A single-domain antibody binding to pcsk9 antigen and a preparation method thereof

Abstract

The present application relates to the antibody technology field of biological medicine, and particularly relates to a single-domain antibody combined with a PCSK9 antigen and a preparation method thereof. The present application obtains a high-quality PCSK9 immune single-domain antibody library by separating peripheral blood mononuclear cells, extracting total RNA, and building a library through reverse transcription and nested PCR. The PCSK9 antigen is coated on an enzyme-labeled plate, and the phage display technology is used to screen the PCSK9 immune single-domain antibody library. The screened single-domain antibody is converted into an Escherichia coli expression system for mass expression, so that a single-domain antibody strain with high affinity to PCSK9 can be obtained in a relatively short time.

Description

Technical Field

[0001] This invention relates to the field of antibody technology in biomedicine, specifically to a single-domain antibody that binds to the PCSK9 antigen and its preparation method. Background Technology

[0002] Currently, the main drugs used to lower cholesterol on the market include statins, cholesterol absorption inhibitors, and probucol. Although statins have shown excellent performance in the treatment of cardiovascular diseases, their widespread use has gradually revealed drawbacks. First, patients treated with statins still have a relatively high residual risk of cardiovascular events, with a 22.4% risk of occurrence within 2 years. Second, a large number of patients cannot tolerate statins, especially those with familial hypercholesterolemia; even with the maximum dose of the most effective statins, they still cannot achieve the goal of lowering low-density lipoprotein cholesterol (LDL-c) levels. Most importantly, statins have a variety of side effects, such as causing abnormal blood sugar, muscle toxicity, and memory and cognitive impairment. The incidence of side effects is as high as 20%, and severe side effects can lead to rhabdomyolysis and acute renal failure. A significant number of patients discontinue treatment because they cannot tolerate the muscle pain caused by the side effects.

[0003] Proprotein convertase subtilisin / kexin type 9 (PCSK9), a novel proprotein convertase belonging to the subtilisin subfamily, is a significant contributing factor to autosomal dominant familial hypercholesterolemia. Studies have found that PCSK9, in addition to affecting plasma cholesterol levels and regulating neuronal apoptosis, is also correlated with inflammatory responses. Current research on PCSK9 primarily focuses on its regulatory function in hepatic lipid metabolism. Previous studies have shown that PCSK9 can regulate hepatic lipid metabolism by promoting the degradation of low-density lipoprotein receptor (LDL-R) in hepatocytes, thereby affecting plasma low-density lipoprotein cholesterol (LDL-c) levels. However, PCSK9 exhibits two mutation types: gain-of-function mutations and loss-of-function mutations. Population studies have shown that several PCSK9 "gain-of-function" mutations commonly occur in individuals with autosomal dominant hypercholesterolemia, while PCSK9 "loss-of-function" mutations are associated with decreased plasma cholesterol. Individuals with PCSK9 loss-of-function mutations have a significantly reduced risk of coronary heart disease. In 2005, Hobbs et al. reported in the Dallas Heart Study that individuals carrying PCSK9 nonsense mutations had LDL-c levels 28% lower than the general population. The Copenhagen Heart Study found that functional loss of the PCSK9 gene reduced LDL-c levels by 11-15% and the incidence of coronary heart disease by 6-46%. Zimbabwe et al. reported that PCSK9 deletion mutations could reduce LDL-c levels by 27% in African women. PCSK9 inhibitors offer a completely new treatment modality to combat LDL-c and are considered the biggest advance in lipid-lowering therapy since statins. The advent of PCSK9 inhibitors has brought good news to patients who experience severe side effects when taking statins, and to patients whose LDL-c target levels cannot be achieved with statin therapy, such as patients with hereditary hypercholesterolemia.

[0004] PCSK9 inhibitors not only prevent LDL-R reuptake but also inhibit NF-κB channels, thereby reducing the risk of acute coronary syndromes such as thrombosis, inflammation, and endothelial cell activation. Current potential research projects in the field of PCSK9 inhibitors include inhibitory protein antibodies, siRNA, antisense oligonucleotides, and small molecule inhibitors. Monoclonal antibody drugs, due to their strong targeting, high specificity, and low toxicity, are currently the main area of ​​research for PCSK9 inhibitors. Animal studies have shown that the addition of neutralizing anti-PCSK9 antibodies significantly increased LDL-R expression levels in mouse livers and decreased LDL-c concentration in the blood by 30%. In primates, PCSK9 monoclonal antibodies also showed significant effects, with the reduction in LDL-c levels lasting for several weeks. To date, no significant toxic side effects have been found in anti-PCSK9 protein monoclonal antibodies; only mild side effects such as local injection reactions, diarrhea, and headaches have been reported. Sanofi's Praluent (Alirocumab), Amgen's Repatha (evolocumab), and Innovent Biologics' IBI-306 (tafolecimab) are currently the only three approved humanized PCSK9 antibodies on the global market.

