CN106928366B - FAP nano antibody Nb67 - Google Patents

Abstract

The invention discloses an FAP nano antibody aiming at FAP polypeptide molecular epitope, also discloses a gene sequence for coding the FAP nano antibody, an expression vector and a host cell capable of expressing the FAP nano antibody, and also discloses application of the FAP nano antibody. The FAP nano antibody, the gene sequence, the host cell and the like disclosed by the invention can be efficiently expressed in escherichia coli, have good specificity and high affinity with FAP immunoreaction, and can be applied to preparation of FAP detection reagents or anti-tumor drugs and the like.

Description

A FAP Nanobody Nb67

technical field

The present invention belongs to the technical field of biomedicine or biopharmaceuticals, and more particularly relates to a nanobody (Nb67) directed against an antigenic epitope molecule of a FAP polypeptide molecule, its coding sequence and use.

Background technique

Fibroblast activation protein (FAP) is a surface antigen specifically expressed by tumor-associated fibroblasts with dipeptidyl peptidase and collagenase activities. The role of immunosuppression. FAP is composed of 761 amino acids and has different levels of glycosylation in different mammalian cells. The relative molecular weight of monomers is 8.8×10 ⁴ , 9.5×10 ⁴ , 9.7×10 ⁴ and other reports. The relative molecular weight of dimers The mass is 1.7×10 ⁵ . The structure of FAP is divided into three parts: the cytoplasmic region, the transmembrane region and the extracellular region. The cytoplasmic region is a short peptide chain composed of the first 6 amino acids, and the transmembrane region is composed of 19 amino acids, which play a fixed role. The hydrophobic transmembrane fragment, however, most of the amino acid sequence 20-761 in the FAP molecule is exposed to the extracellular microenvironment. This part is the extracellular region, which is the key enzyme catalytic region. FAP has both dipeptidyl peptidase activity and collagenase activity, and can degrade dipeptide and type I collagen in the matrix. FAP shares 50% homology with DPPIV (dipeptidyl peptidase IV/CD26) belonging to the dipeptidyl peptidase family, both have exonuclease activity and often form complexes. Therefore, it is speculated that FAP can promote the occurrence and development of tumors by regulating certain peptide growth factors in tumor stroma, modifying active peptides and changing their functions.

In malignant tumors, FAP-positive fibroblasts account for only 2% of the total tumor cells, but when they are removed, both cancer cells and stromal cells undergo rapid necrosis. It can be seen that FAP plays a very important role in the occurrence and development of malignant tumors, and has broad application prospects in tumor treatment. Therapeutic strategies targeting the tumor microenvironment, especially targeting FAP, play a very important role in tumor therapy. In the process of tumor occurrence and development, tumor cells not only passively evade the attack of the immune system, but also actively inhibit the normal function of immune cells in their growth environment. environment and promote the malignant progression of tumors. The existence of these factors provides ideas for exploring the mechanism of tumorigenesis and opening up new tumor immunotherapy regimens.

At present, several FAP-specific monoclonal antibodies have been developed and have begun to be used in clinical diagnosis and treatment, and their affinity for tumor tissue has been improved. However, this traditional method has the disadvantages of poor antibody stability, low sensitivity and high production cost.

Nanobody technology is an antibody engineering revolution carried out by biomedical scientists on the basis of traditional antibodies, using molecular biology technology combined with the concept of nanoparticle science, thereby developing the latest and smallest antibody molecules. .Hamas found in camel blood. Ordinary antibody proteins consist of two heavy chains and two light chains, while the new antibodies discovered from camel blood have only two heavy chains and no light chains. These "heavy chain antibodies" can interact with targets such as antigens like normal antibodies. Tightly bound, but not clumped together like single chain antibodies. Nanobodies based on this "heavy chain antibody" not only have a molecular weight that is only 1/10 of that of ordinary antibodies, but also have more flexible chemical properties, good stability, high solubility, easy expression and availability, and easy coupling to other molecules. However, no suitable nanobody has been developed for FAP in the prior art.

SUMMARY OF THE INVENTION

The technical problem to be solved by the present invention is to provide a FAP nanobody, a DNA molecule encoding the FAP nanobody and the application of the nanobody, aiming at the deficiency of the nanobody against the FAP epitope in the prior art.

The technical solution adopted by the present invention to solve the technical problem is as follows: In the first aspect of the present invention, a FAP nanobody is provided, and the FAP nanobody is a nanobody against the FAP epitope, including two such as SEQ ID NO: 9 The VHH chain of the indicated amino acid sequence. In the present invention, the FAP Nanobody can be abbreviated as Nb67.

