A VHH chain of an anti-PCSK9 nanobody and its application

Abstract

The present invention discloses a VHH chain of a nanobody against PCSK9 and its application. The VHH chain of the nanobody against PCSK9 includes a framework region FR and a complementary determining region CDR. The framework region FR includes FR1, FR2, FR3 and FR4, and the complementary determining region CDR includes: CDR1, CDR2 and CDR3; the specific amino acid sequence is as follows: FR1 is SEQ ID NO: 1, CDR1 is SEQ ID NO: 2, FR2 is SEQ ID NO: 3, CDR2 is SEQ ID NO: 4, FR3 is SEQ ID NO: 5, CDR3 is SEQ ID NO: 6, and FR4 is SEQ ID NO: 7. The nanobody against PCSK9 of the present invention has the following advantages: (1) small molecule and drug delivery. (2) easy to manufacture and express. (3) high affinity and strong specificity. (4) stable performance and good plasticity. (5) low immunogenicity and good metabolic characteristics.

Description

Technical Field

[0001] The present invention relates to the technical field of nano antibodies, and in particular to a VHH chain of an anti-PCSK9 nano antibody and applications thereof. Background Art

[0002] The prevention and treatment of cardiovascular disease (CVD) continues to face significant challenges, and researchers both domestically and internationally are continuously researching the mechanisms and treatments of CVD. Studies have shown that mutations in the gene encoding proprotein convertase subtilisin kexin type 9 (PCSK9) are the cause of familial hypercholesterolemia, an autosomal dominant genetic disorder. Subsequently, PCSK9 became a major therapeutic target for CVD prevention. Shortly after this discovery, large-scale population-based studies demonstrated that genetic variants can lead to increased PCSK9 activity and elevated LDL cholesterol levels, while loss-of-function mutations can reduce LDL cholesterol levels and cardiovascular disease risk. These findings suggest that inhibiting PCSK9 is a viable approach for treating hypercholesterolemia and reducing CVD risk.

[0003] PCSK9 was first discovered by Seidah et al. during neuronal apoptosis. PCSK9 belongs to the serine protein convertase family and consists of 12 exons and 11 introns. It is located on the short arm of chromosome 1 (1p32.3) and is approximately 22 kb long. The PCSK9 protein has a molecular weight of 74 kDa and contains 692 amino acids. In addition to a signal peptide (amino acids 1-30), PCSK9 is a secreted het-dimeric protein with three domains: a prodomain (31-152), a catalytic domain (153-404), a hinge region (405-454), and a cysteine- and histidine-rich C-terminal domain (452-692). It is primarily expressed in the liver, neural tissue, kidney cells, and small intestinal epithelial cells, with highest expression in the liver and jejunum.

[0004] Numerous clinical trials have shown that PCSK9 inhibitors can robustly and safely lower LDL cholesterol levels, prevent cardiovascular disease risk, and reduce mortality. Two monoclonal antibodies, alirocumab and evolocumab, were approved by the FDA in 2015. Monoclonal antibodies can be administered twice a year or less, reducing treatment costs.

[0005] Nanobodies

[0006] Nanobodies (Nbs) were first reported in Nature in 1993 by Belgian scientist Hamers-Casterman and his team. They were found in the blood of camelids (camels, llamas, alpacas, and their relatives) as "heavy-chain antibodies" lacking light chains. These antibodies consist only of a single heavy-chain variable region (VHH) and two heavy-chain CH2 and CH3 domains (Figure 1). VHHs retain full antigen-binding capacity and are the smallest fragments to retain complete antigen-binding capacity. These fragments are known as single-domain antibodies (SDAs). VHH crystals are 2.5 nm in size, 4 nm in length, and have a molecular weight of only 15 kDa.

[0007] Compared to traditional antibodies, nanobodies offer advantages such as simplified humanization, high affinity, high stability, microbial expression, low immunogenicity, good solubility, strong penetration, and the ability to recognize hidden epitopes. Their unique physical and chemical stability provide new research tools for diagnosis and treatment, and they are gaining increasing attention in antibody drug development, basic medical research, and disease diagnosis and treatment.

[0008] In 2018, the European Union approved caplacizumab, the world's first nanobody drug for the treatment of acquired thrombotic thrombocytopenic purpura in adults. Caplacizumab prevents clotting by blocking the interaction between very large vWF multimers and platelets. In 2021, Envida, a PD-L1 antibody co-developed by Alphamab Oncology, Silvio Pharmaceuticals, and Simcere Pharmaceuticals, was approved for marketing in China, becoming the first nanobody drug approved for marketing in China.