[0005] Antibody drugs are currently one of the main directions of new drug development, and have been widely used in the diagnosis, prevention and treatment of infectious diseases and in bioscience research. To date, more than 100 antibody drugs have been successfully launched, and four of the top ten best-selling drugs globally in 2021 were antibody drugs. Since Hamers et al. discovered naturally occurring heavy chain antibodies lacking the light chain and constant region I (CH1) in alpaca blood in 1993, the variable region of heavy chain antibodies, also known as nanobody (Nb), has gradually replaced other small antibodies and become a hot topic in the development of novel antibody drugs. Nitrosamine leukocytes (Nb) are typically only about 15 kDa, approximately one-tenth the size of traditional antibodies. They contain internal disulfide bonds and have numerous hydrophilic residues on their surface, exhibiting strong resistance to heat and pH. The absence of an Fc fragment and light chain in Nb allows them to recognize cryptic or small epitopes that traditional antibodies cannot, while avoiding complement reactions. Furthermore, single-domain antibodies offer numerous advantages, including high stability, low toxicity, high solubility, ease of target screening, and direct expression in prokaryotic microorganisms, making them cost-effective. Sequence homology analysis shows that the VHH germline gene sequence of alpaca Nb is highly homologous to human VH3, but CDR1 and CDR3 are slightly longer than in humans, with CDR3 protruding outwards in the tertiary structure, suggesting higher antigen-binding specificity and affinity. Given these advantages, Nb is being gradually developed as a monoclonal antibody drug for disease diagnosis and treatment, with widespread applications in the development of enzyme inhibitors and biological inhibitors for tumors, infections, and inflammation. However, while the small size of single-domain antibodies offers many advantages for their therapeutic function, these small proteins are easily eliminated in vivo. Genetic engineering to modify Nb into target enzymes, transmembrane proteins, or to divalent Nb can effectively improve antibody activity and stability for research purposes. Studies on inhibiting viral replication have found that divalent single-domain antibodies are at least 60 times more effective than monovalent single-domain antibodies, and their duration of action in animals is longer, effectively delaying mortality. Antibody drugs hold great promise, but the domestic antibody drug development is still in its early stages. Therefore, developing domestically produced, low-cost PCSK9 antibody inhibitors to meet the urgent needs of the Chinese population for antibody drugs is of profound and positive significance.

[0006] Current technologies for the development of PCSK9 single-domain antibodies focus on traditional murine antibodies. Traditional antibodies are difficult to express in large quantities or to humanize, which is time-consuming, costly, and has a low effective antibody yield. This severely limits the development of PCSK9 antibody inhibitors. In particular, domestic antibody drugs are still in their infancy and cannot meet the needs of CVD patients. Summary of the Invention

[0007] The purpose of this invention is to provide a single-domain antibody that binds to the PCSK9 antigen and a method for preparing the same, so as to solve the problems mentioned in the background art.

[0008] To achieve the above objectives, the present invention provides the following technical solution:

[0009] A single-domain antibody that binds to the PCSK9 antigen, the single-domain antibody comprising a heavy chain antibody variable region (VHH), the heavy chain antibody variable region (VHH) comprising a frame region (FR) and a complementarity-determining region (CDR), wherein the complementarity-determining region (CDR) comprises complementarity-determining region 1 (CDR1) with the sequence GRTFSDYA (SEQ ID NO: 1); complementarity-determining region 2 (CDR2) with the sequence IGWSGGQT (SEQ ID NO: 2); and complementarity-determining region 3 (CDR3) with the sequence AASFLVIPGTVKTRYDS (SEQ ID NO: 3).

[0010] The amino acid sequence of the variable region (VHH) of the heavy chain antibody is shown in SEQ ID NO: 4.

[0011] The polynucleotide sequence includes nucleotide sequences encoding the complementarity-determining regions shown in SEQ ID NO: 1 to SEQ ID NO: 3.

[0012] The polynucleotide sequence includes a nucleotide sequence encoding the variable region (VHH) of the heavy chain antibody shown in SEQ ID NO: 4.

[0013] The polynucleotide sequence is shown in SEQ ID NO: 5.

[0014] An expression vector containing a polynucleotide sequence.

[0015] A host cell containing an expression vector capable of expressing a single-domain antibody that specifically binds to the PCSK9 antigen.

[0016] The pharmaceutical composition comprises a single-domain antibody, and a pharmaceutically acceptable carrier, diluent, or excipient.

[0017] The method includes: transforming an expression vector containing a multinucleotide sequence into an expression host cell, culturing the cell, and performing large-scale expression and purification of the single-domain antibody;

[0018] Preferably, the expression vector is the pMECS plasmid, and the host cell is Escherichia coli strain HB2151;

[0019] Preferably, the expression vector is the pPICZα plasmid, and the host cell is yeast strain X33;

[0020] Preferably, the expression vector is pCDNA3.4 plasmid, and the host cell is HEK293F cell line.

[0021] The single-domain antibody is used in the preparation of anti-PCSK9 protein monoclonal antibody drugs, or in the immunological detection of PCSK9 for non-disease diagnostic and therapeutic purposes.

[0022] Compared with the prior art, the beneficial effects of the present invention are:

[0023] This invention involves isolating peripheral blood mononuclear cells, extracting total RNA, and constructing a library using reverse transcription and nested PCR to obtain a high-quality PCSK9 immune single-domain antibody library. PCSK9 antigen is coated onto an ELISA plate, and phage display technology is used to screen the PCSK9 immune single-domain antibody library. The selected single-domain antibodies are then transformed into an E. coli expression system for mass expression, thereby enabling the acquisition of high-affinity PCSK9 monoclonal single-domain antibody strains in a relatively short time. Attached Figure Description

[0024] Appendix Figure 1 This is a schematic diagram illustrating the principle of the binding assay between PCSK9 and its single-domain antibody VHHH12 in Example 4 of the present invention.

