CN113583125A

CN113583125A - Antibody, binding agent containing antibody, immunoadsorption material and application of immunoadsorption material

Info

Publication number: CN113583125A
Application number: CN202110777660.0A
Authority: CN
Inventors: 房鑫; 徐云霞
Original assignee: Suzhou Shengji Pharmaceutical Co ltd
Current assignee: Suzhou Shengji Pharmaceutical Co ltd
Priority date: 2021-07-09
Filing date: 2021-07-09
Publication date: 2021-11-02
Anticipated expiration: 2041-07-09
Also published as: CN116120449A; CN113583125B

Abstract

The invention discloses an antibody targeting a protein comprising an amino acid sequence as set forth in SEQ ID No. 1, said antibody comprising a heavy chain variable region comprising a CDR1, a CDR2 and a CDR3 as set forth herein. The invention also provides a binding agent containing the antibody, an immunoadsorption material and application thereof. The antibody of the invention has better binding activity to protein molecules containing the sequence shown as SEQ ID NO. 1, and has better thermal stability and better alkali resistance; the binding agent and the immunoadsorption material containing the same can purify protein molecules containing sequences shown in SEQ ID NO. 1, such as FSH, hCG, TSH, hLH, FSH-CTP and the like under mild conditions, and have high purification efficiency, high purity of the obtained product, good quality of the product, lower purification cost, more convenient use method and simple operation.

Description

Antibody, binding agent containing antibody, immunoadsorption material and application of immunoadsorption material

Technical Field

The invention relates to the technical field of biology, in particular to an antibody, a binding agent containing the antibody, an immunoadsorption material containing the antibody and application of the antibody.

Background

Affinity chromatography is based on the principle of affinity adsorption between biomolecules and other ligand molecules (e.g., antigens and antibodies, enzymes and substrates, hormones and receptors, complementary strands in nucleic acids, polysaccharide and protein complexes, etc.). The protein and the ligand on the chromatographic carrier are combined in a biological specificity way through the actions of covalent bonds, van der waals force, hydrophobic force, electrostatic force and the like to form a complex, and the complex is linked on the functional group ligand on the surface of the carrier in a covalent way. The protein affinity chromatography achieves the purpose of separation and purification by the adsorption of specific affinity between target protein and ligand, has the characteristic of biological specificity, and has high purification efficiency.

Currently, affinity chromatography is widely used for purifying antibody by using ProA, and a nickel column is used for purifying His tag protein. However, in terms of pharmaceutical recombinant proteins, the recombinant proteins usually do not carry a purification tag in consideration of safety and the like, so that the purification difficulty is high, and the loss of target proteins in the purification process is large. For the purification of some complex proteins, such as heterodimeric proteins FSH (follicle stimulating hormone), hCG (human chorionic gonadotropin), Thyroid Stimulating Hormone (TSH), human luteinizing hormone (hLH), FSH-CTP (follicle stimulating hormone-CTP fusion protein), etc., the proteins themselves are unstable, easy to depolymerize, and are not acid-resistant, and currently, purification processes such as anion exchange chromatography, etc. are generally used. The purification steps are complicated, the operation is carried out under the condition of non-mild pH, the loss in the purification process is more, the yield is far lower than that of affinity chromatography, generally lower than 50 percent, and the purity is not high. Only one commercially available filler for affinity chromatography is currently available for purification of heterodimeric proteins comprising an alpha chain (SEQ ID NO:1), such as FSH, hCG, TSH, hLH, FSH-CTP, and the like, at high cost, resulting in high purification cost of the corresponding drug product.

Therefore, there is a high necessity for an immunoadsorbent material and a purification method that are easy to handle, have high purity of the purified product, and are inexpensive to purify heterodimeric proteins comprising an α chain (SEQ ID NO:1), such as FSH, hCG, TSH, hLH, FSH-CTP, with high efficiency and low cost.

Disclosure of Invention

The invention provides an antibody, a binding agent containing the antibody, an immunoadsorption material and application of the antibody, and aims to overcome the defects of complicated steps, low purification efficiency, more loss, higher cost, lower purity of the obtained product and the like when the heterodimer protein containing an alpha chain (SEQ ID NO:1), such as FSH, hCG, TSH, hLH, FSH-CTP and the like, is purified in the prior art. The antibody of the invention has better binding activity to protein molecules (such as FSH, hCG, TSH, hLH, FSH-CTP and the like) containing the sequence shown as SEQ ID NO. 1, and has better thermal stability and better alkali resistance; the binding agent and the immunoadsorption material containing the same can purify protein molecules such as FSH, hCG, TSH, hLH, FSH-CTP and the like containing a sequence shown in SEQ ID NO. 1 under mild conditions (such as neutral pH conditions), and have high purification efficiency, high purity of the obtained product, good quality of the product, lower purification cost, more convenient use method and simple operation; in addition, the binding agent and the immunoadsorbent material containing the antibody can still maintain better performance after being repeatedly used for many times.

In order to solve the above technical problem, the present invention provides in a first aspect an antibody targeting a protein comprising an amino acid sequence as set forth in SEQ ID No. 1, said antibody comprising a heavy chain variable region (VHH) comprising the following CDRs numbered according to IMGT or mutations thereof: CDR1 having an amino acid sequence as set forth in SEQ ID NO 5 or 19; CDR2 having an amino acid sequence as set forth in

SEQ ID NO

6 or 20; and, CDR3 having an amino acid sequence set forth in SEQ ID NO. 7 or 21.

Wherein the above mutations are insertions, deletions or substitutions of 3, 2 or 1 amino acids in the amino acid sequence of the CDR.

In a certain preferred embodiment, the VHH comprises the following Complementarity Determining Regions (CDRs) numbered according to IMGT, or mutations thereof: VHCDR1 having the amino acid sequence shown in SEQ ID NO. 5; VH CDR2 having an amino acid sequence shown in SEQ ID NO. 6; and/or VH CDR3 having an amino acid sequence shown in SEQ ID NO. 7.

In a certain preferred embodiment, the VHH comprises the following Complementarity Determining Regions (CDRs) numbered according to IMGT, or mutations thereof: CDR1 having an amino acid sequence shown in SEQ ID NO. 19, CDR2 having an amino acid sequence shown in SEQ ID NO. 20, and CDR3 having an amino acid sequence shown in SEQ ID NO. 21.

It is well known to those skilled in the art that CDRs of antibodies can be defined by a variety of methods in the art, such as Chothia (Chothia et Al, (1989) Nature 342: 877-Asn 883, Al-Lazikani et Al, "Standard constraints for the structural organization of immunology", Journal of Molecular Biology,273, 927-Asn 948(1997)), Kabat (Kabat et Al, Sequences of Proteins of Immunological Interest, 4 th edition, U.S. Demontent of Health and Human Services, National instruments of Health (1987)), Abstract of Molecular Interest (university of Molecular Biology), and Mass propagation of biological domains (Mass. I.S. International) using the general crystal of the general family. In the present application, the amino acid sequences of the above listed CDRs are shown according to the IMGT definition rules, but it will be understood by those skilled in the art that, unless otherwise specified, the terms "CDR" and "complementarity determining region" of a given antibody or region thereof (e.g., variable region) will be understood to encompass complementarity determining regions as defined by any one of the CDR definition rules known to those skilled in the art. Although the scope of the claims is based on the sequence shown in the IMGT definition rules, the amino acid sequences corresponding to other CDR definition rules should also fall within the scope of the invention.

