CN106928366A - A kind of FAP nano antibodies Nb67 - Google Patents

Abstract

The invention discloses a FAP nanobody directed at the epitope of the FAP polypeptide molecule, and also discloses the gene sequence encoding the FAP nanobody and the expression vector and host cell capable of expressing the FAP nanobody, and discloses the FAP nanobody Uses of Nanobodies. Through the FAP nanobody, gene sequence and host cell disclosed in the present invention, the FAP nanobody can be efficiently expressed in Escherichia coli, and its immune reaction with FAP has good specificity and high affinity, and can be applied to the preparation of FAP detection reagents or anti-tumor drugs etc.

Description

A FAP Nanobody Nb67

technical field

The invention belongs to the technical field of biomedicine or biopharmaceuticals, and more specifically relates to a nanobody (Nb67) directed at the epitope molecule of the FAP polypeptide molecule, its coding sequence and application.

Background technique

Fibroblast activation protein (fibroblast activation protein, FAP) is a surface antigen specifically expressed by tumor-associated fibroblasts. It has dipeptidyl peptidase and collagenase activities. Immunosuppressive effect. FAP is composed of 761 amino acids. The glycosylation levels in different mammalian cells are different. The relative molecular weight of the monomer is 8.8×10 ⁴ , 9.5×10 ⁴ , 9.7×10 ⁴ and other reports. The relative molecular weight of the dimer 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 segment, however, most of the amino acid sequence 20-761 in the FAP molecule is exposed to the extracellular microenvironment, and this part is the extracellular region, which is the key enzyme catalytic region. FAP has both dipeptidyl peptidase activity and collagenase activity, which can degrade dipeptide and type I collagen in the matrix. FAP has 50% homology with DPPIV (dipeptidyl peptidase IV/CD26), which belongs to the dipeptidyl peptidase family, both have exopeptidase 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 therapy. 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. The existence of these factors provides ideas for exploring the mechanism of tumorigenesis and opening up new tumor immunotherapy programs.

At present, several FAP-specific monoclonal antibodies have been developed and started to be applied in clinical diagnosis and treatment, and their affinity to tumor tissues has been improved. However, this traditional method has disadvantages such as poor antibody stability, low sensitivity, and high production cost.

Nanobody technology is the latest and smallest antibody molecule developed by biomedical scientists on the basis of traditional antibodies by using molecular biology technology combined with the concept of nanoparticle science. It was originally developed by Belgian scientist Lehmann . Hamas found in camel blood. Ordinary antibody proteins are composed 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 bind to targets such as antigens like normal antibodies Binds tightly, but does not stick to each other and aggregate into clumps like single-chain antibodies. Nanobodies based on this "heavy chain antibody" not only have a molecular weight of only 1/10 of ordinary antibodies, but also have more flexible chemical properties, good stability, high solubility, easy expression and acquisition, and easy coupling to other molecules. However, there is no applicable nanobody developed for FAP in the prior art.

Contents 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 use of the nanobody, etc. in view of the lack of nanobodies against the FAP epitope in the prior art.

The technical scheme adopted by the present invention to solve its technical problems is: the first aspect of the present invention provides a FAP nanobody, which is a nanobody directed at the FAP epitope, including two such as SEQ ID NO: 9 VHH chains of the amino acid sequences shown. In the present invention, the FAP Nanobody can be referred to as Nb67 for short.

In a second aspect, the present invention provides a VHH chain directed against a FAP Nanobody, including a framework region FR and a complementarity determining region CDR, and the framework region FR includes the amino acid sequence of FRs in 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 CDR of the complementarity determining region includes the amino acid sequence of the CDR in the following group: 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 a role in 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.

In the third aspect of the present invention, a DNA molecule is provided for encoding a protein selected from the group consisting of the FAP Nanobody Nb67 of the present invention, or the VHH chain of the FAP Nanobody of the present invention.

Preferably, said 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 through phage amplification or genetic engineering recombinant expression. Phage amplification means that the phage displaying the FAP nanobody Nb67 is mass-produced to produce phage particles displaying the FAP nanobody Nb67 through biological amplification. The method of genetic engineering recombinant expression refers to the large-scale preparation of the nanobody in the form of protein expression by cloning the gene encoding the FAP nanobody Nb67 into an expression vector.

The fourth aspect of the present invention provides an expression vector containing 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 the preparation of FAP detection reagents. The FAP nanobody Nb67 of the present invention can replace traditional FAP antibodies to prepare FAP detection reagents for detecting FAP polypeptide molecules. The 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 anti-tumor drug, including the aforementioned FAP nanobody Nb67.

The seventh aspect of the present invention provides the use of the FAP Nanobody Nb67 of the present invention in non-therapeutic immunological detection. The type of immunological detection includes enzyme-linked immunosorbent assay, colloidal gold immunochromatography, immunospot hybridization and other immunological analysis detection types 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 the form of protein by prokaryotes or eukaryotes. Perform immunological assays.

The eighth aspect of the present invention provides the use of the FAP Nanobody Nb67 of the present invention in the preparation of reagents for binding and adsorbing FAP. For example, the FAP nanobody of the present invention can be made into an immunoaffinity column to adsorb and capture FAP, which can be used for further research after enrichment.

Implementing the present invention has the following beneficial effects: the present invention first synthesizes the FAP polypeptide and makes it immunogenic, then couples the FAP molecule to a microtiter plate to display the correct spatial structure of the protein, and utilizes the antigen in this form The 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. The nanobody strain of the nanobody; the FAP nanobody screened by the method of the present invention has the characteristics of immunoreactivity with FAP, and has good specificity and high affinity, and can be applied to the preparation of FAP detection reagents or anti-tumor drugs.