[0025] Appendix Figure 2 This is a schematic diagram of the detection results of the binding assay between PCSK9 and its single-domain antibody VHHH12 in Example 4 of the present invention;

[0026] Appendix Figure 3 This is a schematic diagram showing the results of the affinity assay between the single-domain antibody VHHH12 and PCSK9 using Biacore T200 in Example 4 of this invention. Detailed Implementation

[0027] The present invention will now be described in further detail with reference to specific embodiments and accompanying drawings. In the following embodiments, many details are described to enable a better understanding of the invention. However, those skilled in the art will readily recognize that some features may be omitted in different circumstances, or may be replaced by other elements, materials, or methods.

[0028] Furthermore, the features, operations, or characteristics described in the specification can be combined in any suitable manner to form various embodiments. At the same time, the steps or actions in the method description can be rearranged or adjusted in a manner obvious to those skilled in the art. Therefore, the various orders in the specification and drawings are only for the clear description of a particular embodiment and do not imply a necessary order, unless otherwise stated that a particular order must be followed.

[0029] This invention first immunizes alpacas with PCSK9 antigen expressed in CHO cells (Chinese hamster ovary cells), then isolates peripheral blood cells (PBMCs) from the immunized alpacas, and a heavy chain variable region (VHH) library targeting the PCSK9 antigen is amplified from these cells. Redundant background interference is removed, significantly improving the efficiency of obtaining effective antibodies. Secondly, this invention combines phage display technology, enabling more intuitive acquisition of antibody affinity information and obtaining high-affinity PCSK9 single-domain antibody genes in a shorter time. Furthermore, this invention provides a preparation method for the aforementioned PCSK9 single-domain antibody. Because the pMECS (phage display vector) contains an amber terminator (TAG) between the HA tag and the M13 GIII gene, ordinary expression systems cannot effectively recognize this terminator, thus failing to effectively express the single-domain antibody protein. This invention optimizes the prokaryotic expression system, enabling large-scale expression and purification of the PCSK9 single-domain antibody. The single-domain antibody, verified by ELISA and the Biacore T200 system, exhibits high specificity and high affinity for PCSK9, indicating that the PCSK9 single-domain antibody obtained by this invention has further development value.

[0030] Alpacas were immunized with PCSK9 antigen expressed in CHO cells. Peripheral blood cells (PBMCs) were collected from immunized alpacas, and PCSK9-affinity lymphocytes were isolated from them. Total RNA was extracted, and the variable region (V region) of the alpaca heavy chain antibody was cloned using Nest-PCR technology. This region was then inserted into a phage plasmid to construct a phage expression library. Subsequently, the PCSK9 antigen was screened multiple times using phage display technology. Finally, the high-affinity antibodies obtained from the screening were expressed and purified in large quantities in prokaryotic cells, and the affinity and binding constant of the obtained single-domain antibodies were verified by ELISA and Biacore T200.

[0031] In one embodiment of the present invention, the amino acid sequence of the single-domain antibody VHHH12, which specifically binds to the PCSK9 antigen, is as follows:

[0032] QVQLQESGGGLVQAGGSLRLSCAASGRTFSDYAVGWFRQAPGKEREFVAGIGWSGGQTTYADSVKGRFTISRDNAKDTVYLQMNSLKPEDTAVYVCAASFLVIPGTVKTRYDSWGQGTQVTVPA (SEQ ID NO: 4).

[0033] The sequence of frame region 1 is QVQLQESGGGLVQAGGSLRLSCAAS (SEQ ID NO: 6), the sequence of frame region 2 is VGWFRQAPGKEREFVAG (SEQ ID NO: 7), the sequence of frame region 3 is TYADSVKGRFTISRDNAKDTVYLQMNSLKPEDTAVYVC (SEQ ID NO: 8), the sequence of frame region 4 is WGQGTQVTVPA (SEQ ID NO: 9), the sequence of complementarity-determining region 1 is GRTFSDYA (SEQ ID NO: 1), the sequence of complementarity-determining region 2 is IGWSGGQT (SEQ ID NO: 2), and the sequence of complementarity-determining region 3 is AASFLVIPGTVKTRYDS (SEQ ID NO: 3).

[0034] As is well known, the specific binding properties of an antibody are determined by its complementarity-determining region (CDR). Therefore, one aspect of this invention claims protection for a single-domain antibody that specifically binds to the PCSK9 antigen, comprising a heavy chain variable region (VHH), which is composed of a frame region (FR) and a complementarity-determining region (CDR). The CDR includes complementarity-determining region 1 (CDR1) with the sequence GRTFSDYA (SEQ ID NO: 1); complementarity-determining region 2 (CDR2) with the sequence IGWSGGQT (SEQ ID NO: 2); and complementarity-determining region 3 (CDR3) with the sequence AASFLVIPGTVKTRYDS (SEQ ID NO: 3). In a preferred embodiment, a single-domain antibody that specifically binds to the PCSK9 antigen has a heavy chain variable region (VHH) sequence as shown in SEQ ID NO: 4.