For example, the VHH comprises the following Complementarity Determining Regions (CDRs) numbered according to Kabat or mutations thereof: CDR1 with an amino acid sequence shown as SEQ ID NO. 2, CDR2 with an amino acid sequence shown as SEQ ID NO. 3 and/or CDR3 with an amino acid sequence shown as SEQ ID NO. 4; alternatively, the VHH comprises the following Complementarity Determining Regions (CDRs) numbered according to Chothia or mutations thereof: VH CDR1 with an amino acid sequence shown as SEQ ID NO. 8, VH CDR2 with an amino acid sequence shown as SEQ ID NO. 9 and/or VH CDR3 with an amino acid sequence shown as SEQ ID NO. 10; these should also fall within the scope of the present invention.

For example, the VHH comprises the following Complementarity Determining Regions (CDRs) numbered according to Kabat or mutations thereof: CDR1 with an amino acid sequence shown as SEQ ID NO. 25, CDR2 with an amino acid sequence shown as SEQ ID NO. 26 and/or CDR3 with an amino acid sequence shown as SEQ ID NO. 27; alternatively, the VHH comprises the following Complementarity Determining Regions (CDRs) numbered according to Chothia or mutations thereof: VH CDR1 with an amino acid sequence shown as SEQ ID NO. 22, VH CDR2 with an amino acid sequence shown as SEQ ID NO. 23, and/or VH CDR3 with an amino acid sequence shown as SEQ ID NO. 24; these should also fall within the scope of the present invention.

In the present application, "amino acid mutation" in the analogous "insertion, deletion or substitution with 3, 2 or 1 amino acids" means that there is a mutation of amino acids in the sequence of the variant as compared with the original amino acid sequence, including the occurrence of insertion, deletion or substitution of amino acids on the basis of the original amino acid sequence. An exemplary explanation is that the mutations to the CDRs may comprise 3, 2 or 1 amino acid mutations, and that the CDRs may optionally be mutated by selecting the same or different number of amino acid residues between them, for example, 1 amino acid mutation to CDR1 and no amino acid mutations to CDR2 and CDR 3.

In the present application, the mutations may include mutations that are currently known to those skilled in the art, for example, mutations that may be made to the antibody during the production or use of the antibody, for example, mutations at sites that may be present, particularly post-transcriptional modifications (PTMs) of CDR regions, including aggregation of the antibody, deamidation sensitivity (site (NG, NS, NH, etc.), aspartic acid isomerization (DG, DP) sensitive sites, N-glycosylation (N- { P } S/T) sensitive sites, oxidation sensitive sites, and the like.

Preferably, the heavy chain variable region further comprises a framework region (FWR) of the alpaca antibody and/or human antibody.

Preferably, the amino acid sequence of the heavy chain variable region is the amino acid sequence shown as SEQ ID NO. 11, SEQ ID NO. 17 or a mutation thereof.

The above mutation is a deletion, substitution or addition of one or more amino acid residues in the amino acid sequence of the heavy chain variable region, and the mutated amino acid sequence has at least 85% sequence identity with the amino acid sequence of the heavy chain variable region, and maintains or improves the binding of the antibody to a heterodimeric protein comprising an alpha chain (SEQ ID NO:1), such as FSH, hCG, TSH, hLH, FSH-CTP, etc.; the at least 85% sequence identity is preferably at least 90% sequence identity, more preferably at least 95%, 96%, 97%, 98% sequence identity, and most preferably at least 99% sequence identity.

Preferably, the antibody is a VHH, heavy chain antibody, bispecific antibody or multispecific antibody, or a monoclonal or polyclonal antibody made from such an antibody.

In the present application, the term "multispecific antibody" is used in its broadest sense to encompass antibodies having polyepitopic specificity. These multispecific antibodies include, but are not limited to: an antibody comprising a heavy chain variable region (VH), wherein the VH unit has polyepitopic specificity; an antibody having two or more VH regions, each VH unit binding to a different target or to a different epitope of the same target; an antibody having two or more single variable regions, each single variable region binding to a different target or a different epitope of the same target; full length antibodies, antibody fragments, bispecific antibodies (diabodies), and triabodies (triabodies), antibody fragments linked together covalently or non-covalently, and the like.

In the present application, the monoclonal antibody or mAb or Ab refers to an antibody obtained from a single clonal cell line, and the cell line is not limited to eukaryotic, prokaryotic, or phage clonal cell lines.

In this application, the term "heavy chain antibody" refers to an antibody comprising only one heavy chain variable region (VHH) and two conventional CH2 and CH3 regions, also known as HCAbs.

In the present application, the term "VHH (single domain antibody)", also called "nanobody", refers to a VHH structure cloned from a heavy chain antibody, which is the smallest unit known to bind to a target antigen.

Preferably, the "protein comprising an amino acid sequence shown as SEQ ID NO. 1" is one or more of FSH, hCG, TSH, hLH and FSH-CTP.

Preferably, the antibody is a VHH, such as VHH-sgs-HHHHHHHHHHHH, VHH-sgs-HHHHHHHHHHHHHH-C, or VHH-sgs-HHHHHHHHHHHHHHHHHHHHHH-CC.

In order to solve the above technical problem, the second aspect of the present invention provides an isolated nucleic acid encoding the antibody according to the first aspect of the present invention.

The preparation method of the nucleic acid is a preparation method which is conventional in the field, and preferably comprises the following steps: obtaining the nucleic acid molecule for coding the antibody by a gene cloning technology, or obtaining the nucleic acid molecule for coding the antibody by an artificial complete sequence synthesis method.

Those skilled in the art know that the base sequence encoding the amino acid sequence of the above antibody may be appropriately introduced with substitutions, deletions, alterations, insertions or additions to provide a polynucleotide homolog. The polynucleotide homologue of the present invention may be prepared by substituting, deleting or adding one or more bases of a gene encoding the antibody sequence within a range in which the activity of the antibody is maintained.

In order to solve the above technical problems, the third aspect of the present invention provides a recombinant expression vector comprising the isolated nucleic acid according to the second aspect of the present invention.

The recombinant expression vector can be obtained by methods conventional in the art, namely: the nucleic acid molecules described herein are constructed by ligating them to various expression vectors. The expression vector is any vector conventionally used in the art so long as it can carry the aforementioned nucleic acid molecule.

Preferably, the recombinant expression vector is a plasmid, cosmid, phage, or viral vector, preferably a retroviral vector, a lentiviral vector, an adenoviral vector, or an adeno-associated viral vector.

In order to solve the above technical problems, the fourth aspect of the present invention provides a transformant comprising the recombinant expression vector according to the third aspect of the present invention in a host cell.