Description of drawings

The present invention will be further described below in conjunction with accompanying drawing and embodiment, in the accompanying drawing:

Fig. 1 is the amino acid numbering and structural representation of FAP nanobody;

Fig. 2 is the DNA electrophoresis figure of FAP nanobody;

Fig. 3 is the electrophoresis figure of SDS-PAGE after FAP nanobody is purified by nickel column resin gel affinity chromatography.

detailed description

In order to make the object, technical solution and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with 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 obtain a phage clone that can be efficiently expressed in Escherichia coli Expressed Nanobody strain, namely FAP Nanobody Nb67.

Below in conjunction with specific embodiment, further illustrate the present invention.

Example 1: Construction of a Nanobody library for FAP:

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

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

(3) Synthesize cDNA and amplify VHH by nested PCR;

(4) Use restriction enzymes 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 connect the two fragments; (5) Transform the ligated product into electroporation In state cell TG1, the FAP nanobody library was constructed and the library capacity was determined, and the library size was 1.85×10 ⁸ .

Embodiment 2: Nanobody screening process against FAP:

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

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

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

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

(5) Use 100mM TEA (triethylamine) to dissociate the phage that specifically binds to FAP, and infect Escherichia coli TG1 in logarithmic phase growth, culture at 37°C for 1h, produce and purify the phage for the next round For screening, the same screening process was repeated for 3-5 rounds to gradually obtain enrichment.

Embodiment 3: use the enzyme-linked immunosorbent method (ELISA) of phage to screen specificity single positive clone:

(1) From the cell culture dish containing phage after the above 3-5 rounds of selection, pick 96 single colonies and inoculate them in TB medium containing 100 micrograms per milliliter of ampicillin (1 liter of TB medium contains 2.3 grams of phosphoric acid Potassium dihydrogen, 12.52 grams of dipotassium hydrogen phosphate, 12 grams of peptone, 24 grams of yeast extract, 4 milliliters of glycerol), after growing to the logarithmic phase, add isopropylthiogalactoside ( IPTG), cultivate overnight at 30-35°C.

(2) Use the infiltration method to obtain the crudely extracted antibody, and transfer the antibody to an antigen-coated ELISA plate, and place it at room temperature for 1-1.5 hours.

(3) Unbound antibodies were washed away with PBST, and a primary mouse anti-HAtag antibody (mouse anti-HAtag antibody, purchased from Beijing Kangwei Century Biotechnology Co., Ltd.) was added, and left 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 Amicate 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 at a wavelength of 405 nm on the ELISA instrument.

(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) Shake the bacteria of the positive clone wells in LB liquid containing 100 micrograms per milliliter so as to extract the plasmid and perform sequencing.

Through the above experiments, the present invention has obtained 6 positive clone wells showing more than 3 times the binding force to the antigen, which are regarded as positive clones in the primary screening. According to the sequence comparison software Vector NTI, the gene sequence of each primary screened positive clone is analyzed, and the strains with the same CDR1, CDR2, and CDR3 sequences are regarded as the same clone, while the strains with different sequences are regarded as different clones, and a group can 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 structural schematic diagram are shown in FIG. 1 .

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

Example 4: Expression and purification of nanobodies in host bacteria Escherichia coli:

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

(2) Select a single bacterial colony and inoculate it in 5 mL of LB culture solution containing ampicillin, and culture it on a shaking table at 37°C overnight;

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

(4) centrifuge, collect bacteria;

(5) Obtain antibody crude extract by osmosis method;

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

Please refer to Figure 3, which is the electrophoresis diagram of SDS-PAGE after the FAP nanobody was purified 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 showed that the size of the FAP nanobody Nb67 was about 15KDa, and the purity of the FAP nanobody Nb67 could reach more than 95% after the above purification process.

The present invention also performed a kinetic affinity analysis on the purified FAP Nanobody Nb67 in Example 4 above. 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 it is difficult to dissociate between antibody antigens, and its affinity is stronger.

The present invention has been described based on specific embodiments, but those skilled in the art will understand that various changes and equivalent substitutions can be made without departing from the scope of the present invention. In addition, many modifications may be made to adapt the technique to a particular situation or material without departing from its scope. Therefore, it is intended that the invention not be limited to the particular embodiments disclosed herein, but include all embodiments falling within the scope of the appended claims.

Claims (10)

1. A FAP Nanobody, characterized in that, the FAP Nanobody is a Nanobody directed at the FAP epitope, comprising two VHH chains of the amino acid sequence shown in SEQ ID NO:9. 2. A VHH chain directed against a FAP nanobody, comprising a framework region FR and a 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 CDR of the complementarity determining region includes the amino acid sequence of the CDR in the following group: SEQ ID NO: 5 CDR1 shown in SEQ ID NO:6, CDR2 shown in SEQ ID NO:7, CDR3 shown in SEQ ID NO:7. 3. The VHH chain against FAP Nanobody according to claim 2, characterized in that it has the amino acid sequence shown in SEQ ID NO:9. 4. A DNA molecule characterized in that it is used to encode a protein selected from the group consisting of the FAP Nanobody according to claim 1, or the VHH against the FAP Nanobody according to claim 2 or 3 chain. 5. The DNA molecule according to claim 4, characterized in that it has the nucleotide sequence shown in SEQ ID NO:8. 6. An expression vector, characterized in that it contains the nucleotide sequence shown in SEQ ID NO:8. 7. A host cell, characterized in that it can express the FAP nanobody of claim 1. 8. The FAP nanobody of claim 1 is used in the preparation of FAP detection reagents or antitumor drugs. 9. A FAP detection reagent or anti-tumor drug, characterized in that it comprises the FAP nanobody according to claim 1. 10. The application of the FAP Nanobody according to claim 1 in the preparation of FAP reagents bound and adsorbed.