CN117024529B

CN117024529B - Antibody binding proteins and uses thereof

Info

Publication number: CN117024529B
Application number: CN202310875829.5A
Authority: CN
Inventors: 肖秀孝; 许可; 刘鹏飞; 郑长龙; 赵占勇
Original assignee: Beijing Dacheng Biotechnology Co ltd
Current assignee: Beijing Dacheng Biotechnology Co ltd
Priority date: 2023-07-17
Filing date: 2023-07-17
Publication date: 2024-01-26
Anticipated expiration: 2043-07-17
Also published as: CN117024529A

Abstract

The invention discloses an antibody binding protein, the amino acid sequence of which is obtained by mutating the amino acid sequence shown in SEQ ID NO. 1, wherein the mutation at least comprises that an asparagine residue is mutated into other amino acids except a glutamine residue. The invention also provides multimers, nucleic acids, recombinant vectors, host cells, matrices, methods of IgG isolation and kits. The antibody binding egg has better alkali resistance and IgG affinity.

Description

Antibody binding proteins and uses thereof

Technical Field

The invention relates to the related technical field of protein mutants. More specifically, the present invention relates to an antibody binding protein and uses thereof.

Background

Antibodies are one of the very important glycoproteins produced by vertebrate plasma cells and are used by the immune system to recognize and neutralize foreign substances such as bacteria and viruses. In 1975, kohler and millstein developed hybridoma technology and produced the first monoclonal antibody. Since then, antibodies have become a very important tool for molecular level research. Furthermore, since antibodies recognize and bind to their antigens with high affinity and specificity, they are used for sensitive and selective detection of antigens (immunoassays). Unlike conventional analytical methods, which require large and expensive instruments, such as liquid chromatography and mass spectrometry, the immunoassay has significant advantages of convenience, simplicity, and rapid operation. However, there is a constant need to further improve the performance of immunoassays, for example to detect targets with higher sensitivity in a shorter time to achieve difficult but demanding tasks such as early cancer diagnosis.

In addition, recently, antibodies have been developed at a very rapid rate, and are a key drug for the treatment of various cancers, rheumatoid arthritis and other refractory diseases. Today, many pharmaceutical companies are focusing on the development of antibody drug antibodies. Humanized antibodies and human antibodies are of particular interest because they are considered valuable for therapeutic applications, avoiding the human anti-mouse antibody responses often observed with rodent antibodies. However, the purity of biological agents, including human antibodies, as a drug is a challenging problem for the biopharmaceutical industry.

For purification of antibodies, affinity chromatography is typically used. As affinity ligands, the most widely used are antibody binding proteins, i.e. protein a and protein G. Since these antibody binding proteins bind to the various subclasses of IgG via non-covalent bonds, they are unlikely to abrogate the function of the antibody. Protein a is the major cell wall component protein of staphylococcus aureus and protein G is an immunoglobulin binding protein expressed in group C and group G streptococci, very similar to protein a, but with different specificities. It is a 65kDa (G148 protein G) and 58kDa (C40 protein G) cell surface protein, protein G consisting of approximately 600 amino acid residues. The carboxy-terminal half contains three immunoglobulin G (IgG) binding domains, called domains C1, C2 and C3, each containing 55 amino acid residues with two "gaps," i.e., 16 amino acids Dl and D2. It has affinity to the Fab and Fc fragments of human IgG through separate and spaced binding sites. Shows a great affinity for almost all mammalian immunoglobulin G (IgG) classes, including all human IgG subclasses (IgG 1, igG2, igG3 and IgG 4) as well as rabbit, mouse and goat IgG. Protein G binds to all test monoclonal from mouse IgG1, igG2a and IgG3 and rat IgG2a, igG2b and IgG2cIgG. In addition, polyclonal IgG from humans, cattle, rabbits, goats, rats and mice bind to protein G, while chicken IgG does not. NH of protein G ₂ The terminal half of the domain can bind Human Serum Albumin (HSA) but is structurally separated from the site of the IgG binding region. Protein G binds to the IgG subclass more widely than staphylococcal protein a. This applies to polyclonal IgG from bovine, rat, goat, human and rabbit sources, as well as several rat and mouse monoclonal antibodies. Protein G is therefore a powerful reagent for detecting IgG and thus is also the antigen against which these antibodies are directed. It is used in western blot analysis to detect various antigen-antibody complexes on nitrocellulose membranes. In addition, protein G is widely used as a ligand coupled to a resin in affinity chromatography for antibody purification. However, most protein-based affinity chromatography media exhibit a weakness to alkaline conditions. This is a major problem in many industrial applications where the ability to remove contaminants from chromatographic media is critical, typically achieved by an integrated cleaning-in-place (CIP) scheme. Sodium hydroxide in the concentration range of 0.1 to 1M is the most commonly used reagent in this scheme. When they are used as binding ligands, the sensitivity of proteins to the extremely harsh environment of high pH is a disadvantage.