The second aspect of the present invention provides a VHH chain against a FAP Nanobody, comprising a framework region FR and a complementarity determining region CDR, wherein the framework region FR includes the amino acid sequence of the following group of FRs: FR1 shown in SEQ ID NO: 1 , FR2 shown in SEQ ID NO: 2, FR3 shown in SEQ ID NO: 3, and FR4 shown in SEQ ID NO: 4; the complementarity determining region CDRs include the amino acid sequences of the following CDRs: SEQ ID NO: CDR1 shown in 5, CDR2 shown in SEQ ID NO:6, CDR3 shown in SEQ ID NO:7. The structure of the framework region is relatively conservative and mainly plays the role of maintaining the protein structure; the structure of the complementarity determining region is relatively diverse and is mainly responsible for the recognition of antibodies.

Preferably, the VHH chain of the nanobody against FAP has the amino acid sequence shown in SEQ ID NO:9.

The third aspect of the present invention provides a DNA molecule for encoding a protein selected from the group consisting of the FAP nanobody Nb67 of the present invention, or the VHH chain against the FAP nanobody of the present invention.

Preferably, the DNA molecule has the nucleotide sequence shown in SEQ ID NO:8.

The FAP nanobody provided by the present invention can be produced in large quantities by means of phage amplification or recombinant expression through genetic engineering. Phage amplification refers to mass propagation of phages displaying FAP nanobody Nb67 through biological amplification to produce phage particles displaying FAP nanobody Nb67. The way of genetic engineering recombinant expression refers to the gene encoding FAP nanobody Nb67 is cloned into an expression vector, and the nanobody is produced in large quantities in the form of protein expression.

The fourth aspect of the present invention provides an expression vector, which contains the nucleotide sequence shown in SEQ ID NO:8.

The fifth aspect of the present invention provides a host cell that can express the FAP nanobody Nb67 of the present invention.

The sixth aspect of the present invention provides the use of the FAP nanobody Nb67 of the present invention in preparing a FAP detection reagent. The FAP nanobody Nb67 of the present invention can replace the traditional FAP antibody to prepare a FAP detection reagent for detecting FAP polypeptide molecules. The present invention also provides the use of the FAP nanobody Nb67 in the preparation of antitumor drugs. The present invention also correspondingly provides a FAP detection reagent or an anti-tumor drug, including the FAP nanobody Nb67 as described above.

The seventh aspect of the present invention provides the use of the FAP nanobody Nb67 of the present invention in immunological detection for non-therapeutic purposes. The types of immunological detection include enzyme-linked immunosorbent assay, colloidal gold immunochromatography, immunospot hybridization and other types of immunological analysis and detection based on antigen-antibody specific reaction. When the FAP nanobody Nb67 of the present invention is applied, the phage particles displaying the FAP nanobody Nb67 obtained by phage amplification can be directly used for analysis and detection, and the FAP nanobody Nb67 can be expressed in prokaryotes or eukaryotes in the form of protein Perform immunological assays.

The eighth aspect of the present invention provides the application of the FAP nanobody Nb67 of the present invention in the preparation of a binding and adsorbing FAP reagent. For example, the FAP nanobody of the present invention can be made into an immunoaffinity column, and the FAP can be adsorbed and captured for further research after enrichment.

The implementation of the present invention has the following beneficial effects: the present invention first synthesizes the FAP polypeptide and makes it immunogenic, and then couples the FAP molecule on the ELISA plate to display the correct spatial structure of the protein, and the antigen in this form utilizes Phage display technology screened the immune nanobody gene library (camel heavy chain antibody phage display gene library) to obtain the FAP-specific nanobody gene, which was transferred to E. coli, thereby establishing a highly efficient expression in E. coli The nanobody strain of FAP obtained by the method of the present invention has the characteristics of immunoreactivity with FAP, and has good specificity and high affinity, and can be used for preparing FAP detection reagents or antitumor drugs.

Description of drawings

The present invention will be further described below in conjunction with the accompanying drawings and embodiments, in which:

Figure 1 is a schematic diagram of the amino acid numbering and structure of the FAP Nanobody;

Fig. 2 is the DNA electrophoresis picture of FAP Nanobody;

Figure 3 is an electrophoresis image of SDS-PAGE after purification of FAP Nanobody by nickel-column resin gel affinity chromatography.