The preparation method of the transformant may be a preparation method conventional in the art, for example: transforming the recombinant expression vector into a host cell. The host cell of the transformant is a variety of host cells which are conventional in the art, as long as the recombinant expression vector is stably self-replicating and the nucleic acid carried by the recombinant expression vector can be efficiently expressed. Preferably, the host cell is a yeast such as Saccharomyces Cerevisiae (Saccharomyces Cerevisiae) or other yeast, mold, bacteria, or cellular expression system. The recombinant expression plasmid is transformed into a host cell to obtain a recombinant expression transformant preferred in the present invention. Wherein the transformation method is a transformation method conventional in the art, preferably a chemical transformation method, a thermal shock method or an electric transformation method.

Furthermore, the present invention provides a chimeric antigen receptor comprising an antibody according to the first aspect of the present invention.

In addition, the present invention provides a genetically modified cell comprising a chimeric antigen receptor as described above.

Preferably, the genetically modified cell is a eukaryotic cell, preferably an isolated human cell; more preferably immune cells such as T cells, or NK cells.

In addition, the invention also provides a preparation method of the antibody, which comprises the following steps: culturing the transformant according to the fourth aspect of the present invention, and obtaining the antibody from the culture.

Furthermore, the present invention provides an antibody drug conjugate comprising a cytotoxic agent, and an antibody according to the first aspect of the invention.

Furthermore, the present invention provides a pharmaceutical composition comprising an antibody according to the first aspect of the invention, and/or an antibody drug conjugate as described above.

In addition, the invention also provides the application of the antibody of the first aspect of the invention, the chimeric antigen receptor, the genetically modified cell, the antibody drug conjugate and/or the pharmaceutical composition in preparing medicines for treating and/or preventing diseases such as cancer.

Furthermore, the invention provides a kit comprising an antibody according to the first aspect of the invention, a chimeric antigen receptor as described above, a genetically modified cell as described above, or an antibody drug conjugate as described above or a pharmaceutical composition as described above.

Preferably, the kit further comprises (i) a device for administering the antibody or antigen-binding fragment thereof or antibody drug conjugate or pharmaceutical composition; and/or (ii) instructions for use.

The present inventors have made many attempts to find that when the above-mentioned antibody is used for the preparation of an immunoadsorbent material and used as an affinity filler, the purification efficiency is high when the antibody is used for purifying a heterodimeric protein comprising an α chain (SEQ ID NO:1), such as FSH, hCG, TSH, hLH, FSH-CTP, and the purity of the obtained target protein is high and the product quality is good.

In order to solve the above technical problem, a fifth aspect of the present invention provides a binding agent comprising the antibody according to the first aspect of the present invention.

In the present invention, the binding agent is a component that specifically binds to a target molecule (e.g., a protein of the present invention comprising an amino acid sequence as set forth in SEQ ID NO:1) with a desired binding affinity.

In the present invention, the binding agent may be a monospecific binding agent, and may further comprise other components that specifically bind to a target molecule (e.g., a protein of the present invention having a sequence as shown in SEQ ID NO: 1). As the name implies, a monospecific binding agent is specific for a single epitope or ligand.

In the present invention, the binding agent may be derived from any suitable source organism, or may be prepared synthetically or by genetically modified organisms. For example, the binding agent may have a modification at its N-terminus that enables the antibody to be expressed initially, e.g., may have one or more methionine at its N-terminus. For example, the binding agent may have a tag at its C-terminus for detection of properties such as activity or for purification, e.g., a 6 × histidine tag (6 × His tag). The 6 × histidine tag is preferably linked at its C-terminus via a linker peptide such as SGS. Further, one or more cysteines and the like may also be included.

In a preferred embodiment of the invention, the antibody of the binding agent is in the form of a VHH molecule, which may include, but is not limited to, the following structures: VHH-sgs-HHHHHHHHHH, VHH-sgs-HHHHHHHHHHHH-C, VHH-sgs-HHHHHHHHHHHHHH-CC, and the like.

In the present invention, the binding agent specifically binds to a protein molecule comprising an amino acid sequence shown in SEQ ID NO. 1, such as FSH, hCG, TSH, hLH, FSH-CTP, etc.

In order to solve the above technical problems, a sixth aspect of the present invention provides an immunoadsorbent material comprising a binding agent according to the fifth aspect of the present invention.

In the present invention, the immunoadsorbent material may preferably be a composition comprising a population of homogeneous binding agents (a composition comprising monospecific binding agents).

Immunoadsorbent material, which can be used, for example, as affinity purification medium (or also called packing material), is understood in the present invention to mean a combination of a support and a binding agent immobilized on the support. The support may be any material that can be used for immobilization of the binding agent. Suitable examples are matrix materials encapsulating the binding agent, cell surfaces displaying the binding agent and polymers that can be covalently linked to the binding agent. Suitable supports such as Agarose (Agarose), which may be commercial supports, e.g., Sepharose, PureCube, Superflow, Purabead, etc., are well known to those skilled in the art of affinity chromatography. The binding agent may be covalently linked or linked by other interactions to a suitable support. Preferably immobilized on a support by covalent bonds.

Preferably, the immunoadsorbent material further comprises a carrier. Examples of suitable supports include porous solid supports (materials) such as agarose, magnetic beads of agarose, polystyrene, controlled pore glass, cellulose, dextran, diatomaceous earth, synthetic polymers (e.g., Sepharose)^TM) Porous amorphous silica. The support material may be in any suitable form, such as granules, powders, flakes, beads, filters, membranes, and the like. A further list of suitable carrier materials is disclosed, for example, in EP-A-434317.

In the present invention, the binding agent may be immobilized on a support and may be pre-activated in a manner conventional in the art. Preferably, the carrier is a carrier pre-activated with CNBr and/or NHS, so that the antibody can be immobilized on the carrierOn the carrier. There are a variety of methods available for rapid, easy and safe immobilization of ligands (e.g., the antibodies described above) via selected functional groups. The correct choice of coupling method depends on the substance to be immobilized. For example, the following commercially known Sepharose^TMThe derivatives make it possible to conveniently immobilize proteins thereon: CNBr activated Sepharose^TM4B enables ligands containing primary amino groups to be immobilized rapidly by spontaneous reactions. AH-Sepharose^TM4B and CH-Sepharose^TM4B both have spacer arms six carbons in length and allow coupling via carboxyl and amino groups, respectively. Flexible spacers are suitable where the flexibility of the target molecule is limited, or where the 3-dimensional structure of the target requires some degree of flexibility of the binding agent so that optimal binding occurs. Activated CH-Sepharose^TM4B provides a six carbon long spacer and an active ester for spontaneous coupling via an amino group. Preferably by-NH 2, -SH (cysteine) or-COOH, SH being a site-directed cross-linking, -COOH, -NH2 being a random cross-linking.

In a preferred embodiment, in the immunoadsorbent material, the carrier is agarose; the amino acid sequence of the antibody in the binding agent is shown as SEQ ID NO. 11 or 17.

In a preferred embodiment, in the immunoadsorbent material, the carrier is agarose; the amino acid sequence of the antibody in the binding agent is shown in SEQ ID NO. 11 or 17, the N terminal of the antibody is provided with methionine, and the C terminal of the antibody is connected with a 6 × histidine tag through a connecting peptide SGS.