Therefore, it is necessary to design a technical solution that can overcome the above-mentioned drawbacks to a certain extent.

Disclosure of Invention

An object of the present invention is to provide an antibody binding protein having a good alkali resistance and IgG affinity, and uses thereof.

To achieve these objects and other advantages and in accordance with the purpose of the invention, there is provided an antibody binding protein having an amino acid sequence obtained by mutating the amino acid sequence shown in SEQ ID NO. 1, wherein the mutation includes at least a mutation of an asparagine residue to an amino acid other than a glutamine residue.

Further, the mutation also includes mutation of glycine residues, aspartic acid residues, threonine residues to other amino acids than glutamine residues.

Further, the antibody binding protein has an amino acid sequence shown as SEQ ID NO. 2 or SEQ ID NO. 3.

The invention also provides multimers consisting of two or more repeat units that are said antibody binding proteins.

The invention also provides nucleic acids encoding the antibody binding proteins.

The invention also provides a recombinant vector comprising the nucleic acid.

The invention also provides host cells comprising the recombinant vectors.

The invention also provides a matrix for affinity purification, said matrix being loaded with said antibody binding protein or said multimer.

The invention also provides a method for isolating antibody IgG using said antibody binding protein or said multimer or said matrix.

The invention also provides kits comprising said antibody binding proteins or said multimers.

The invention at least comprises the following beneficial effects:

the antibody binding protein is obtained by mutating the amino acid sequence shown in SEQ ID NO. 1, and through mutation, the original affinity and specificity are maintained, the alkaline tolerance is improved, the antibody binding protein can bear in-situ cleaning for a long time, the detection and purification of industrial antibodies are facilitated, and the production cost is reduced.

Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.

Drawings

FIG. 1 shows a map of plasmid pET-C3 (N7A/G13A/N34T/N36E/D45E) -Cys, the gene encoding C3 (N7A/G13A/N34T/N36E/D45E) -Cys.

FIG. 2 shows a map of plasmid pET-C3 (N7A/T17D/D21E/N34T/N36E) -CyS, the gene encoding C3 (N7A/T17D/D21E/N34T/N36E) -CyS.

FIG. 3 shows a map of plasmid pET-C3 (N7A/G13A/N34T/N36E/D45E) tetramer-CyS, the gene encoding C3 (N7A/G13A/N34T/N36E/D45E) tetramer-CyS.

FIG. 4 shows a map of plasmid pET-C3 (N7A/T17D/D21E/N34T/N36E) tetramer-CyS, the gene encoding C3 (N7A/T17D/D21E/N34T/N36E) tetramer-CyS.

FIG. 5 shows the results of a comparison of an antibody binding protein with an labile C3 domain protein by alkali treatment (washing in situ).

Detailed Description

The present invention is described in further detail below with reference to the drawings to enable those skilled in the art to practice the invention by referring to the description.

It will be understood that terms, such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.