Detailed ways

In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

The present invention utilizes phage display technology to screen single-domain heavy-chain antibody (VHH) phage clones that can specifically bind to the target molecule FAP antigen from the camel-derived immunized single-domain heavy chain antibody library, and obtains a single-domain heavy chain antibody (VHH) phage clone that is highly efficient in Escherichia coli The expressed Nanobody strain, the FAP Nanobody Nb67.

The present invention will be further described below in conjunction with specific embodiments.

Example 1: Construction of Nanobody library against FAP:

(1) First, synthesize FAP polypeptide, mix 1 mg of FAP with an equal volume of Freund's adjuvant, and immunize a Xinjiang dromedary camel once a week for a total of 7 times to stimulate B cells to express antigen-specific nanobodies;

(2) After 7 immunizations, extract 100 mL of camel peripheral blood lymphocytes and extract total RNA;

(3) synthesizing cDNA and using nested PCR to amplify VHH;

(4) Use restriction endonucleases PstI and NotI to digest 20ug pComb3 phage display vector (supplied by Biovector China Plasmid Vector Strain Cell Gene Collection Center) and 10ug VHH and ligate the two fragments; (5) Transform the ligated product into electrocompetence In the state cell TG1, the FAP nanobody library was constructed and the storage capacity was determined, and the storage capacity was 1.85×10 ⁸ .

Example 2: Nanobody screening process against FAP:

(1) 20ug FAP dissolved in 100mM NaHCO ₃ , pH 8.2 was coupled to a NUNC ELISA plate and placed at 4°C overnight;

(2) Add 100uL of 0.1% casein the next day, block at room temperature for 1-3h;

(3) After 1-3h, add 100uL of phage (5×10 ¹¹ tfu immunized camel nanobody phage display gene library), and act for 1-2h at room temperature;

(4) Wash 4-6 times with 0.05% PBS+Tween-20 to wash off unbound phage;

(5) Dissociate the phage that specifically binds to FAP with 100 mM TEA (triethylamine), infect E. coli TG1 in log phase growth, and cultivate at 37°C for 1 h to generate and purify the phage for the next round of Screening, the same screening process was repeated for 3-5 rounds, and enrichment was gradually obtained.

Example 3: Screening of specific single positive clones with phage-enzyme-linked immunosorbent assay (ELISA):

(1) From the cell culture dishes containing phage after 3-5 rounds of selection above, select 96 single colonies and inoculate them in TB medium containing 100 μg/mL ampicillin (1 liter of TB medium contains 2.3 g phosphoric acid) Potassium dihydrogen, 12.52 g of dipotassium hydrogen phosphate, 12 g of peptone, 24 g of yeast extract, 4 ml of glycerol), after growth to logarithmic phase, add a final concentration of 1 mmol of isopropyl thiogalactoside ( IPTG), incubated overnight at 30-35°C.

(2) Obtain crude antibody by permeation method, transfer the antibody to an antigen-coated ELISA plate, and place at room temperature for 1-1.5 hours.

(3) Wash off unbound antibodies with PBST, add primary anti-mouse anti-HA tagantibody (purchased from Beijing Kangwei Century Biotechnology Co., Ltd.), and place at room temperature for 1-1.5 hours.

(4) Wash off unbound antibodies with PBST, add anti-mouse alkaline phosphatase conjugate (goat anti-mouse alkaline phosphatase-labeled antibody, purchased from Ai Meijie Technology Co., Ltd.), and place at room temperature for 1-1.5 hours.

(5) Wash off the unbound antibody with PBST, add alkaline phosphatase chromogenic solution, and read the absorbance value on an ELISA instrument at a wavelength of 405 nm.

(6) When the OD value of the sample well is more than 3 times greater than the OD value of the control well, it is judged as a positive clone well.

(7) The bacteria in the positive clone wells were shaken in LB liquid containing 100 micrograms per milliliter to extract plasmids and sequenced.

Through the above experiments, the present invention obtained 6 positive clone wells that showed more than 3 times the binding force to the antigen, which were regarded as the primary screening positive clones. According to the sequence alignment software Vector NTI, the gene sequences of each positive clone strain were analyzed, and the strains with the same CDR1, CDR2 and CDR3 sequences were regarded as the same clone strain, and the strains with different sequences were regarded as different clone strains, and a group of clones could be obtained. The amino acid sequence of the VHH chain of the antibody is shown in SEQ ID NO:9. The FAP nanobody is numbered as Nb67, and its amino acid numbering and schematic diagram are shown in Figure 1.

