffer and mix, centrifuge to remove the supernatant, add 1mL of activation buffer and mix by sonication, then add activator (EDC NHS), shake and mix in the dark for 20min, centrifuge to remove the supernatant, add coupling buffer with the same volume as the microspheres, mix by sonication, add 0.2-0.4mg of labeled antibody, shake and mix in the dark for 3h, finally add blocking buffer for blocking, shake and mix in the dark for 45min, terminate labeling, centrifuge to remove the supernatant, re-dissolve the microspheres with microsphere preservation solution, mix by sonication, and store at 4℃ for use.

2) Prepare microsphere working solution: dilute the antibody marker to a final concentration of 10-20% with microsphere diluent, and then spray the marker onto the glass fiber using a spray pad.

3) Prepare the dried microsphere pad: Place the sprayed fluorescent pad in a 50°C oven and dry for more than 2 hours.

4) Sample pad treatment: dilute the blocking agent to 0.4 mg/ml using sample pad diluent and spread it on the glass fiber, then put it in a 50°C oven to dry overnight.

5) NC membrane coating: dilute the coating antibody to 1.0 mg/ml using coating diluent and then coat; dry in a 50°C oven overnight.

6) Preparation of fluorescent chromatography strips: Cut the fluorescent chromatography strips with a strip cutter, assemble them and add samples for detection.

7) Detection (1) Quality control material: recombinant antigen, diluted to the corresponding concentration using sample diluent, and then tested with paired antibodies (2) Detection method: 15 minutes after adding the sample, perform instrument judgment.

Deviation = (thermal test activity -4°C, 7-day activity) / -4°C, 7-day activity;

Average deviation = total deviation/number of deviation groups.

Table 9: Stability of mouse anti-IgG2a antibodies

The results showed that the average deviation of mouse IgG2a wild-type antibody at 45℃ for 7 days and 5 freeze-thaw cycles reached 24.04% and 22.7% respectively, exceeding 10%, and failed the assessment. The average deviation of mutant group 3 and mutant group 5 under all assessment conditions was within ±10%, and passed the assessment. The stability of the mutant groups was improved compared with the wild type.

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.

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 (10)

1. A conjugate of a polypeptide of the present invention, characterized by comprising the following steps: an antibody and at least one conjugate moiety attached to the antibody; wherein the antibody comprises a CH1 fragment and a CL fragment; the CH1 and CL fragments of the antibodies have cysteine sites selected from any of the following groups:

1) The 67 th site of the CH1 fragment and the 28 th site of the CL fragment, or equivalent sites thereof;

2) The 9 th site of the CH1 fragment and the 17 th site of the CL fragment, or equivalent sites thereof;

3) The 9 th site of the CH1 fragment and the 14 th site of the CL fragment, or equivalent sites thereof;

4) The 54 th site of the CH1 fragment and the 55 th site of the CL fragment, or equivalent sites thereof;

5) The 53 rd position of the CH1 fragment and the 55 th position of the CL fragment, or equivalent positions thereof;

6) The 14 th site of the CH1 fragment and the 12 th site of the CL fragment, or equivalent sites thereof;

7) The 14 th site of the CH1 fragment and the 102 th site of the CL fragment, or equivalent sites thereof.

2. The conjugate according to claim 1, wherein the CH1 fragment at positions 9, 14, 53, 54, 67 is numbered sequentially from the N-terminus to the C-terminus with the amino acid sequence shown in CH1 of murine wild-type IgHG; the 12 th, 14 th, 17 th, 28 th, 55 th and 102 th positions of the CL fragment are sequentially numbered from the N end to the C end by the amino acid sequence shown by the CL of the murine wild-type IGLK;

alternatively, the antibody has a CH1 fragment and a CL fragment selected from any one of the following groups or having 90% or more identity thereto:

CH1 fragment CL fragment 1 SEQ ID NO:13 SEQ ID NO:14 2 SEQ ID NO:15 SEQ ID NO:16 3 SEQ ID NO:15 SEQ ID NO:17 4 SEQ ID NO:18 SEQ ID NO:19 5 SEQ ID NO:20 SEQ ID NO:19 6 SEQ ID NO:21 SEQ ID NO:22 7 SEQ ID NO:21 SEQ ID NO:23 8 SEQ ID NO:46 SEQ ID NO:14 9 SEQ ID NO:47 SEQ ID NO:16 10 SEQ ID NO:47 SEQ ID NO:17

Optionally, the antibody further comprises an Fc fragment;

optionally, the C-terminus of the CH1 fragment is linked to the N-terminus of the Fc fragment;

Optionally, the antibody further comprises a hinge region;