As shown in fig. 1-5, embodiments of the present application provide antibody binding proteins having an amino acid sequence that is mutated from the amino acid sequence shown in SEQ ID No. 1, including at least the mutation of an asparagine residue to an amino acid other than a glutamine residue; SEQ ID NO. 1 is the C3 domain of the parent antibody binding protein G, the amino acid sequence of which is TYKLVINGKTLKGETTTKAVDAETAEKAFKQYANDNGVDGV WTYDDATKTFTVTEKPEHHHHHHC; the antibody binding protein is capable of binding to the Fc region of an immunoglobulin (IgG) antibody molecule; the asparagine residue may be mutated to one of the following amino acids: glycine (Gly), alanine (apla), valine (Val), leucine (Leu), isoleucine (Ile), serine (Ser), threonine (Thr), cysteine (Cys), methionine (Met), phenylalanine (Phe), tyrosine (Tyr), tryptophan (Trp), glutamic acid (Glu), arginine (Arg), histidine (His), lysine (Lys) or proline (Pro), but not mutated to glutamine residues; amino acids other than asparagine residues may also be mutated, including substitution, deletion or addition of one or more amino acids, while having the ability to bind to and enhance alkali resistance properties of the Fc region (Fc segment, i.e., a crystalline fragment consisting of CH2 and CH 3 domains, fc segment having no antigen binding activity, being the site of interaction with effector molecules or cells) of an immunoglobulin antibody molecule; optionally, cysteine (Cys) is added.

In another embodiment, the mutation further comprises mutation of glycine residues, aspartic acid residues, threonine residues to other amino acids than glutamine residues; alternatively, glycine residues have been mutated to alanine residues, aspartic acid residues have been mutated to glutamic acid residues, and threonine residues have been mutated to aspartic acid residues.

In another embodiment, the antibody binding protein has the amino acid sequence shown as SEQ ID NO. 2 or SEQ ID NO. 3; the amino acid sequence shown in SEQ ID NO. 2 is TYKLVIAGKTLKAETTTKAVDAETA EKAFKQYATDEGVDGVWTYEDATKTFTVTEKPEHHHHHHC; compared to the sequence SEQ ID NO. 1, the asparagine residue at position 7 has been mutated to an alanine residue, the glycine residue at position 13 has been mutated to an alanine residue, the asparagine residue at position 34 has been mutated to a threonine residue, the asparagine residue at position 36 has been mutated to a glutamic acid residue, and the aspartic acid residue at position 45 has been mutated to a glutamic acid residue (N7A/T17D/D21E/N34T/N36E);

the amino acid sequence shown in SEQ ID NO. 3 is: TYKLVIAGKTLKGETTDKAVEAETAEKAFKQ YATDEGVDGVWTYDDATKTFTVTEKPEHHHHHHC; in contrast to the sequence SEQ ID NO 1, the asparagine residue at position 7 has been mutated to an alanine residue, the threonine residue at position 17 has been mutated to an aspartic acid residue, the aspartic acid residue at position 21 has been mutated to a glutamic acid residue, the asparagine residue at position 34 has been mutated to a threonine residue, and the asparagine residue at position 36 has been mutated to a glutamic acid residue.

Embodiments of the present application also provide multimers consisting of two or more repeat units that are the antibody-binding proteins; alternatively, it may be a dimer, trimer, tetramer, pentamer or hexamer, wherein the repeating units may be the same or different; alternatively, the repeat unit of the multimer is the amino acid sequence shown as SEQ ID NO. 2 or SEQ ID NO. 3; alternatively, the multimer is a multimer comprising the mutations N7A/G13A/N34T/N36E/D45E and

tetramers of the C3 domain of SPG of N7A/T17D/D21E/N34T/N36E are shown in SEQ ID NO. 4 and SEQ ID NO: 5. The amino acid sequence shown in SEQ ID NO. 4 is:

TYKLVIAGKTLKAETTTKAVDAETAEKAFKQYATDEGVDGVWTYEDATKTFTVTE

KPEVIDASELTPAVT

TYKLVIAGKTLKAETTTKAVDAETAEKAFKQYATDEGVDGVWTYEDATKTFTVTE

KPEVIDASELTPAVT

TYKLVIAGKTLKAETTTKAVDAETAEKAFKQYATDEGVDGVWTYEDATKTFTVTE

KPEVIDASELTPAVT

TYKLVIAGKTLKAETTTKAVDAETAEKAFKQYATDEGVDGVWTYEDATKTFTVTEKPEHHHHHHC；

the amino acid sequence shown in SEQ ID NO. 5 is:

TYKLVIAGKTLKGETTDKAVEAETAEKAFKQYATDEGVDGVWTYDDATKTFTVTE

KPEVIDASELTPAVT

TYKLVIAGKTLKGETTDKAVEAETAEKAFKQYATDEGVDGVWTYDDATKTFTVTE

KPEVIDASELTPAVT

TYKLVIAGKTLKGETTDKAVEAETAEKAFKQYATDEGVDGVWTYDDATKTFTVTE

KPEVIDASELTPAVT

TYKLVIAGKTLKGETTDKAVEAETAEKAFKQYATDEGVDGVWTYDDATKTFTVTEKPEHHHHHHC。

embodiments of the present application also provide nucleic acids encoding the antibody binding proteins, i.e., useful for expression in recombinant hosts using existing biotechnology methods to produce antibody binding proteins.

