CN115746140A - VHH chain of PCSK 9-resistant nano antibody and application thereof - Google Patents

Abstract

The invention discloses a VHH chain of a PCSK 9-resistant nano antibody and application thereof. The VHH chain of the nanobody against PCSK9 comprises a framework region FR and a complementarity determining region CDR, wherein the framework region FR comprises FR1, FR2, FR3 and FR4, and the complementarity determining region CDR comprises: 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, FR4 is SEQ ID NO. 7. The PCSK9 resisting nano antibody has the following beneficial effects: (1) small molecule, drug delivery. (2) easy to produce and express. And (3) the affinity is high, and the specificity is strong. And (4) the performance is stable, and the plasticity is good. And (5) the immunogenicity is low, and the metabolic characteristics are good.

Description

VHH chain of PCSK 9-resistant nano antibody and application thereof

Technical Field

The invention relates to the technical field of nano antibodies, in particular to a VHH chain of a PCSK 9-resistant nano antibody and application thereof.

Background

Cardiovascular disease (CVD) is one of the most serious diseases threatening humans in the world today, and its morbidity and mortality have jumped first over neoplastic disease. Chinese CVD patients are about 2.9 million. The work of preventing and treating CVD still faces serious challenges, and researchers at home and abroad have never been interrupted on the generation mechanism and treatment of CVD. Studies have shown that the mutation of the gene coding for Proprotein convertase subtilisin Kexin-9 (protein convertase subtilisin Kexin type 9, PCSK 9) is identified as the cause of autosomal dominant hereditary diseases such as familial hypercholesterolemia. Subsequently, PCSK9 became the main therapeutic target for prevention of CVD. Shortly after this finding, large-scale human studies have shown that genetic variation leads to increased PCSK9 activity and increased LDL cholesterol levels, while loss-of-function mutations reduce low density lipoprotein cholesterol levels and cardiovascular disease risk. These findings indicate that inhibition of PCSK9 is a viable approach to treating hypercholesterolemia, and can reduce CVD risk.

PCSK9 was first discovered by Seidah et al during neuronal apoptosis. PCSK9 belongs to the serine protein convertase family, consists of 12 exons and 11 introns, is located on the short arm of chromosome 1 (1p32.3), and is about 22kb in length. The PCSK9 protein has a molecular weight of 74kDa and comprises 692 amino acids. In addition to the signal peptide (amino acids 1-30), PCSK9 is a secreted het-dimer protein with 3 domains, which includes 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). Mainly expressed in liver, nervous tissue, kidney cells and small intestine epithelial cells, with highest expression in liver and jejunum.

Many clinical trials have shown that PCSK9 inhibitors are able to robustly and safely reduce LDL cholesterol levels, prevent cardiovascular disease risk and reduce mortality. Two monoclonal antibodies, alirocumab and evolocumab, were FDA approved in 2015, and monoclonal antibodies were administered twice a year or less and treatment costs were reduced.

Nanobodies

Nanobodies (Nbs) were first reported in nature journal by bimers-Casterman and his group in 1993 by belgium scientists to find that in camelids (camel, llama, alpaca and their close relatives) blood a part of the antibody is a "heavy chain antibody" lacking a light chain, which antibody comprises only one heavy chain variable region (VHH) and two heavy chain CH2 and CH3 regions (fig. 1). VHH retains the full antigen binding capacity, is the smallest fragment retaining the whole antigen binding, and is called Single domain antibodies (Single domain antibodies), VHH crystal is 2.5nm, length is 4nm, and molecular weight is only 15kDa.

Compared with the traditional antibody, the nano antibody also has the advantages of simple humanization, high affinity, high stability, microbial expression, low immunogenicity, good solubility, strong permeability, capability of identifying hidden epitopes and the like. The unique physical and chemical stability of the nano antibody provides a new research tool for diagnosis and treatment. There is an increasing interest in antibody drug development, basic medical research, and disease diagnosis and treatment.

The European union approved the first global nano antibody drug Capacizumab for treating adult acquired thrombotic thrombocytopenic purpura in 2018, and prevents coagulation by blocking the interaction between the oversized vWF multimer and platelets. In 2021, the PD-L1 antibody Envida which is developed by the cooperation of Canning Jirui pharmacy, didi medicine and Misheng medicine is approved to be marketed in China and is the first nano antibody drug approved to be marketed in China.

Accordingly, the prior art is deficient and needs improvement.

Disclosure of Invention

The technical problem to be solved by the invention is as follows: provides a VHH chain of the PCSK 9-resistant nano antibody and application thereof, and meets the production requirement and application of the PCSK 9-resistant nano antibody.

The technical scheme of the invention is as follows: provided is a VHH chain of a nanobody against PCSK9, comprising a framework region FR and a complementarity determining region CDR, wherein the framework region FR comprises FR1, FR2, FR3 and FR4, and the complementarity determining region CDR comprises: 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, FR4 is SEQ ID NO. 7.