[0035] In one embodiment of the present invention, the nucleotide sequence encoding the PCSK9 single-domain antibody VHHH12 is as follows:

[0036] 5'-caggtgcagctgcaggagtctggaggaggattggtgcaggctgggggctctctgagactctcctgtgcagcctctggacgcacgttcagcga ctatgccgtgggctggttccgccaggctcccgggaaggagcgtgagtttgtagctggtataggctggagtggcggtcaaacaacctatgcagactc cgtgaagggccgattcaccatctccagagacaacgccaaagacacggtgtatctgcaaatgaacagcctgaaacctgaggacacggccgtttatg tctgtgcagcttcatttttggttatccccggcaccgtgaaaactcggtatgacagctggggccagggaacccaggtcaccgttccagcg-3'(SEQ ID NO: 5).

[0037] However, considering the degeneracy of the coding gene and the fact that the specific binding characteristics of the antibody are determined by the complementarity-determining region (CDR), one aspect of this invention claims a polynucleotide sequence encoding a PCSK9 single-domain antibody VHHH12, comprising a nucleotide sequence encoding the CDR shown in SEQ ID NO: 1 to SEQ ID NO: 3. Due to the degeneracy of the coding gene, the base sequence of such a polynucleotide sequence can vary, as long as it encodes the CDR shown in SEQ ID NO: 1 to SEQ ID NO: 3. In a preferred embodiment, the polynucleotide sequence is as shown in SEQ ID NO: 5.

[0038] In one embodiment of the present invention, an expression vector containing the polynucleotide sequence of the present invention is provided. Those skilled in the art will understand that, within the spirit of the present invention, pET series vectors, pCYT, pMECS, pMG36e, pPICZα, pFUSE series, or pCDNA series vectors, etc., can all be used as expression vectors for the polynucleotide sequence of the present invention. In a preferred embodiment, the expression vector is the phage display vector pMECS (preserved in this laboratory).

[0039] In one embodiment of the present invention, a host cell is provided containing the expression vector of the present invention, capable of expressing a single-domain antibody that specifically binds to the PCSK9 antigen. Those skilled in the art will understand that, within the spirit of the present invention, numerous cell expression systems such as *Escherichia coli* HB2151, *Lactobacillus* NZ9000, yeast X33, plant cells, insect cells, or mammalian cells HEK293F can all serve as host cells for the expression vector of the present invention. In a preferred embodiment, the host cell is *Escherichia coli* HB2151 strain (preserved in our laboratory).

[0040] In one embodiment of the present invention, a pharmaceutical composition is provided comprising the single-domain antibody of the present invention, and a pharmaceutically acceptable carrier, diluent, or excipient.

[0041] The pharmaceutical compositions of the present invention can be prepared by methods well known in the art (e.g., Remington: The Science and Practice of Pharmacy, 19th ed. (1995), A. Gennaro et al., Mack Publishing Co.), and comprise a single-domain antibody as disclosed in the present invention and one or more pharmaceutically acceptable carriers, diluents or excipients.

[0042] In one embodiment of the present invention, a method for preparing the single-domain antibody of the present invention is provided, comprising: transforming an expression vector containing the polynucleotide sequence of the present invention into an expression host cell, culturing, and performing large-scale expression and purification of the single-domain antibody. In a preferred embodiment, the expression vector is a phage display vector pMECS plasmid, and the host cell is Escherichia coli HB2151 strain.

[0043] The single-domain antibody of the present invention can be used to prepare anti-PCSK9 protein monoclonal antibody drugs and can also be used for immunological detection of PCSK9. Therefore, in one embodiment of the present invention, the use of the single-domain antibody of the present invention in the preparation of anti-PCSK9 protein monoclonal antibody drugs, or in the immunological detection of PCSK9 for non-disease diagnostic and therapeutic purposes, is provided.

[0044] This invention utilizes eukaryotically expressed PCSK9 antigen to immunize alpacas. Peripheral blood mononuclear cells are isolated, total RNA is extracted, and a high-quality PCSK9 immune single-domain antibody library is obtained through reverse transcription and nested PCR library construction. The PCSK9 antigen is coated onto an ELISA plate, and the PCSK9 immune single-domain antibody library is screened using phage display technology. The selected single-domain antibodies are then transformed into an E. coli expression system for mass expression, thereby enabling the acquisition of high-affinity PCSK9 monoclonal single-domain antibody strains in a relatively short time.

[0045] The technical solutions of the present invention are described in detail below through embodiments. It should be understood that the embodiments are merely exemplary and should not be construed as limiting the scope of protection of the present invention.

[0046] Example 1: Construction of PCSK9 single-domain antibody phage display library

[0047] (1) PCSK9 immunized alpaca

[0048] Mix 500 μL of PCSK9 (50 μg) with an equal volume of Freund's adjuvant to make 1 mL, and inject subcutaneously into the alpaca's neck at 3-5 points. Collect blood from the marginal ear vein before immunization. Immunize monthly for a total of 4 injections; collect 10 mL of peripheral blood from the alpaca at each immunization. During blood collection, fix the alpaca's head to one side, shave the skin at the blood collection site, disinfect with 75% alcohol, and allow it to dry before collecting blood. Apply pressure to the jugular vein groove with your finger until the vein bulges, then disinfect the collection site and insert the needle to collect 10 mL of blood into an EDTA anticoagulant tube. Immediately and continuously shake slowly to mix thoroughly, place on ice, and transport back to the laboratory.