In a preferred embodiment, the initial mass to volume ratio of the antibody to the carrier, e.g., agarose, when coupled is 5-40 mg/mL, e.g., 18 mg/mL.

In a preferred embodiment, the support, e.g., agarose, is pre-activated by treatment with CNBr or NHS.

In order to solve the above technical problems, the seventh aspect of the present invention provides a method for purifying a protein comprising an amino acid sequence shown as SEQ ID NO:1, the method comprising the steps of:

1) contacting a protein (a protein to be purified comprising a protein of interest) with an immunoadsorbent material as described above;

2) washing away unbound protein with a wash solution;

3) eluting the protein combined with the immunoadsorption material by eluent to obtain the purified target protein.

In step 1), the protein to be purified comprising the protein of interest may generally be a protein comprising many other impurities (e.g., proteins) in addition to the protein of interest (e.g., the protein comprising the sequence shown in SEQ ID NO: 1). The conditions of this contacting step are such that binding of the binding agent in the immunoadsorbent material to the protein of interest occurs.

In step 2), the washing solution may be conventional in the art, and may be, for example, Tris-HCl buffer or phosphate buffer, preferably at a concentration of 20-50mM, and preferably at a pH of 6-8, for example 7.2. In one embodiment, the wash solution is Tris HCl buffer at pH 7.2, preferably at 20 mM. It is preferred to wash the loaded material until the non-specific immunoadsorbent material or binding agent is eluted.

Step 3) is the desorption of the protein of interest. This desorption is preferably performed by changing the conditions such that the antibody or fragment no longer binds to the target molecule (protein of interest). Elution may be achieved by varying conditions involving pH, salt, temperature, or any other suitable measure. The elution method in which desorption is usually carried out may be elution with an eluent. Preferably, the eluent may be an acidic buffer, the pH of which may preferably be 2-4.5, e.g. 3-4; the eluent is preferably acetic acid buffer solution or glycine buffer solution, and the concentration of the eluent is preferably 20-50 mM; in one embodiment, the eluent is 50mM acetic buffer, pH 3-4. Alternatively, the eluent may also be a salt ion buffer, such as a neutral salt ion buffer. In a certain embodiment, the eluent is 20mM Tris, 2.0M MgCl, pH7.4₂And (4) a buffer solution.

In order to solve the above technical problems, an eighth aspect of the present invention provides the use of an antibody with low affinity for the preparation of a binding agent and/or an immunoadsorbent material; or, the invention provides the use of an antibody with low affinity for the purification of a protein comprising the amino acid sequence shown in SEQ ID NO 1; preferably, the low affinity antibody is an antibody according to the first aspect of the invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods, devices, and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods, devices, and materials are now described.

The three letter and one letter codes for amino acids used herein are known to those skilled in the art, or described in j.biol.chem,243, p3558 (1968).

As used herein, the terms "comprising" or "including" are intended to mean that the compositions and methods include the recited elements but do not exclude other elements, but also include the case of "consisting of … …" as the context dictates.

The term "nucleotide" or "polynucleotide" means a deoxyribonucleotide, deoxyribonucleoside, ribonucleoside, or ribonucleotide, and polymers thereof, in either single-or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have binding properties similar to the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specifically limited, the term also means oligonucleotide analogs, which include PNAs (peptide nucleic acids), DNA analogs used in antisense technology (phosphorothioates, phosphoramidates, and the like). Unless otherwise specified, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (including, but not limited to, degenerate codon substitutions) and complementary sequences as well as the sequence explicitly specified. In particular, degenerate codon substitutions may be achieved by generating sequences in which the 3rd position of one or more selected (or all) codons is substituted with mixed base and/or deoxyinosine residues (Batzer et al, Nucleic Acid Res.19:5081 (1991); Ohtsuka et al, J.biol.chem.260: 2605-S2608 (1985); and Cassol et al (1992); Rossolini et al, Mol cell.Probes8:91-98 (1994)).

The terms "polypeptide" and "protein" are used interchangeably herein to mean a polymer of amino acid residues. That is, the description for a polypeptide applies equally to the description of a peptide and to the description of a protein, and vice versa. The terms apply to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues are a non-naturally encoded amino acid. As used herein, the term encompasses amino acid chains of any length, including full-length proteins (i.e., antigens), in which the amino acid residues are linked via covalent peptide bonds.

The term "host cell" means a cell comprising a nucleotide of the invention, regardless of the method used for insertion to produce a recombinant host cell, e.g., direct uptake, transduction, f-pairing or other methods known in the art. The exogenous polynucleotide may remain as a non-integrating vector, such as a plasmid, or may integrate into the host genome. The host cell may be a prokaryotic cell or a eukaryotic cell.

The term "transformation" means the process of introducing a heterologous DNA sequence into a host cell or organism.

The term "expression" means the transcription and/or translation of an endogenous gene or transgene in a cell.

The positive progress effects of the invention are as follows:

the antibody of the invention has better binding activity to protein molecules (such as FSH, hCG, TSH, hLH, FSH-CTP and the like) containing the sequence shown as SEQ ID NO. 1, and has better thermal stability and better alkali resistance; the binding agent and the immunoadsorption material containing the same can purify protein molecules such as FSH, hCG, TSH, hLH, FSH-CTP and the like containing a sequence shown in SEQ ID NO. 1 under mild conditions (such as neutral pH conditions), and have high purification efficiency, high purity of the obtained product, good product quality, lower purification cost, more convenient use method and simple operation. In addition, the binding agent and the immunoadsorbent material containing the antibody can still maintain better performance after being repeatedly used for many times. In a preferred embodiment, the purification yield is as high as 80% and the purity is as high as 98% or more.

Drawings

FIG. 1: SDS-PAGE results of the purification of the different sequences are shown, in which lanes 1-8 correspond to the results of SEQ ID NO 11 to 18, respectively.

FIG. 2: is a result chart of purity detection of purified protein after coupling and fixing different proteins.

FIG. 3: is a graph showing the results of SDS-PAGE detection after heat-stability treatment of SEQ ID NO. 11 and 17.

FIG. 4: results of binding activity assay after thermal stability treatment of

SEQ ID NO

11 and 17 are shown.

FIG. 5: results of binding activity assays after different alkali treatment times for

SEQ ID NO

11 and 17 are shown.

FIG. 6: the result graph of protein detection for different coupling modes.

FIG. 7: for the IEF profile of the purified protein, the control was the drug Ezier (Ovidrel).

FIG. 8: dynamic loading test of affinity filler every 15 uses.

FIG. 9: affinity fillers the total amount of protein obtained from 8mg of protein per 15 purifications.

FIG. 10: affinity filler protein HCP content assay obtained every 15 purifications.

FIG. 11: and the purity of the protein SEC obtained by purifying the affinity packing every 15 times is detected.

FIG. 12: affinity packing the resulting protein was checked with CE-SDS after every 15 purifications.

FIG. 13: purifying the SDS map of hCG protein by using the affinity filler; lane 1 shows the protein purification results of the affinity filler using the protein of SEQ ID NO. 11 as a ligand, and lane 2 shows the protein purification results of the affinity filler using the protein of SEQ ID NO. 17 as a ligand.