Optionally, the N-terminus of the hinge region is linked to the C-terminus of the CH1 fragment, and the C-terminus of the hinge region is linked to the N-terminus of the Fc fragment;

Optionally, the antibody comprises a heavy chain constant region shown in any one of SEQ ID NO 26-SEQ ID NO 30 and SEQ ID NO 50-SEQ ID NO 51, or an amino acid sequence with more than 90% of identity with the heavy chain constant region;

optionally, the antibody further comprises a heavy chain variable region and/or a light chain variable region;

Optionally, the C-terminus of the heavy chain variable region is linked to the N-terminus of the CH1 fragment and the C-terminus of the light chain variable region is linked to the N-terminus of the CL fragment;

optionally, the antibody has a heavy chain and a light chain selected from any of the following groups:

heavy chain Light chain 1 SEQ ID NO:33 SEQ ID NO:34 2 SEQ ID NO:35 SEQ ID NO:36 3 SEQ ID NO:35 SEQ ID NO:37 4 SEQ ID NO:38 SEQ ID NO:39 5 SEQ ID NO:40 SEQ ID NO:39 6 SEQ ID NO:41 SEQ ID NO:42 7 SEQ ID NO:41 SEQ ID NO:43 8 SEQ ID NO:78 SEQ ID NO:79 9 SEQ ID NO:80 SEQ ID NO:81

Optionally, the conjugate moiety is attached to a reactive group of the antibody;

Optionally, the reactive group comprises at least one of an amino group, an imino group, a sulfhydryl group, a hydroxyl group, and a carboxyl group;

optionally, the conjugate moiety is selected from at least one of a carrier, a tag;

optionally, the carrier comprises at least one of a solid phase carrier, a labeling carrier, an affinity substance, a fluorescent substance, a luminescent substance, a radioisotope, a gold colloid, and a colored substance;

optionally, the affinity substance comprises biotin/avidin;

Optionally, the tag comprises at least one of a His tag, a Flag tag, a GST tag, an MBP tag, a SUMO tag, and a C-Myc tag;

Optionally, further comprising a linker molecule, said conjugate moiety being attached to said reactive group via a linker molecule;

optionally, the linker molecule is attached to the sulfhydryl groups of two cysteine residues paired in the antibody;

Optionally, the linker molecule forms a sulfur-carbon bridge linkage with the antibody between chains;

optionally, the antibody conjugate comprises the following structure:

Wherein n and m are each independently selected from any integer between 0 and 24;

A is the conjugate moiety;

B is the antibody;

alternatively, a is a carrier;

alternatively, n=4, m=4.

3. An antibody, characterized in that it is an antibody as defined in any one of claims 1 to 2; optionally, the antibody comprises at least one selected from the group consisting of a polyclonal antibody, a full length monoclonal antibody, a Fab 'antibody, a F (ab') ₂ antibody, a F (ab) ₂ antibody, a single chain antibody.

4. A nucleic acid molecule, vector, cell or host, wherein the nucleic acid molecule encodes an antibody in the conjugate of any one of claims 1-2 or an antibody of claim 3; the vector comprising the nucleic acid molecule described above the cell or host comprises:

The nucleic acid molecule, the vector or the vector

Expressing an antibody in a conjugate according to any one of claims 1-2 or an antibody according to claim 3.

5. Use of the antibody of claim 3, the nucleic acid molecule, the vector, the cell or the host of claim 4 in the preparation of a conjugate or a kit.

6. A method of preparing a conjugate comprising:

The conjugate is obtained by performing a first conjugation reaction with an antibody in the conjugate according to any one of claims 1 to 2 or an antibody according to claim 3 and a conjugation moiety.

7. A reagent or a kit of parts, characterized by comprising the following steps:

The conjugate of any one of claims 1-2 or the antibody of claim 3.

8. Use of the conjugate of any one of claims 1-2, the antibody of claim 3, the nucleic acid molecule, vector, cell or host of claim 4 or the reagent or kit of claim 7 in an immunoassay or in the preparation of an immunoassay product.

9. A method of immunoassays is provided, characterized by comprising the following steps:

Contacting a sample to be detected with the conjugate of any one of claims 1 to 2 or the antibody of claim 3 to form an immune complex;

optionally, determining whether the sample to be detected contains a target substance or the content of the target substance based on the signal of the immune complex;

Optionally, the signal comprises a fluorescent signal.

10. A method of increasing the activity, stability or sensitivity of a conjugate comprising an antibody and at least one conjugate moiety attached to the antibody, comprising:

introducing a CH1 fragment and a CL fragment of the antibody into one or more sets of cysteine sites, which are identical to the cysteine sites defined for the conjugate according to any of claims 1-2.