[0049] (2) Blood lymphocyte sample separation

[0050] Lymphocytes were isolated from blood samples collected before and after each immunization, using the following methods:

[0051] i. Add 7 mL of Ficoll lymphocyte separation medium to a 15 mL centrifuge tube;

[0052] ii. Add an equal volume of PBS (1×) or physiological saline to fresh whole blood that has been treated with an anticoagulant (EDTA) to dilute the blood and mix thoroughly.

[0053] iii. In the centrifuge tube containing the lymphocyte separation medium, carefully and slowly add an equal volume (7 mL) of diluted blood using a 1 mL pipette, ensuring that the mixture is above the surface of the lymphocyte separation medium (i.e., do not mix the two liquids and maintain a clear interface), and centrifuge at 3000g for 20 min.

[0054] iv. Carefully transfer the supernatant (plasma sample) to a 1.5 mL cell cryopreservation tube using a 1 mL pipette, write the animal number and the word "plasma" on it, place it in a small cloth bag with a drawstring, and store it in a liquid nitrogen container.

[0055] v. Carefully separate the white blood cell layer into a 15 mL centrifuge tube using a 1 mL pipette; fill the tube with PBS (1×) to 15 mL; wash the white blood cells with PBS (1×), centrifuge (3000 g for 20 min), carefully discard the supernatant, without disturbing the cell clumps at the bottom of the tube, and collect the white blood cells in the remaining 0.1-0.2 mL of PBS;

[0056] vi. Add 5 times the volume of RNA later, gently mix the cell clumps, divide into 2 portions and place into 1.5 mL cell cryopreservation tubes, then store in liquid nitrogen.

[0057] (3) Total RNA extraction and cDNA synthesis

[0058] Take a sample of frozen lymphocytes, add 1 mL of Trizol, let stand at room temperature for 10 min, then add 0.2 mL of chloroform, shake vigorously, let stand at room temperature until the solution separates into layers (about 10 min), centrifuge at 12,000 rpm, collect the upper aqueous phase, add an equal volume of isopropanol, mix well, let stand at room temperature for 15 min until nucleic acid precipitation, centrifuge at high speed to remove the supernatant, add 1 mL of 75% ethanol (prepared with DEPC water) to the RNA precipitate, centrifuge at high speed to remove the supernatant, drain the water, dissolve the RNA in nuclease-free water, and take 1 μL for concentration and purity determination;

[0059] Take an appropriate amount (7–20 μg) of RNA and use SuperScript. TM III. First-Strand Synthesis SuperMix (Invitrogen) kit was used for cDNA synthesis. Oligo dT primers were used for reverse transcription. The synthesized cDNA was stored at -20°C.

[0060] (4) Construction of phage display library

[0061] PCR amplification: Using the synthesized cDNA as a template, Nest-PCR was used to amplify the V region (VHH) of the alpaca heavy chain antibody. Table 1 shows the names and sequences of the Nest-PCR primers.

[0062] Table 1. Primer information used for alpaca VHH fragment amplification.

[0063]

[0064] The PCR reaction system is as follows:

[0065] Round 1: cDNA 2 μL; 2×Master Mix 12.5 μL; CALL001 0.5 μL; CALL002 0.5 μL; CALL005 0.5 μL; CALL006 0.5 μL; water to a final volume of 25 μL. 2×Master Mix (purchased from KAPABiosystems).

[0066] Reaction conditions: 95℃ for 5 min; 94℃ for 1 min; 57℃ for 1 min; 72℃ for 1 min per cycle; 72℃ for 7 min; amplification for 35 cycles.

[0067] Second round: Template (first round product) 40ng; 2*Master Mix 25μL; VHH-For (10μM) 1μL; VHH-Back (10μM) 1μL; Water to make up to 50μL.

[0068] Reaction conditions: 95℃ for 5 min; 94℃ for 45 s; 60℃ for 45 s; 72℃ for 45 s per cycle; 72℃ for 7 min for 25 cycles of amplification.

[0069] After the PCR reaction was completed, the PCR products were detected by 1.5% agarose gel electrophoresis. The target gene fragment in the first round of PCR was located at 700 bp. The target band was cut and recovered. In the second round of PCR, the target gene fragment was located at 500 bp. The target band, i.e., the VHH fragment, was cut and recovered.

[0070] The VHH fragment and the vector (pMECS plasmid, stored in our laboratory) were double-digested using NEB restriction endonucleases Not I and Pst I, respectively. The reaction system is as follows:

[0071] Vector digestion system: 20 μg vector; 10 μL Pst I; 20 μL Not I; 50 μL Cutsmart (10× buffer, purchased from NEB); add H2O to 500 μL.

[0072] Fragment digestion system: VHH fragment 5 μg; PstI 7 μL; NotI 14 μL; Cutsmart (10× buffer) 50 μL; add H2O to 500 μL.

[0073] Digestion was performed overnight at 37°C. After agarose gel electrophoresis, the digested products of the vector and VHH fragment were removed from the gel. The digested products were mixed and ligated overnight at 16°C using NEB ligase.

[0074] (5) Construction of phage display library

[0075] After purification of the ligation product using a PCR Purification Kit (purchased from Beijing Tiangen Biotech), 1 μL was used to transform TG competent cells. The cells were then incubated at 37°C for 2 hours and serially diluted to 10¹, 10², and 10³. 300 μL of each product was plated and incubated overnight at 37°C. The clone count was calculated to be approximately 10⁵ clones per plate.