FIG. 14: purifying SDS map of hFSH protein by affinity filling material; lane 1 shows the protein purification results of the affinity filler using the protein of SEQ ID NO. 11 as a ligand, and lane 2 shows the protein purification results of the affinity filler using the protein of SEQ ID NO. 17 as a ligand.

FIG. 15: the SDS map of hLH protein is purified by affinity filling; lane 1 shows the protein purification results of the affinity filler using the protein of SEQ ID NO. 11 as a ligand, and lane 2 shows the protein purification results of the affinity filler using the protein of SEQ ID NO. 17 as a ligand.

Detailed Description

The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.

EXAMPLE 1 Synthesis, expression and purification of sequences

The VHHs of the common alpha chain SEQ ID NO:1 (amino acid sequence is shown in Table 1) of common FSH, hCG, TSH, FSH-CTP and hLH are screened by an immunization method. The alpaca is immunized by using the protein of SEQ ID NO. 1, and a plurality of VHH sequences with the binding activity with the sequence of SEQ ID NO. 1 are obtained by screening through a Phage ELISA method. Of these 6 CDRs, sequences similar but with widely different binding activities were selected for expression (table 2) and tested for their effect as affinity filler ligands. The VHH sequence 1G9E (SEQ ID NO:12) [ PMID:24739391 ] which has been reported to bind to the common alpha chain of FSH, hCG, TSH, FSH-CTP and hLH, SEQ ID NO:1, was also selected. At the same time, a mutant thereof was constructed in which the 59 th tyrosine of 1G9E was mutated to histidine (SEQ ID NO: 11).

The codon optimization of Escherichia coli preference is entrusted to the SEQ ID NO. 11-18 by Nanjing Kingsler Biotech Co., Ltd, N-terminal methionine and C-terminal 6 × histidine tag (VHH-sgs-HHHHHHHHHHHHHHH) are added, polynucleotide sequences encoding the SEQ ID NO. 11-18 (amino acid sequences are shown in the table 1) are synthesized and inserted into a pET32a expression vector, and the recombinant plasmid for polypeptide expression is obtained after the sequencing is correct. The prepared recombinant plasmid was electrically transformed into E.coli BL21 Star (DE3) and inoculated onto LB agarose plates containing 100. mu.g/ml ampicillin. After culturing at 37 ℃ overnight until colonies grew out, individual colonies were picked up and inoculated into 3ml of LB medium containing 100. mu.g/ml ampicillin and cultured at 37 ℃ overnight at 250 rpm. The overnight culture was inoculated into 50ml of LB medium containing 100. mu.g/ml ampicillin, cultured at 37 ℃ until OD600 reached 0.4 to 0.6, and then 0.1mM IPTG was added to continue the culture overnight. And finally, centrifugally collecting the bacterial precipitates of the culture, carrying out ultrasonic cell breaking treatment after the bacterial precipitates are resuspended to 100g/L by PBS, and centrifugally collecting supernatant after cell breaking. The supernatant was purified by nickel column. The purified protein was exchanged into PBS (pH7.0) using an ultrafiltration centrifuge tube at a concentration of greater than 20 mg/mL. The purity of the obtained protein was evaluated by SDS-PAGE, and the results in FIG. 1 show that the purity of 8 sequences is equal to or greater than 97%.

Table 1: amino acid sequences corresponding to different SEQ ID NOs

Example 2 evaluation of binding Activity

The ELISA plate was washed with 2. mu.g/ml hCG

Coating at 2-8 ℃ overnight; then, the plate was washed 3 times with PBST (PBS containing 0.05% Tween 20, pH7.4), and the washed plate was blocked with 300. mu.l of a blocking solution (PBST containing 1% BSA) at 37 ℃ for 2hr, and then washed with PBST.

Sample dilution: samples were diluted to 400ng/ml starting, 2-fold for 11 spots, and loaded into 96-well plates at 100. mu.l/well duplicate wells.

Incubating the ELISA plate at 37 deg.C for 1h with shaking, washing with PBST, adding 40ng/ml Anti-6 XHis tag antibody (HRP) (cat # AB1187, Abcam) into the plate, incubating at 37 deg.C for 1h with shaking, washing with PBST, adding TMB (tetramethyllbenzidine) as chromogenic substrate for HRP, incubating for 15min, and incubating with 2N H₂SO₄And (6) terminating. Absorbance at 450nm was measured with a microplate reader.

Table 2: binding Activity of 8 sequence-corresponding proteins to hCG

Injecting: the ELISA detection of the 1G9E, 1G9E mutant and SEQ ID NO 17 samples does not form a complete 4-parameter curve, the OD value at a high concentration point is far lower than that of other VHHs, saturation is not achieved, and NO EC50 data exist.

The detection results are shown in Table 2, wherein the 1G9E mutant and the protein SEQ ID NO. 17 have NO detectable combination, and the optimization method carries out antigen combination confirmation on the two proteins.

The ELISA plates were washed with 10. mu.g/ml of hCG

And FSH

Coating at 2-8 deg.C overnight. Samples were diluted to 1mg/ml starting, 2 fold diluted for 7 spots, and loaded into 96 well plates at 100 μ l/well replicates, respectively. Anti-6 × His tag antibody (HRP) (cat # AB1187, Abcam) was diluted 1: 5000. The results show (Table 3) that at higher protein concentrations, both proteins of

SEQ ID NO

11, 17 are able to bind both antigens.

Table 3: binding of proteins of

SEQ ID NO

11, 17 to antigen

Example 3 ligand selection

The purified proteins corresponding to SEQ ID NO. 11-18 are immobilized, and the advantages and disadvantages of eight proteins as ligands are compared. The filler is pre-activated by a CNBr coupling mode, and the protein corresponding to SEQ ID NO. 11-18 is fixed on agarose microspheres according to 5mg VHH protein per ml of the filler to prepare the immunoadsorption filler. The immunoadsorption filler is used for purifying protein, and the method comprises the following specific steps:

1) loading a culture supernatant of CHO cells stably expressing hCG to combine the hCG with an immunoadsorption filler, and loading the hCG protein on the filler;

2) washing the loaded immunoadsorbent packing with 20mM Tris HCl buffer pH 7.2 to remove non-specific binding;

3) elution was performed with 50mM acetate buffer pH 3 to finally obtain purified hCG protein.

Collecting the final eluted protein, and detecting the purity of the purified protein by SDS-PAGE. FIG. 2 shows that the purified bands are more specific for the antibodies with

SEQ ID NO

11, 12, 16, 17, 18 as ligands. The antigen is difficult to elute after being combined with the high affinity antibody

SEQ ID NO

12, 16, 18 fillers. The filler elution bands of the coupling of

SEQ ID NO

13, 14 and 15 are relatively miscellaneous. The low affinity antibody

SEQ ID NO

11 and 17 has better filler purification effect. Thus, the proteins of

SEQ ID NO

11, 17 were selected as ligands for subsequent affinity filler preparation tests.

Example 4 thermal stability test

The proteins of

SEQ ID NO

11 and 17 were tested for thermostability. 7 portions were equally taken and subjected to 7 different treatments, respectively, in the manner shown in Table 4. Mixing 20ul of the sample with 2 xSDS loading buffer solution with the same volume, carrying out water bath at 75 ℃ for 5min, and detecting the size and the purity of the protein by SDS-PAGE; the remaining other samples were subjected to similar binding activity assays as in example 2.