Embodiments of the present application also provide recombinant vectors comprising the nucleic acids; a vector refers to a nucleic acid vehicle into which nucleic acid is inserted and which allows expression of the protein, and the vector may be transformed, transduced or transfected into a host cell to allow expression of the genetic material carried thereby in the host cell, and includes plasmids, phagemids and the like.

Embodiments of the present application also provide host cells, including the recombinant vectors, which are cells harboring the recombinant vectors, capable of expressing the antibody-binding proteins.

Embodiments of the present application also provide a matrix for affinity purification, the matrix carrying the antibody binding protein or the multimer; the matrix comprises an antibody binding protein G as ligand coupled to a solid support, wherein at least three asparagine residues in the protein have been mutated to an amino acid other than glutamine; the matrix of this example shows increased binding capacity during multiple CIP when compared to a matrix consisting of parent molecules as ligands; the mutant protein ligand is preferably a protein that binds to an Fc fragment, preferably the antibody is IgG; the solid support of the matrix may be of any suitable kind; alternatively, the carrier may be e.g. based on polysaccharides such as cellulose, pullulan, cross-linked agarose, etc.; in a preferred embodiment, the solid support is a porous agarose bead; in an advantageous possibility, the carrier has been modified to increase its rigidity, thereby making the matrix more suitable for high flow rates; the ligand may be attached to the carrier by conventional coupling techniques using, for example, mercapto, amino and/or carboxyl groups present in the ligand, tris (2-carboxyethyl) phosphine (TECP), cyanogen bromide, maleimide, ethylene oxide, N-hydroxysuccinimide (NHS), triazine (triazine), periodate, carbonyldiimidazole (carbocyldiimidazole), 2,4, 6-trifluoro-5-chloropyridine (FCP), oxalic acid hydrazide (adipic acid dihydrazide), divinyl sulfone (divinyl lsulfane) and the like are well known coupling reagents. Alternatively, the ligand may be attached to the support via a non-covalent bond; in an advantageous possibility, the ligand is provided with a C-terminal cysteine residue for coupling, the ligand being coupled to the carrier via a thioether bond; affinity for antibodies, i.e. binding properties of the ligand, the loading of the matrix is not substantially changed by treatment with an alkaline reagent, typically NaOH in a concentration of up to 1M, for in-situ washing of the matrix when affinity separation is performed; another way to characterize the matrix of this example is that the above-described mutations will reduce their binding capacity to about 80%, preferably about 88%, after 12h of treatment with 1M NaOH.

Embodiments of the present application also provide methods of isolating antibody IgG using the antibody binding protein or the multimer or the matrix; in particular, the antibodies are isolated from the liquid by adsorption to an antibody binding protein G or multimer or matrix as described above; that is, by passing a solution containing the antibodies through the separation matrix under conditions of retention time and ionic strength, the antibodies are mechanically adsorbed and other components of the solution will pass through substantially unimpeded; the substrate is then washed with PB solution to remove retained or poorly bound impurities, and then an eluent is passed through the substrate to release the antibodies, typically by changing pH, ionic strength, hydrophobicity, etc.

Embodiments of the present application also provide kits comprising the antibody binding protein or the multimer; the kit can be used for preparing affinity purified matrix and then separating antibody IgG.

The following is a description of several specific embodiments.

Example 1: mutation of the C3 Domain (SEQ ID NO: 1), vector construction, expression and purification

The domain C3-Cys gene (linked to cysteine Cys for coupling to the vector) was synthesized from Shanghai Langerhans, and the mutant C3 (N7A/G13A/N34T/N36E/D45E) -Cys and C3 (N7A/T17D/D21E/N34T/N36E) -Cys codons were optimized for gene synthesis and cloned into plasmid pET-30a (+) see FIGS. 1 and 2. Coli strain DH 5. Alpha. Was used during cloning, and BL21 (DE 3) was used for protein expression.