[0076] Using the same transformation method described above, perform large-scale transformations until the number of clones in the library reaches 10⁷ or more. Elute all clones with sterile LB liquid medium, centrifuge at 5,000g for 5 min, resuspend the precipitate in 2 mL of sterile LB liquid medium, add an equal volume of 30% glycerol, and freeze at -80°C.

[0077] (6) Library diversity detection

[0078] Thirty clones from step (5) were randomly selected as templates for clonal PCR. The PCR products were detected by 1.5% agarose gel electrophoresis to verify the recombination rate of the constructed PCSK9 single-domain antibody library. Then, the libraries were sequenced to analyze the diversity of the PCSK9 single-domain antibody library. The sequencing results showed that 15 single clones had 13 different amino acid sequences, indicating that the constructed library had good diversity.

[0079] (7) Phage amplification and rescue

[0080] The PCSK9 single-domain antibody phage library was amplified and rescued using helper phages. The monoclonal library saved in step (5) was inoculated into 100 mL of culture medium and cultured to the logarithmic growth phase. Helper phage M13 (preserved in our laboratory) with an MOI (multiplicity of infection) of 20 was added. The culture was incubated at room temperature for 30 min, centrifuged at low speed, and the precipitate was resuspended in culture medium and inoculated into 300 mL of culture medium. The culture was incubated overnight. The next day, the phage was centrifuged at 3,000 g for 30 min, the supernatant was collected, PEG was added to precipitate the phage, the culture was incubated on ice for 30 min, centrifuged at 3,000 g for 30 min, and the precipitate was the PCSK9 single-domain antibody phage library. After resuspending the precipitate with PBS, its titer was measured to be 2.9 × 10¹² pfu / mL.

[0081] Example 2: Panning PCSK9 single-domain antibodies using phage display technology

[0082] (1) Washing of affinity PCSK9 single-domain antibody phage library

[0083] 100 ng of PCSK9 antigen was used to coat an ELISA plate and incubated overnight at 4°C. The next day, the rescued PCSK9 single-domain antibody phage was added and incubated at room temperature for 2 hours. The wells were washed 10 times with PBST, and 100 μL of triethylamine was added and incubated at room temperature for 30 minutes. The collected phages constituted the PCSK9 single-domain antibody phage library obtained through affinity washing. 10 μL of TG1 E. coli cells were infected and spread onto a plate for determining the number of clones after screening. The remaining selected phages were used for amplification.

[0084] (2) Amplification and rescue of phages after screening

[0085] The amplification and rescue method is the same as step (7) in Example 1. The obtained PBS suspension is the phage after the first round of screening. It is stored at 4°C and used for the next round of screening. The antigen amount is gradually reduced according to the same screening steps as above, and 3-4 rounds of screening are performed.

[0086] (3) ELISA to evaluate the enrichment of specific antibodies

[0087] ELISA plates were coated with 100 ng of PCSK9 antigen and incubated overnight at 4°C. The next day, 2% BSA was added for blocking at room temperature for 1 hour. The experimental group was added with phage amplified after each round of washing, while the control group was added with an equal amount of wild-type phage and incubated at room temperature for 2 hours. The plates were washed 10 times with PBST to remove unbound phage. HRP-labeled anti-M13 antibody was added and incubated at room temperature for 1 hour. The chromogenic solution was added and the plates were reacted in the dark for 10-30 minutes. The absorbance was measured, and the absorbance gradually increased with the number of washes, stabilizing during the third to fourth washes, indicating that the specific antibody was enriched.

[0088] (4) Identification of PCSK9-specific single-domain antibody-positive clones

[0089] ELISA plates were coated with 100 ng of PCSK9 antigen and incubated overnight at 4°C. Thirty-eight single clones were randomly selected from the phage-spread plates obtained in the last round of screening and placed in 1 mL of culture medium. The plates were incubated at 37°C until the logarithmic growth phase, and then induced overnight with 1 mM IPTG. The next day, the bacterial sediment was collected by centrifugation, lysed, and centrifuged at 5,000 g for 15 min. The supernatant was collected. Simultaneously, ELISA plates were blocked with 2% BSA at room temperature for 1 h. The experimental group received single clone lysate supernatant in each well, while the control group received blank TG1 lysate supernatant. The plates were incubated at room temperature for 2 h. After washing 10 times with PBST, mouse anti-HA tag antibody was added, and the plates were incubated at room temperature for 1 h. After washing 3-5 times with PBST, AP-labeled anti-mouse IgG antibody was added, and the plates were incubated at room temperature for 1 h. The substrate was added, and the reaction was allowed to proceed for 5-20 min depending on the actual situation. The absorbance was read on a microplate reader. A positive clone was considered to have an absorbance value greater than 2.1 (baseline) compared to the control well.