[0009] Therefore, the existing technology has defects and needs to be improved. Summary of the Invention

[0010] The technical problem to be solved by the present invention is to provide a VHH chain of an anti-PCSK9 nanobody and its application to meet the production requirements and application of anti-PCSK9 nanobodies.

[0011] The technical solution of the present invention is as follows: Provided is an anti-PCSK9 nanobody, comprising a framework region FR and a complementary determining region CDR, characterized in that the framework region FR includes FR1, FR2, FR3 and FR4, and the complementary determining region CDR includes: CDR1, CDR2 and CDR3; the specific amino acid sequence is as follows: FR1 is SEQ ID NO: 1, CDR1 is SEQ ID NO: 2, FR2 is SEQ ID NO: 3, CDR2 is SEQ ID NO: 4, FR3 is SEQ ID NO: 5, CDR3 is SEQ ID NO: 6, and FR4 is SEQ ID NO: 7.

[0012] The amino acid sequence of the anti-PCSK9 Nanobody is shown in SEQ ID NO:8.

[0013] A preferred embodiment of the anti-PCSK9 Nanobody of the present invention is Nanobody P2-11D.

[0014] The present invention also provides a nucleotide molecule encoding the aforementioned anti-PCSK9 nanobody.

[0015] The nucleotide molecule has a nucleotide sequence as shown in SEQ ID NO:9.

[0016] The present invention also provides a recombinant expression vector comprising the aforementioned nucleotide molecule.

[0017] The present invention also provides a recombinant host cell comprising the aforementioned recombinant expression vector. The host cell is selected from Escherichia coli BL21 (DE3).

[0018] The present invention also provides a method for producing anti-PCSK9 nanobodies, which is prepared by culturing the aforementioned recombinant host cells and inducing the recombinant host cells to express anti-PCSK9 nanobodies.

[0019] The present invention also provides a use of an anti-PCSK9 nanobody in the preparation of a diagnostic reagent for detecting PCSK9, using the aforementioned anti-PCSK9 nanobody.

[0020] Using the above scheme, the present invention provides a VHH chain of an anti-PCSK9 nanobody and its application, which has the following beneficial effects:

[0021] (1) Small molecules and drug delivery. The anti-PCSK9 nanoantibodies of the present invention are small molecules. When used as targeting molecules, they have less conformational impact and steric hindrance on the active site (effector molecule), resulting in higher activity of the effector molecule and can be used in drug delivery systems.

[0022] (2) Easy to manufacture and express: The anti-PCSK9 nanobody of the present invention can be efficiently expressed using Escherichia coli.

[0023] (3) High affinity and strong specificity: The anti-PCSK9 nanobody of the present invention has a VHH chain, which obtains a high affinity for the PCSK9 protein.

[0024] (4) Stable performance and good plasticity: The anti-PCSK9 nanoantibody of the present invention can be coupled to other molecules and can maintain a stable binding ability against PCSK9.

[0025] (5) Low immunogenicity and good metabolic characteristics: The PCSK9 nanobody of the present invention retains the ability of VVH chain antibodies and avoids the introduction of non-human exogenous proteins as much as possible, taking into account the function of effectively binding to anti-PCSK9 and the lowest possible heterogeneity.

[0026] The anti-PCSK9 nanobody provided by the present invention has a unique antigenic determinant recognition site and has specific recognition and binding capabilities for PCSK9 antigens. The nanobody has a high antigen affinity, which can reach 7×10 -8 M, showing excellent detection effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 For titer detection of PCSK9-immunized alpacas according to an embodiment of the present invention;

[0028] Figure 2 This is the SDS-PAGE image of the expression and purification of nanobody P2-11D;

[0029] Figure 3 This is the affinity test curve of nanobody P2-11D (KD = 7×10 -8 M). DETAILED DESCRIPTION

[0030] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0031] Construction of an anti-PCSK9 single domain heavy chain antibody phage library

[0032] Alpacas (Lama pacos) were immunized subcutaneously at multiple sites with 800 μg of PCSK9 protein emulsified in Freund's complete adjuvant. Booster immunizations were performed every two weeks with 800 μg of PCSK9 protein emulsified in Freund's incomplete adjuvant. Venous blood was collected seven days after each immunization, and serum titers were determined by indirect ELISA. Lymphocytes were isolated from the sample with the highest serum titer, and total RNA was extracted and reverse transcribed into cDNA. After two rounds of PCR amplification, antibody sequence amplification products were obtained. The selected vectors were digested with enzymes and ligated. Finally, the amplified products were transformed into TG1 competent Escherichia coli cells to generate a bacterial library. Phage libraries were prepared after infection and induction with helper phages (M13KO7).