All C3 variants were successfully expressed in E.coli and the yield was estimated to be about 110mg/L by gel electrophoresis and Coomassie blue staining. The variant proteins were purified by His affinity chromatography.

Example 2: carrier construction, expression and purification of mutant protein tetramer

After codon optimization of the genes for the mutant proteins C3 (N7A/G13A/N34T/N36E/D45E) tetramer-Cys and C3 (N7A/T17D/D21E/N34T/N36E) tetramer-Cys, the genes were synthesized and cloned into pET-30a (+) vector, see FIGS. 3 and 4. Coli strain DH 5. Alpha. Was used during cloning, and BL21 (DE 3) was used for protein expression.

All C3 variant tetrameric proteins were successfully expressed in E.coli and the yield was estimated to be approximately 125mg/L by gel electrophoresis and Coomassie blue staining. The variant protein tetramer was purified by His affinity chromatography.

Example 3: BIACORE analysis of alkali resistance

To detect the alkaline resistance properties of different muteins and tetramers of muteins, the properties of variants of domain C3 as affinity ligand were analyzed by standard affinity matrices.

The change in binding capacity of each purified recombinant protein to human IgG after exposure to NaOH was monitored using a BIACORE 3000 instrument (GE Healthcare). Human IgG was immobilized on carboxylated dextran surfaces of CM5 sensor chips (GE Healthcare) by amino coupling using N-hydroxysuccinimide (NHS) and N-ethyl-N' - (3-dimethylaminopropyl) carbodiimide (EDC). Immediately after NHS/EDC activation, the excess active carboxyl groups on the chip were blocked with ethanolamine. A1 mg/mL human IgG solution was prepared by dilution in 20mM sodium phosphate (pH 7.4) containing 0.15M NaCl. The IgG solution was further diluted in 10mM sodium acetate (pH 4.5) before being used in the immobilization procedure. To analyze IgG binding affinity, three solutions of different protein concentrations (10 to 1000 nM) were prepared for each protein using running buffer (20 mm NaH2PO4-Na2HPO4, 150mM NaCl,0.005%P-20, ph 7.4) and each protein solution was added to the sensor chip. To analyze their alkali resistance, each protein was adjusted to a common concentration, such as 30 μm, with a specific amount of 1M NaOH, and incubated for a specific time at room temperature. Subsequently, 1M HCl was added to each mixture to neutralize the solution. The solution was further diluted 1:1 in running buffer (20 mM sodium phosphate, 0.15mNaCl,0.005% P-20, pH 7.4). Protein solutions were also prepared in the same manner prior to the alkali treatment. Each protein solution before and after the alkali treatment was applied to the sensor chip at a flow rate of 20. Mu.L/min, and the surface was regenerated using 50mM NaOH. The data were analyzed using BIA evaluation software. Calculation of the binding rate constant k using a 1:1 Langmuir model _on (M ^-1 s ^-1 ) Dissociation rateConstant k _off (s ^-1 ) And a binding constant K _a (M ^-1 ). Global fitting was used to determine the affinity constant of each mutant for IgG and local fitting was used to analyze its alkaline stability. For the alkaline stability analysis, the concentration was set to remain constant before and after treatment in the fitting analysis. By calculating R after alkali treatment of each mutant _max Relative to R before alkali treatment _max The value, i.e., residual IgG binding activity (%), was evaluated for alkali resistance.

After 6h alkali treatment of the control C3-Cys protein, the residual IgG binding activity is only 4%; the affinity of the mutant is 3-4 times higher than that of the control protein, but the residual IgG binding activity is more than 70% after 24h alkali treatment. Specifically, the affinity of mutant C3 (N7A/G13A/N34T/N36E/D45E) -Cys was 3.5 times that of the control protein, and the residual IgG binding activity was 71% after 24h alkali treatment; c3 (N7A/G13A/N34T/N36E/D45E) tetramer-Cys was 24% higher in high affinity than its monomer and after 24h alkali treatment the residual IgG binding activity was 71%; the affinity of mutant C3 (N7A/T17D/D21E/N34T/N36E) -Cys is 3.8 times that of the control protein, and the residual IgG binding activity is 66% after 24h alkali treatment; c3 (N7A/T17D/D21E/N34T/N36E) tetramer-Cys with a high affinity 12% higher than its monomer, residual IgG binding activity after 24h alkali treatment was 75%; the above mutations show extremely strong alkali resistance and higher affinity. These data make C3 (N7A/G13A/N34T/N36E/D45E) and C3 (N7A/T17D/D21E/N34T/N36E) very superior candidates for affinity purified ligands.