[0090] (5) Positive clone sequence analysis

[0091] The DNA from the 30 positive clones obtained in step (4) was extracted and the inserted fragments were verified by PCR. Clones that were positive by PCR were then sequenced. Sequencing results showed two nucleotide sequences. Analysis of their amino acid sequences revealed that one sequence exhibited a typical single-domain antibody structure, consisting of a framework region (FR1, FR2, FR3, and FR4) and a complementarity-determining region (CDR1, CDR2, and CDR3). The nucleotide and amino acid sequences of this single-domain antibody monoclonal strain are as follows:

[0092] PCSK9 single-domain antibody protein VHHH12 has the following amino acid sequence: QVQLQESGGGLVQAGGSLRLSCAASGRTFSDYAVGWFRQAPGKEREFVAGIGWSGGQTTYADSVKGRFTISRDNAKDTVYLQMNSLKPEDTAVYVCAASFLVIPGTVKTRYDSWGQGTQVTVPA (SEQ ID NO: 4). The sequence of frame region 1 is QVQLQESGGGLVQAGGSLRLSCAAS (SEQ ID NO: 6), the sequence of frame region 2 is VGWFRQAPGKEREFVAG (SEQ ID NO: 7), the sequence of frame region 3 is TYADSVKGRFTISRDNAKDTVYLQMNSLKPEDTAVYVC (SEQ ID NO: 8), the sequence of frame region 4 is WGQGTQVTVPA (SEQ ID NO: 9), the sequence of complementarity-determining region 1 is GRTFSDYA (SEQ ID NO: 1), the sequence of complementarity-determining region 2 is IGWSGGQT (SEQ ID NO: 2), and the sequence of complementarity-determining region 3 is AASFLVIPGTVKTRYDS (SEQ ID NO: 3).

[0093] The nucleotide sequence encoding the PCSK9 single-domain antibody protein VHHH12 is as follows:

[0094] 5'-caggtgcagctgcaggagtctggaggaggattggtgcaggctgggggctctctgagactctcctgtgcagcctctggac gcacgttcagcgactatgccgtgggctggttccgccaggctcccgggaaggagcgtgagtttgtagctggtataggctggagtggcggtcaaacaacctatgcagactccgtgaagggccgattcaccatctccagagacaacgccaaag acacggtgtatctgcaaatgaacagcctgaaacctgaggacacggccgtttatgtctgtgcagcttcatttttggttatccccggcaccgtgaaaactcggtatgacagctggggccagggaacccaggtcaccgttccagcg-3'(SEQ ID NO: 5).

[0095] Example 3: Induction and purification of anti-PCSK9 single-domain antibody VHHH12

[0096] (1) Construction of PCSK9 single-domain antibody expression bacteria

[0097] First, the PCSK9 single-domain antibody monoclonal transfer medium was incubated overnight at 37°C. The next day, the plasmid was extracted using the Plasmid mini-extraction kit (purchased from OMEGA). After agarose gel electrophoresis and concentration determination, the plasmid containing the PCSK9 single-domain antibody sequence was transformed into expression bacteria HB2151, plated, and incubated overnight at 37°C.

[0098] (2) Induction of expression of anti-PCSK9 single-domain antibody VHHH12

[0099] The following day, five clones were picked from the plate for clonal PCR to verify whether the plasmid had been transformed into the expression strain. Positive clones were selected and cultured at 37°C until the OD600 reached 0.6-0.8, and IPTG was added to induce expression. The bacterial culture was centrifuged, the bacterial pellet was collected, the pellet was resuspended with lysis buffer, the bacterial cells were sonicated to disrupt the structure, and the supernatant was collected by centrifugation.

[0100] (3) Purification of anti-PCSK9 single-domain antibody VHHH12

[0101] PCSK9 single-domain antibody was obtained by affinity purification using a Ni column. The Ni column was first washed with ultrapure water, then with lysis buffer. The supernatant of the PCSK9 single-domain antibody expressing bacteria was added to the Ni column at a flow rate of 1 mL / min. Impurities were washed away with 5 column volumes of affinity buffer A (20 mM imidazole), followed by an equal volume of affinity buffer B (250 mM imidazole), and the eluent was collected. Finally, the expression and purification of the PCSK9 single-domain antibody were detected by 15% SDS-PAGE gel electrophoresis.

[0102] Example 4: Affinity determination of PCSK9 with its single-domain antibody VHHH12

[0103] (1) ELISA method was used to analyze the binding of PCSK9 single-domain antibody VHHH12. Figure 1 )

[0104] The experimental group was coated with 100 ng of PCSK9 protein onto an ELISA plate (with a reserved PBS blank control group). The no-coating control group was the uncoated antigen control group. The plates were incubated overnight at 4°C. The next day, 2% BSA was added for blocking at room temperature for 1 hour. Purified PCSK9 single-domain antibody was added to the experimental group and PBS to the blank group, and incubated at room temperature for 2 hours. After washing 10 times with 1×PBST, mouse anti-HA tag antibody was added, and the plate was incubated at room temperature for 1 hour. After washing 3-5 times with 1×PBST, AP-labeled anti-mouse IgG antibody was added, and the plate was incubated at room temperature for 1 hour. The substrate was added, and the reaction was allowed to proceed for 10-20 minutes. The absorbance was then read on an ELISA reader. ELISA results ( Figure 2The results showed that the VHHH12 single-domain antibody had good binding to PCSK9, with a binding affinity much higher than that of the control group.

[0105] (2) Biacore T200 analysis of the affinity constant of anti-PCSK9 single-domain antibody VHHH12

[0106] After activating the chip, the PCSK9 antigen was coupled to the CM5 chip used by the Biacore machine, and the coupling reaction was stopped at approximately 790 RU. Then, 150 μl of 1M ethanolamine hydrochloride was added to wash away residual active carboxyl groups. Next, the machine sequentially pumped serially diluted PCSK9 single-domain antibody VHHH12 (218 nM - 109 nM - 54 nM - 27 nM - 13.5 nM) across the chip surface at a rate of 25 μL / min, with binding for 120 s and dissociation for 230 s. After obtaining the data, the results were processed, as shown below. Figure 3 As shown, the parameters for the interaction between the single-domain antibody VHHH12 and the antigen PCSK9 are as follows: Kon(1 / Ms) = 2.221E+5 (binding constant), Koff(1 / s) = 5.844E-4 (dissociation constant), Rmax(RU) = 31.76 (maximum binding response value), and KD(M) = 2.631E-9 (approximately 2.6 nM), which represents the affinity of the antigen-antibody interaction. This indicates that the single-domain antibody interacts well with the PCSK9 antigen and has potential for further development.