[0033] Panning and identification of anti-PCSK9 single domain heavy chain antibodies

[0034] Solid-phase affinity panning was used to select single-domain heavy chain antibodies against PCSK9 from an anti-PCSK9 single-domain heavy chain antibody phage library. 120 μL of PCSK9 diluted in PBS was added to each enzyme-labeled well and coated overnight at 4°C. The coating concentrations for each round of panning were 100, 75, and 50 μg / mL, respectively. The coating solution was aspirated, the plate was washed five times with PBS, and 300 μL of 3% BSA-PBS was added to each well and blocked at 37°C for 2 hours. The plate was washed five times with PBS, and 100 μL of phage antibody library (containing approximately 1×1011 CFU) was added and incubated at 37°C for 2 hours. Unbound phage was aspirated, and the plate was washed 3-5 times (increased by 5 times per round) with PBST (containing 0.5% Tween-20), and then washed 15-25 times with PBS. The plate was washed with 100 μL of elution buffer (glycine-HCl, pH 2 .2) Elute the phage adsorbed to the enzyme-labeled wells and neutralize the eluate with 35μL Tris-HCl (1mol / L, pH 8.0). 10μL is used for titer determination, and the remaining 125μL of the eluate is amplified and used for the next round of panning. After four rounds of panning, randomly selected monoclonal clones are rescued using helper phage KM13 to obtain phage particles displaying the antibody variable regions. The binding activity and specificity of the phage particles are then measured using an indirect phage-ELISA. ELISA-positive clones are sent to a biotechnology service company for sequencing, and the DNA sequence of the insert is obtained, which encodes a single-domain heavy chain antibody targeting PCSK9. The sequence of the resulting monovalent nanobody P2-11D (SEQ ID NO. 8) is:

[0035] DVQLQESGGGLVQPGGSLRLSCAASGFTFRAYDMGWYRQAPGKQRDLVAVISSSGGTPNYADSVKDRFTISRDNDKNTVYLQMNSLKSEDTALYYCNARWESSAYERDYWGRGTQVTVSS, wherein the amino acid sequence at positions 1-25 is FR1, the amino acid sequence at positions 26-33 is CDR1, the amino acid sequence at positions 34-50 is FR2, the amino acid sequence at positions 51-57 is CDR2, the amino acid sequence at positions 58-95 is FR3, the amino acid sequence at positions 96-109 is CDR3, and the amino acid sequence at positions 110-120 is FR4.

[0036] Preparation of anti-PCSK9 single domain heavy chain antibody

[0037] The anti-PCSK9 single domain heavy chain antibody gene fragment (SEQ ID NO.9)

[0038] atgtgcagctgcaagagtccgggggcggcctggtccaacccgggggcagcctgagactgagctgcgccgctagcggcttcaccttcagagcctacgacatgggctggtacagacaagcccccggcaagcagagagacctggtggccgtgatcagcagcggcggcacccccaactacgccgacagcgtgaaggacagattcaccatcagcagagacaacgacaagaacaccgtgtacctgcagatgaacagcctgaagagcgaggacaccgccctgtactactgcaacgctagatgggagagcagcgcctacgagagagactactggggcagaggcacccaagtgaccgtgagcag was cloned into the expression vector pET22b, and the expression vector of the anti-PCSK9 single domain heavy chain antibody was constructed and identified by PCR and enzyme digestion. The expression vector was transformed into Escherichia coli BL21, and the single clone colonies were picked to 5 ml of LB medium containing antibiotics, shake the bacteria at 37℃ for 5 h; add 5 ml of the bacterial solution to 500 ml of LB medium containing antibiotics to expand the culture; when the OD value of the bacterial solution reaches 0.6-0.8, add 0.5 mM IPTG and induce at 18℃ overnight. The bacterial pellet was collected by centrifugation at 4000 rpm for 5 min; the pellet was resuspended in a solution containing 20 mM Tris-HCl, 150 mM NaCl, 1 mM PMSF, and 5 mM imidazole and sonicated at 300 W for 20 min; the supernatant was collected by centrifugation at 18000 rpm for 15 min; the supernatant was mixed with 1 ml Ni resin and incubated for 30 min; the supernatant was discarded by centrifugation at 1000 rpm for 2 min, and the pellet was resuspended in a buffer containing 20 mM imidazole and loaded onto a gravity column; impurities were washed with 20 mM, 40 mM, and 60 mM imidazole solutions in sequence, and the target protein was eluted with 6 ml of a solution containing 500 mM imidazole and directly transferred to a concentrator tube. Samples were taken for gel analysis and concentrated to 1 ml at 3000 rpm for 10 min / time; the pellet was passed through a molecular sieve and frozen after gel analysis.