TABLE 1 kinetic data

Example 4: preparation of affinity matrices

To finally demonstrate the reliability of the preferred muteins, according to the experimental results of the BIACORE 3000 instrument on the kinetics and alkali resistance of the muteins, C3-Cys was selected as control ligand, C3 (N7A/G13A/N34T/N36E/D45E) tetramer-Cys and C3 (N7A/T17D/D21E/N34T/N36E) tetramer-Cys as preferred ligands were first produced by fermentation, the thallus was harvested and purified.

And (3) preparing an affinity filler. The SulfoLink coupling resin is a porous crosslinked 6% microbead agarose that has been activated with iodoacetic acid groups for effecting covalent immobilization of cysteine proteins and other thiol molecules. Incubation with a solution of C3-Cys containing reduced cysteine residues and preferably a variant protein, the SulfoLink resin iodoacetic acid groups react specifically and efficiently with the exposed sulfhydryl groups (-SH) to form covalent and irreversible thioether linkages that permanently link the mutein to the resin. The matrix was packed in a column to give a seed volume of 5ml.

The following buffers were filtered with a 0.45 μm filter and sonicated before use.

Binding/washing buffer: 0.15M NaCl,20mM Na2HPO4,pH7.0

Elution buffer: 0.1M Glycine, pH 3.0

Neutralization buffer: 1M Tris-HCl, pH 8.5

Stop buffer: 50mM Tris-HCl, pH 7.5

Regeneration buffer: 1M NaOH

Human IgG1 was formulated in binding/wash buffer and injected over-load onto the affinity column. Standard affinity chromatography procedures were performed on a purifier for 24 cycles. One CIP step is integrated between each cycle. The affinity column was washed with regeneration buffer, with a contact time of 60 minutes for each wash, resulting in a total contact time of 24 hours. Eluted material was detected at 280 nm. The amount of hIgG1 eluted was measured after each cycle to determine the total capacity of the column. Referring to FIG. 5, the results show that C3 (N7A/G13A/N34T/N36E/D45E), C3 (N7A/T17D/D21E/N34T/N36E), C3 (N7A/G13A/N34T/N36E/D45E) tetramer-Cys and C3 (N7A/T17D/D21E/N34T/N36E) tetramer-Cys all showed better alkali resistance than C3-Cys.

The number of equipment and the scale of processing described herein are intended to simplify the description of the present invention. The use, modification and variation of the antibody binding proteins of the invention will be apparent to those skilled in the art.

Although embodiments of the present invention have been disclosed above, it is not limited to the details and embodiments shown and described, it is well suited to various fields of use for which the invention would be readily apparent to those skilled in the art, and accordingly, the invention is not limited to the specific details and illustrations shown and described herein, without departing from the general concepts defined in the claims and their equivalents.

Claims

1. An antibody binding protein, characterized in that the antibody binding protein has an amino acid sequence as shown in SEQ ID NO. 2 or SEQ ID NO. 3.

2. A multimer comprising two or more repeat units, wherein the repeat units are the antibody binding protein of claim 1.

3. A nucleic acid encoding the antibody binding protein of claim 1.

4. A recombinant vector comprising the nucleic acid of claim 3.

5. A host cell comprising the recombinant vector of claim 4.

6. A matrix for affinity purification, characterized in that the matrix is loaded with the antibody binding protein according to claim 1 or the multimer according to claim 2.

7. A method for isolating antibody IgG, characterized in that the isolation is performed using an antibody binding protein according to claim 1 or a multimer according to claim 2 or a matrix according to claim 6.

8. A kit comprising the antibody binding protein of claim 1 or the multimer of claim 2.