Please refer to Figure 2 for the DNA electropherogram of the FAP Nanobody. The DNA band of the first gel hole marked M in the figure is: 1000bp DNA molecular marker, and the DNA bands of the gel holes marked 1-24 from left to right are all FAP nanobody DNA electrophoresis strips bring. The DNAs in these 1-24 gel wells are all PCR products of the FAP Nanobody DNA in the aforementioned positive clones, and the PCR product band is about 500 bp.

Example 4: Nanobody expression and purification in host bacteria Escherichia coli:

(1) Electrotransform the plasmid of the cloned strain whose amino acid sequence obtained by the previous sequencing analysis as shown in SEQ ID NO: 9 into Escherichia coli WK6, and spread it on LA+glucose, that is, a culture plate containing ampicillin and glucose incubate at 37°C overnight;

(2) Select a single colony and inoculate it in 5 mL of LB medium containing ampicillin, and cultivate overnight at 37°C on a shaker;

(3) Inoculate 1 mL of overnight bacterial strain into 330 mL of TB culture solution, cultivate at 37°C on a shaker, cultivate until the OD value reaches 0.6 to 1, add IPTG, and culture at 30-35°C on a shaker overnight;

(4) centrifugation to collect bacteria;

(5) utilize osmosis method, obtain antibody crude extract;

(6) Proteins with a purity of more than 90% can be prepared by nickel column ion affinity chromatography.

Please refer to FIG. 3 , which is the electropherogram of SDS-PAGE after purification of FAP nanobody by nickel-column resin gel affinity chromatography. The symbol M represents a protein molecular marker, and the symbol Nb67 represents the SDS-PAGE band of the FAP nanobody Nb67 obtained in Example 4 of the present invention, and the unit in the figure is KDa. The results show that the size of the FAP nanobody Nb67 is about 15KDa, and the purity of the FAP nanobody Nb67 can reach more than 95% after the above purification process.

The present invention also carried out kinetic affinity analysis on the FAP nanobody Nb67 purified in the above Example 4. The experimentally measured association rate constant is 4.21×10 ⁴ (unit M ^-1 S ^-1 ), the dissociation rate constant is 2.87×10 ^-3 (unit S ^-1 ), and the equilibrium dissociation constant is calculated to be 6.82×10 ^-8 (unit M). Compared with the affinity data of other clones selected from the phage library, the equilibrium dissociation constant of the FAP nanobody Nb67 is lower, which means that the antibody and antigen are difficult to dissociate, and its affinity is stronger.

The present invention has been described in terms of specific embodiments, but it will be understood by those skilled in the art that various changes and equivalents may be made without departing from the scope of the invention. In addition, many modifications may be made to adapt the present invention to a particular situation or material to which it is intended without departing from its scope. Therefore, the present invention is not limited to the specific embodiments disclosed herein, but includes all embodiments falling within the scope of the claims.

Claims (9)

1. A FAP Nanobody, characterized in that, the FAP Nanobody is a Nanobody against FAP epitope, and the amino acid sequence of its VHH chain is shown in SEQ ID NO:9. 2. A VHH chain against FAP Nanobody, comprising framework region FR and complementarity determining region CDR, characterized in that the framework region FR comprises the amino acid sequence of the FR of the following group: FR1 shown in SEQ ID NO: 1, FR2 shown in SEQ ID NO: 2, FR3 shown in SEQ ID NO: 3, FR4 shown in SEQ ID NO: 4; the complementarity determining region CDRs include the amino acid sequences of the following CDRs: SEQ ID NO:5 CDR1 shown, CDR2 shown in SEQ ID NO:6, CDR3 shown in SEQ ID NO:7. 3. A DNA molecule, characterized in that it is used to encode a protein selected from the group consisting of the FAP Nanobody of claim 1, or the VHH chain of claim 2 against the FAP Nanobody. 4 . The DNA molecule according to claim 3 , wherein the nucleotide sequence is shown in SEQ ID NO: 8. 5 . 5. An expression vector, characterized in that it contains the nucleotide sequence shown in SEQ ID NO:8. 6. A host cell, characterized in that it can express the FAP nanobody of claim 1. 7. Use of the FAP nanobody of claim 1 in the preparation of a FAP detection reagent or an antitumor drug. 8 . A FAP detection reagent or anti-tumor drug, characterized in that it comprises the FAP nanobody of claim 1 . 9. The application of the FAP nanobody of claim 1 in the preparation of binding and adsorbing FAP reagents.

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