Table 4: thermal stability test treatment mode

Group of	Protein concentration	Sample volume (ul)	Treatment method
				1	22mg/ml	200	Standing at 2-8 ℃ for 7 days
2	22mg/ml	200	Standing at 37 deg.C for 3 days
				3	22mg/ml	200	Standing at 37 deg.C for 5 days
4	22mg/ml	200	Standing at 37 deg.C for 7 days
				5	22mg/ml	200	Freeze thawing for 1 time
6	22mg/ml	200	Freeze thawing for 3 times
				7	22mg/ml	200	Freeze thawing for 5 times

The results in FIGS. 3 and 4 show that the VHH protein corresponding to SEQ ID NO. 11 is more thermostable; the VHH protein corresponding to SEQ ID NO. 17 has better freeze-thaw stability.

Example 5 alkali resistance test

The filler preparation process included alkaline conditioning, thus subjecting the selected proteins of SEQ ID NO:11 and 17 to an alkaline resistance test. Mixing the purified protein with 0.4M sodium bicarbonate of the same volume (pH of about 11), standing for 1hr, 2hr, 4hr and 6hr respectively, and adjusting pH to 7.0 with acetic acid. 20ul of each alkali treatment sample is mixed with 2 xSDS loading buffer solution with the same volume, and then the mixture is bathed in water at 75 ℃ for 5min, and the size and the purity of the protein are detected by SDS-PAGE. The remaining samples were subjected to similar binding activity assays as in example 2.

The results showed NO decrease in activity with different alkaline treatment times (FIG. 5), indicating that the aglucon proteins of SEQ ID NO:11 and 17 are more alkaline resistant.

EXAMPLE 6 preparation of affinity purification Medium

The agarose filler is pre-activated by CNBr, NHS and epoxy. The initial amount of conjugated protein was increased to 18mg protein per ml of filler. Proteins of

SEQ ID NO

11 and 17 were immobilized on agarose microspheres. The coupling efficiencies of the different methods are shown in table 5.

The results show that the coupling amount of the filler coupled by CNBr is 16.55mg/ml and 15.57mg/ml corresponding to the proteins SEQ ID NO. 11 and 17, and the coupling efficiency is 91.9% and 86.5%.

Table 5: coupling efficiency of different fillers

Example 7 binding load test

The filler prepared in example 6 was tested for binding loading using a purification procedure similar to that of example 3. And (3) overload sample loading is carried out on CHO cell culture supernatant which stably expresses hCG, all flow-through is collected, the concentration of the flow-through protein is detected, and the total amount of the sample loading protein and the total amount of the flow-through protein are calculated according to the volume.

Binding capacity (total amount of loading protein-total amount of flow-through protein)/column volume

The epoxidized filler incorporation was found to be somewhat lower, while the filler incorporation was higher for both coupling modes, with filler incorporation by CNBr reaching about 9mg/ml (table 6).

Table 6: binding carriers of different coupling modes

Bound loading (mg/ml)	Protein of SEQ ID NO 11	17 protein of SEQ ID NO
			CNBr	9.58	8.83
NHS	7.66	8.16
			Epoxy resin	4.9	5.9

Example 8 affinity purification of hCG

The filler obtained in example 6 was subjected to a purification effect test by a purification method similar to that in example 3. The culture supernatants of CHO cells stably expressing hCG were loaded at 70% of the binding capacity. The purified product was subjected to SDS-PAGE, host cell protein residue (HCP) and IEF. The results show that the yields of the filler purified proteins obtained by the CNBr and NHS modes are both high, wherein the final yield is as high as 80 percent (table 7) and the purity is as high as more than 98 percent (figure 6) by the CNBr coupling method. And the purified product had a similar IEF profile compared to the control drug (Merck Serono esque) (fig. 7). The purified protein was subjected to host cell protein residue (HCP) content detection using CHO HCP RESUPPLY 3rd Generation ELISA Kit (Cygnus Technologies, cat # F550-1). The results show that the residual value of the purified protein host cell protein is far lower than the HCP residual minimum limit (100-500PPM) of the hCG drug, which indicates that the filler purified product of the invention has low HCP residual and high safety (Table 7).

Table 7: purification effect of different fillers

Example 9: study on cleaning effect of affinity filler cleaning buffer

The filler obtained by pre-activation of CNBr was subjected to an affinity filler washing buffer effect test similar to the purification method in example 3, and after elution of the target protein, 4 column volumes were washed with Gly, pH 2.0. The purification experiment was repeated 60 times, and the content of the target protein and the residual amount of HCP in the washing solution were measured 15 times at a time.

And (3) detecting the content of the target protein:

coating the ELISA plates with 2 mu g/ml of hCG alpha antibody (hCG-alpha) (cat: AB20712, abcam) at 2-8 ℃ overnight; then, the plate was washed 3 times with PBST (PBS containing 0.05% Tween 20, pH7.4), and the washed plate was blocked with 300. mu.l of a blocking solution (PBST containing 1% BSA) at 37 ℃ for 2hr, and then washed with PBST.

Sample dilution: the elution wash samples were undiluted, diluted 10-fold, and 100-fold with PBST, and loaded into a 96-well plate at 100. mu.l/well. The standard substance is diluted by recombinant human chorionic gonadotrophin injection (Ezeria) in a dilution range of 100 ng/ml-0.098 ng/ml in a gradient manner by 2 times.

Detecting an antibody: 0.1ug/ml of Anti-hCG beta antibody-C-terminal (cat # AB171549, abcam) was added to the microplate and incubated.

The ELISA plate was incubated at 37 ℃ for 1 hour with shaking, washed with PBST, and then 40ng/ml goat anti-rabbit lgG (H + L) antibody (HRP) (cat # A0208, Biyunyan) was added to the plate and incubated at 37 ℃ for 1 hour with shaking. After washing with PBST, TMB (tetramethyle) was addedlbenzidine) as chromogenic substrate for HRP for 15min, 2N H₂SO₄And (6) terminating. Absorbance at 450nm was measured with a microplate reader.

Table 8: cleaning buffer cleaning effect of different fillers

As can be seen from the above table, the content of the target protein in the solution obtained by washing was very low and no residual amount of HCP remained. Therefore, 0.1MGly, pH2.0 can be used as a wash solution for affinity chromatography.

Example 10: study on the Life of affinity Filler

Affinity filler lifetime studies were performed on the fillers obtained by CNBr preactivation using a purification method similar to that in example 3 and using the affinity filler washing buffer of example 9. The purification experiment is repeated for 60 times, and the eluted target protein is taken every 15 times for detection, wherein the detection indexes comprise HPLC-SEC purity, CE-SDS (capillary electrophoresis), exogenous DNA residue and affinity ligand residue. And dynamic loading was determined for chromatographic column performance.