[0107] SEQ ID NO: 1

[0108] GRTFSDYA

[0109] SEQ ID NO: 2

[0110] IGWSGGQT

[0111] SEQ ID NO: 3

[0112] AASFLVIPGTVKTRYDS

[0113] SEQ ID NO: 4

[0114] QVQLQESGGGLVQAGGSLRLSCAASGRTFSDYAVGWFRQAPGKEREFVAGIGWSGGQTTYADSVKGRFTISRDNAKDTVYLQMNSLKPEDTAVYVCAASFLVIPGTVKTRYDSWGQGTQVTVPA

[0115] SEQ ID NO: 5

[0116] 5’-caggtgcagctgcaggagtctggaggaggattggtgcaggctgggggctctctgagactctcctgtgcagcctctggacgcacgttcagcgactatgccgtgggctggttccgccaggctcccgggaaggagcgtgagtttgtagctggtataggctggagtggcggtcaaacaacctatgcagactccgtgaagggccgattcaccatctccagagacaacgccaaagacacggtgtatctgcaaatgaacagcctgaaacctgaggacacggccgtttatgtctgtgcagcttcatttttggttatccccggcaccgtgaaaactcggtatgacagctggggccagggaacccaggtcaccgttccagcg-3’

[0117] SEQ ID NO:6

[0118] QVQLQESGGGLVQAGGSLRLSCAAS

[0119] SEQ ID NO:7

[0120] VGWFRQAPGKEREFVAG

[0121] SEQ ID NO:8

[0122] TYADSVKGRFTISRDNAKDTVYLQMNSLKPEDTAVYVC

[0123] SEQ ID NO:9

[0124] WGQGTQVTVPA

[0125] SEQ ID NO:10

[0126] GTCCTGGCTGCTCTTCTACAAGG

[0127] SEQ ID NO:11

[0128] GGTACGTGCTGTTGAACTGTTCC

[0129] SEQ ID NO:12

[0130] TGGTGGCAGGTCCCCAAGGT

[0131] SEQ ID NO: 13

[0132] TTCTTGGTGGCAGTAGCCGCAGT

[0133] SEQ ID NO: 14

[0134] GATGTGCAGCTGCAGGAGTCTGGRGGAGG

[0135] SEQ ID NO: 15

[0136] CTAGTGCGGCCGCTGGAGACGGTGACCTGGGT

[0137] The above examples illustrate the present invention only to aid in understanding it and are not intended to limit the scope of the invention. Those skilled in the art can make various simple deductions, modifications, or substitutions based on the principles of this invention.

Claims

1. A single-domain antibody that binds to the PCSK9 antigen, characterized in that: The single-domain antibody includes a heavy chain antibody variable region (VHH), which is composed of a frame region (FR) and a complementarity-determining region (CDR). The complementarity-determining region (CDR) includes complementarity-determining region 1 (CDR1) with the sequence GRTFSDYA (SEQ ID NO: 1); complementarity-determining region 2 (CDR2) with the sequence IGWSGGQT (SEQ ID NO: 2); and complementarity-determining region 3 (CDR3) with the sequence AASFLVIPGTVKTRYDS (SEQ ID NO: 3).

2. The single-domain antibody according to claim 1, characterized in that: The amino acid sequence of the variable region (VHH) of the heavy chain antibody is shown in SEQ ID NO:

4.

3. A polynucleotide molecule encoding the single-domain antibody of claim 1 or 2, characterized in that: The nucleotide sequence of the polynucleotide molecule includes the nucleotide sequence encoding the complementarity-determining regions shown in SEQ ID NO: 1 to SEQ ID NO:

3.

4. The polynucleotide molecule according to claim 3, characterized in that: The nucleotide sequence of the polynucleotide molecule includes a nucleotide sequence encoding the variable region (VHH) of the heavy chain antibody shown in SEQ ID NO:

4.

5. The polynucleotide molecule according to claim 3 or 4, characterized in that: The nucleotide sequence of the polynucleotide molecule is shown in SEQ ID NO:

5.

6. An expression vector, characterized in that: The expression vector contains the polynucleotide molecule as described in any one of claims 3-5.

7. A host cell, characterized in that: The host cell contains the expression vector of claim 6, which is capable of expressing a single-domain antibody that specifically binds to the PCSK9 antigen.

8. A method for preparing the single-domain antibody according to claim 1 or 2, characterized in that: The method includes: transforming an expression vector containing the polynucleotide molecule of any one of claims 3-5 into an expression host cell, culturing the cell, and performing large-scale expression and purification of the single-domain antibody; The expression vector is the pMECS plasmid, and the host cell is Escherichia coli HB2151 strain; or the expression vector is the pPICZα plasmid, and the host cell is yeast X33 strain; or the expression vector is the pCDNA3.4 plasmid, and the host cell is HEK293F cell line.

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