[0039] Affinity determination of anti-PCSK9 nanobodies

[0040] The affinity of the anti-PCSK9 single domain heavy chain antibody prepared above was determined using the intermolecular interaction test method SPR (surface plasmon resonance).

[0041] Affinity KD(M) = kdis(1 / s) / kon(1 / Ms). The results of the test were as follows: kdis(1 / s) = 0.009213; kon(1 / Ms) = 129500; KD(M) = kdis(1 / s) / kon(1 / Ms) = 7× 10 -8 M.

[0042] See also Figure 1 , Figure 1 This is a titer test of PCSK9-immunized alpacas according to an embodiment of the present invention; it can be seen from the figure that the antigen immunization of alpacas of the present invention is effective, which is the key to obtaining high-affinity and specific anti-PCSK9 nanoantibodies, and provides a basis for obtaining effective anti-PCSK9 nanoantibodies.

[0043] Figure 2 This is the SDS-PAGE diagram of the expression and purification of nanobody P2-11D; Figure 2 As can be seen from the figure, the nanobody has a small molecular weight.

[0044] Figure 3 This is the affinity test curve of the nanobody P2-11D (KD = 7×10-8 M). It can be seen from the figure that the nanobody has high affinity and good specificity.

[0045] In summary, the present invention provides a VHH chain of an anti-PCSK9 nanobody and its application, which has the following beneficial effects:

[0046] (1) Small molecules and drug delivery. The anti-PCSK9 nanoantibodies of the present invention are small molecules. When used as targeting molecules, they have less conformational impact and steric hindrance on the active site (effector molecule), resulting in higher activity of the effector molecule and can be used in drug delivery systems.

[0047] (2) Easy to manufacture and express: The anti-PCSK9 nanobody of the present invention can be efficiently expressed using Escherichia coli.

[0048] (3) High affinity and strong specificity: The anti-PCSK9 nanobody of the present invention has a VHH chain, which obtains a high affinity for the PCSK9 protein.

[0049] (4) Stable performance and good plasticity: The anti-PCSK9 nanoantibody of the present invention can be coupled to other molecules and can maintain a stable binding ability against PCSK9.

[0050] (5) Low immunogenicity and good metabolic characteristics: The PCSK9 nanobody of the present invention retains the ability of VVH chain antibodies and avoids the introduction of non-human exogenous proteins as much as possible, taking into account the function of effectively binding to anti-PCSK9 and the lowest possible heterogeneity.

[0051] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A nanobody against PCSK9, comprising a framework region FR and a complementary determining region CDR, characterized in that: The framework regions FR include FR1, FR2, FR3 and FR4, and the complementarity determining regions CDR include: CDR1, CDR2 and CDR3; the specific amino acid sequences are as follows: FR1 is SEQ ID NO: 1, CDR1 is SEQ ID NO: 2, FR2 is SEQ ID NO: 3, CDR2 is SEQ ID NO: 4, FR3 is SEQ ID NO: 5, CDR3 is SEQ ID NO: 6, and FR4 is SEQ ID NO:

7.

2. An anti-PCSK9 nanobody according to claim 1, characterized in that Its amino acid sequence is shown in SEQ ID NO:

8.

3. A nucleotide molecule, characterized in that It encodes the anti-PCSK9 Nanobody as described in claim 1 or 2.

4. A nucleotide molecule according to claim 3, characterized in that The nucleotide sequence is shown in SEQ ID NO:

9.

5. A recombinant expression vector, characterized in that: It comprises the nucleotide molecule according to claim 3 or 4.

6. A recombinant host cell, characterized in that It comprises the recombinant expression vector according to claim 5.

7. A method for producing an anti-PCSK9 nanobody, characterized in that It is prepared by culturing the recombinant host cell as claimed in claim 6 and inducing the recombinant host cell to express the anti-PCSK9 nanobody.

8. Use of an anti-PCSK9 nanobody in the preparation of a diagnostic reagent for detecting PCSK9, characterized in that: The anti-PCSK9 nanobody according to claim 1 or 2 is used.

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