Dynamic load detection: 0.5mg/ml hCG using AKTA Avant protein purification apparatus (GE) as the test device

To test the solution. The hCG solution was first tested for UV280 absorption at 0.5mg/ml hCG solution and then the UV absorption curve of the hCG solution on different packing. The retention time was 5 minutes, and the loading volume through which 10% of the sample flowed was recorded and the dynamic adsorption loading of the packing was calculated. Calculating the formula: dynamic loading is the loading volume x loading protein concentration/column volume.

Detecting exogenous DNA residues: the CHO residual DNA detection kit (PCR-fluorescent probe method) of Huzhou Shenke biotechnology limited company is adopted to detect the content of exogenous DNA in the purified protein.

And (3) detecting the residue of the affinity ligand: the residual amount of affinity ligand was measured by ELISA similar to that of example 2. The final affinity ligand residue formula is: the residual amount (ppm) of the test HCG affinity ligand (measured as the test HCG affinity ligand content (ng/ml) × dilution factor)/the test protein content (mg/ml).

The results show that the affinity filler was used 60 times and the dynamic loading of the filler was more consistent (figure 8). The amount of protein eluted was not significantly changed (fig. 9). The HCP content was below 30ppm for each assay (FIG. 10). The SEC purity and the CE-SDS content of the target protein were both greater than 99% (FIG. 11 and FIG. 12). The detection results of the exogenous DNA residue and the affinity ligand residue are both less than the detection limit. The research shows that the affinity filler can ensure relatively consistent purification effect after being repeatedly used for 60 times.

Example 11: effect of affinity filler on purification of different proteins containing common alpha subunit

The filler obtained by pre-activation of CNBr was purified by a similar purification method as in example 3 for different proteins including hCG, hFSH, hLH. After purification, the eluted protein samples were tested and the purification effect was shown by SDS mapping, and FIG. 13, FIG. 14, FIG. 15 correspond to hCG, hFSH, hLH, respectively. Wherein Lane 1 shows the result of protein purification by affinity packing using the protein of SEQ ID NO. 11 as a ligand, and Lane 2 shows the result of protein purification by affinity packing using the protein of SEQ ID NO. 17 as a ligand. The purified protein has single band and the purity is higher than 90%.

The applicant states that the present invention is illustrated in detail by the above examples, but the present invention is not limited to the above detailed methods, i.e. it is not meant that the present invention must rely on the above detailed methods for its implementation. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.

SEQUENCE LISTING

<110> Suzhou Cheng Ji pharmaceutical Co., Ltd

<120> antibody, binding agent containing same, immunoadsorbent material and application thereof

<130> P20014942C

<160> 27

<170> PatentIn version 3.5

<210> 1

<211> 92

<212> PRT

<213> Artificial Sequence

<220>

<223> alpha chain

<400> 1

Ala Pro Asp Val Gln Asp Cys Pro Glu Cys Thr Leu Gln Glu Asn Pro

1 5 10 15

Phe Phe Ser Gln Pro Gly Ala Pro Ile Leu Gln Cys Met Gly Cys Cys

20 25 30

Phe Ser Arg Ala Tyr Pro Thr Pro Leu Arg Ser Lys Lys Thr Met Leu

35 40 45

Val Gln Lys Asn Val Thr Ser Glu Ser Thr Cys Cys Val Ala Lys Ser

50 55 60

Tyr Asn Arg Val Thr Val Met Gly Gly Phe Lys Val Glu Asn His Thr

65 70 75 80

Ala Cys His Cys Ser Thr Cys Tyr Tyr His Lys Ser

85 90

<210> 2

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR1-Kabat of protein 11

<400> 2

Thr Tyr Asp Met Gly

1 5

<210> 3

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2-Kabat of protein 11

<400> 3

Ala Ile Asn Trp Asp Ser Ala Arg Thr His Tyr Ala Ser Ser Val Arg

1 5 10 15

Gly

<210> 4

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR3-Kabat of protein 11

<400> 4

Gly Glu Gly Gly Thr Trp Asp Ser

1 5

<210> 5

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR1-IMGT of protein 11

<400> 5

Gly Arg Thr Gly Ser Thr Tyr Asp

1 5

<210> 6

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2-IMGT of protein 11

<400> 6

Ile Asn Trp Asp Ser Ala Arg Thr

1 5

<210> 7

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR3-IMGT of protein 11

<400> 7

Gly Ala Gly Glu Gly Gly Thr Trp Asp Ser

1 5 10

<210> 8

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR1-Chothia of protein 11

<400> 8

Gly Arg Thr Gly Ser Thr Tyr

1 5

<210> 9

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2-Chothia of protein 11

<400> 9

Asn Trp Asp Ser Ala Arg

1 5

<210> 10

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR3-Chothia of protein 11

<400> 10

Gly Glu Gly Gly Thr Trp Asp Ser

1 5

<210> 11

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> protein 11

<400> 11

Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Gly Ser Thr Tyr

20 25 30

Asp Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Ser Val

35 40 45

Ala Ala Ile Asn Trp Asp Ser Ala Arg Thr His Tyr Ala Ser Ser Val

50 55 60

Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Lys Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Thr Cys

85 90 95

Gly Ala Gly Glu Gly Gly Thr Trp Asp Ser Trp Gly Gln Gly Thr Gln

100 105 110

Val Thr Val Ser Ser

115

<210> 12

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> protein 12

<400> 12

Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Gly Ser Thr Tyr

20 25 30

Asp Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Ser Val

35 40 45

Ala Ala Ile Asn Trp Asp Ser Ala Arg Thr Tyr Tyr Ala Ser Ser Val

50 55 60

Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Lys Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Thr Cys

85 90 95

Gly Ala Gly Glu Gly Gly Thr Trp Asp Ser Trp Gly Gln Gly Thr Gln

100 105 110

Val Thr Val Ser Ser

115

<210> 13

<211> 119

<212> PRT

<213> Artificial Sequence

<220>

<223> protein 13

<400> 13

Gln Val Gln Val Ala Glu Ser Gly Gly Gly Leu Val Gln Ser Gly Gly

1 5 10 15

Ser Met Asn Leu Ser Cys Thr Ala Ser Gly Asn Leu Val Ser Val Asp

20 25 30

Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Glu Glu Arg Glu Ala Val

35 40 45

Ala Ala Ile Met Trp Ser Gly Asp Ser Thr Tyr Tyr Ala Asn Ala Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Arg Leu Gln Glu Glu Gly Trp Trp Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Gln Val Thr Val Ser Ser

115

<210> 14

<211> 119

<212> PRT

<213> Artificial Sequence

<220>

<223> protein 14

<400> 14

Gln Val Gln Val Ala Glu Ser Gly Gly Gly Leu Val Gln Ser Gly Gly

1 5 10 15

Ser Met Asn Leu Ser Cys Thr Ala Ser Gly Arg Thr Phe Ser Ser His

20 25 30

Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Glu Glu Arg Glu Ala Val

35 40 45

Ala Ala Ile Met Trp Ser Gly Asp Ser Thr Tyr Tyr Ala Asn Ala Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Arg Leu Gln Glu Glu Gly Trp Trp Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Gln Val Thr Val Ser Ser

115

<210> 15

<211> 121

<212> PRT

<213> Artificial Sequence

<220>

<223> protein 15

<400> 15

Gln Val Gln Val Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Asp

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Arg Thr Gly Ser Phe Ser Ser Tyr

20 25 30

Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Ala Val

35 40 45

Ala Ala Ile Thr Trp Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Thr Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Arg Asp Arg Ala Asp Trp Tyr Ser Trp Trp Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 16

<211> 121

<212> PRT

<213> Artificial Sequence

<220>

<223> protein 16

<400> 16

Gln Val Gln Val Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Asp

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Tyr

20 25 30

Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Ala Val

35 40 45

Ala Ala Ile Thr Trp Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Thr Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Arg Asp Arg Ala Asp Gly Ser Ser Trp Trp Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 17

<211> 121

<212> PRT

<213> Artificial Sequence

<220>

<223> protein 17

<400> 17

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Thr Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Tyr

20 25 30

Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Glu Glu Arg Glu Ala Val

35 40 45

Ala Ala Leu Ser Trp Ser Gly Gly Ser Thr Tyr Tyr Ala Ala Ser Val

50 55 60

Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Arg Asp Arg Ala Asp Gly Ser Ser Trp Trp Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 18

<211> 121

<212> PRT

<213> Artificial Sequence

<220>

<223> protein 18

<400> 18

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Thr Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Tyr

20 25 30

Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Glu Glu Arg Glu Ala Val

35 40 45

Ala Ala Ile Thr Trp Ser Gly Gly Ser Thr Tyr Tyr Ala Ala Ser Val

50 55 60

Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Arg Asp Arg Ala Asp Gly Ser Ser Trp Trp Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 19

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR1-IMGT of protein 17

<400> 19

Gly Arg Thr Phe Ser Ser Tyr Ala

1 5

<210> 20

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2-IMGT of protein 17

<400> 20

Leu Ser Trp Ser Gly Gly Ser Thr

1 5

<210> 21

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR3-IMGT of protein 17

<400> 21

Ala Ala Arg Asp Arg Ala Asp Gly Ser Ser Trp Trp Asp Tyr

1 5 10

<210> 22

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR1-Chothia of protein 17

<400> 22

Gly Arg Thr Phe Ser Ser Tyr

1 5

<210> 23

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2-Chothia of protein 17

<400> 23

Ser Trp Ser Gly Gly Ser

1 5

<210> 24

<211> 12

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR3-Chothia of protein 17

<400> 24

Arg Asp Arg Ala Asp Gly Ser Ser Trp Trp Asp Tyr

1 5 10

<210> 25

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR1-Kabat of protein 17

<400> 25

Ser Tyr Ala Met Gly

1 5

<210> 26

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2-Kabat of protein 17

<400> 26

Ala Leu Ser Trp Ser Gly Gly Ser Thr Tyr Tyr Ala Ala Ser Val Gln

1 5 10 15

Gly

<210> 27

<211> 12

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR3-Kabat of protein 17

<400> 27

Arg Asp Arg Ala Asp Gly Ser Ser Trp Trp Asp Tyr

1 5 10

Claims

1. An antibody targeting a protein comprising the amino acid sequence shown as SEQ ID NO. 1,

the antibody comprises a heavy chain variable region (VHH) comprising the following CDRs numbered according to IMGT: CDR1 having an amino acid sequence as set forth in SEQ ID NO 5 or 19; CDR2 having an amino acid sequence as set forth in SEQ ID NO 6 or 20; and, CDR3 having an amino acid sequence set forth in SEQ ID NO. 7 or 21.

2. The antibody of claim 1, wherein the VHH comprises the following CDRs numbered according to IMGT: CDR1 having an amino acid sequence set forth in SEQ ID NO. 5; CDR2 having an amino acid sequence set forth in SEQ ID NO. 6; and, CDR3 having an amino acid sequence set forth in SEQ ID NO. 7; alternatively, the VHH comprises the following CDRs numbered according to IMGT: CDR1 having an amino acid sequence shown in SEQ ID NO. 19, CDR2 having an amino acid sequence shown in SEQ ID NO. 20, and CDR3 having an amino acid sequence shown in SEQ ID NO. 21;

preferably, the heavy chain variable region further comprises a framework region (FWR) of an alpaca antibody or a human antibody;

more preferably, the amino acid sequence of the heavy chain variable region is the amino acid sequence shown as SEQ ID NO 11 or 17.

3. The antibody of claim 1 or 2, wherein the antibody is a VHH, a heavy chain antibody, a bispecific antibody or a multispecific antibody.

4. The antibody of any one of claims 1-3, wherein the protein comprising the amino acid sequence set forth in SEQ ID NO. 1 is selected from one or more of FSH, hCG, TSH, FSH-CTP and hLH; and/or, the antibody is a VHH.

5. An isolated nucleic acid encoding the antibody of any one of claims 1-4.

6. A recombinant expression vector comprising the isolated nucleic acid of claim 5;

7. A transformant comprising the recombinant expression vector of claim 6 in a host cell;

preferably, the host cell is a yeast such as saccharomyces cerevisiae, a mold, a bacterium, or a cellular expression system.

8. A binding agent comprising the antibody of any one of claims 1-4;

preferably:

the binding agent is a monospecific binding agent;

and/or, the antibody is a genetically modified antibody, preferably having a modification at its N-terminus to allow for initial expression of the antibody and/or a tag at its C-terminus, e.g. a methionine at its N-terminus, and/or a 6 × histidine tag at its C-terminus, said 6 × histidine tag preferably being linked to its C-terminus by a linking peptide, e.g. SGS.

9. An immunoadsorbent material comprising the binding agent of claim 8;

preferably:

the immunoadsorbent material further comprises a support, preferably a porous solid support, such as agarose, Sepharose beads, polystyrene, controlled pore glass, cellulose, dextran, diatomaceous earth, synthetic polymers such as Sepharose^TMOr porous amorphous silica; and/or, the carrier is preferably a CNBr and/or NHS pre-activated carrier;

more preferably:

the carrier is agarose, and the initial mass-to-volume ratio of the antibody to the agarose when coupled is preferably 5-40 mg/mL, such as 18 mg/mL.

10. A method of purifying a protein comprising an amino acid sequence as set forth in SEQ ID No. 1, comprising the steps of:

1) contacting the protein with the immunoadsorbent material of claim 9;

2) washing away unbound protein with a wash solution;

3) eluting the protein bound to the immunoadsorbent material with an eluent;

preferably:

the washing solution is Tris hydrochloric acid buffer solution or phosphate buffer solution, the concentration of the washing solution is preferably 20-50mM, and the pH of the washing solution is preferably 6-8, such as 7.2;

and/or, the eluent is an acidic buffer, preferably having a pH of 2-4.5, e.g. 3-4; the eluent is preferably acetic acid buffer solution or glycine buffer solution, and the concentration of the eluent is preferably 20-50 mM; alternatively, the eluent is a salt buffer, preferably a neutral salt buffer, preferably at a concentration of 20mM, for example 20mM Tris, 2.0M MgCl, pH7.4₂Salt ion buffer.

11. Use of an antibody with low affinity for the preparation of a binding agent and/or an immunoadsorption material, or for the purification of a protein comprising an amino acid sequence as shown in SEQ ID NO 1; preferably, the low affinity antibody is the antibody of claims 1-4.