The amino acid sequence of the VHH chain of the nanometer antibody for resisting PCSK9 is shown as SEQ ID NO. 8.

The invention also provides an anti-PCSK 9 nanobody, which comprises: VHH chains of the aforementioned nanobodies against PCSK 9. One preferred example is nanobody P2-11D.

The invention also provides a gene sequence encoding a VHH chain of the anti-PCSK 9 nanobody of claim 1 or 2 or encoding an anti-PCSK 9 nanobody of claim 3.

The gene sequence has a nucleotide sequence shown as SEQ ID NO. 9.

The invention also provides a nucleotide construct which comprises the gene sequence.

The invention also provides a recombinant expression vector which comprises the nucleotide construct.

The invention also provides a recombinant host cell comprising the nucleotide construct or the recombinant expression vector. The host cell is Escherichia coli BL21 (DE 3)

The invention also provides a method for producing the anti-PCSK 9 nanobody, which is prepared by culturing the recombinant host cell mentioned above and inducing the recombinant host cell to express the form of the anti-PCSK 9 nanobody.

The invention also provides application of the diagnostic reagent for detecting PCSK9, wherein the VHH chain of the anti-PCSK 9 nanobody or the anti-PCSK 9 nanobody is adopted.

By adopting the scheme, the invention provides the VHH chain of the PCSK 9-resistant nano antibody and the application thereof, and the VHH chain has the following beneficial effects:

(1) Small molecule, drug delivery. The anti-PCSK 9 nano antibody of the invention has small molecules, when being used as a targeting molecule, the nano antibody has smaller conformational influence and steric hindrance on an active site (effector molecule), and the effector molecule has higher activity, and can be applied to a drug delivery system.

(2) Easy to manufacture and express: the PCSK 9-resistant nano antibody can be efficiently expressed by utilizing escherichia coli.

(3) High affinity and strong specificity: the anti-PCSK 9 nanobody has a VHH chain, and obtains high affinity to PCSK9 protein.

(4) The performance is stable, and the plasticity is good: the anti-PCSK 9 nanobody can be coupled with other molecules, and can keep the stable binding capacity against PCSK 9.

(5) Low immunogenicity and good metabolic characteristics: the nanometer antibody of PCSK9 of the invention retains the ability of VVH chain antibody, avoids the introduction of non-human exogenous protein as much as possible, and combines the functions of effectively binding to PCSK9 and the heterogeneity as low as possible.

The anti-PCSK 9 nano antibody provided by the invention has unique antigenic determinant recognition sites, has specific recognition and binding capacity on PCSK9 antigens, has high antigen affinity, and the affinity can reach 7 multiplied by 10 ^-8 M, shows excellent detection effect.

Drawings

Figure 1 is a potency assay for PCSK9 immune alpaca according to embodiments of the present invention;

FIG. 2 is a SDS-PAGE picture of expression and purification of the nanobody P2-11D;

FIG. 3 is a graph of the affinity test of the nanobody P2-11D (KD = 7X 10) ^-8 M)。

Detailed Description

The invention is described in detail below with reference to the figures and the specific embodiments.

Construction of anti-PCSK 9 single-domain heavy-chain antibody phage library

800 mu g of PCSK9 protein is emulsified with Freund's complete adjuvant, and the alpaca (Lama pacos) is immunized by subcutaneous multipoint injection. The enhanced immunization is carried out by emulsifying 800 mu g of PCSK9 protein with Freund incomplete adjuvant at intervals of 2 weeks, taking blood from veins 7 days after each immunization, measuring serum titer by indirect ELISA, selecting a sample with the highest serum titer to separate lymphocytes, extracting total RNA, carrying out reverse transcription to cDNA, carrying out two-round PCR amplification to obtain an antibody sequence amplification product, selecting a carrier for enzyme digestion and then connecting, finally transforming to TG1 escherichia coli competent cells to obtain a bacterial library, and infecting and inducing by an auxiliary phage library (M13 KO 7) to prepare the phage library.

Panning and identification of anti-PCSK 9 single domain heavy chain antibodies

Panning single domain heavy chain antibodies against PCSK9 from an anti-PCSK 9 single domain heavy chain antibody phage library using a solid phase affinity panning method. Adding 120 mu L of PCSK9 diluted by PBS into each enzyme-labeled well, coating at 4 ℃ overnight, wherein the coating concentration of each round of panning is 100, 75 and 50 mu g/mL respectively; aspirate the coating solution, wash the plate 5 times with PBS, add 300 μ L of 3% BSA-PBS per well, block at 37 ℃ for 2h; the plate was washed 5 times with PBS, 100. Mu.L phage antibody library (containing about 1X 1011 CFU) was added, incubated at 37 ℃ for 2h; the unbound phage were aspirated, washed 3-5 times (5 rounds by round) with PBST (containing 0.5% Tween-20) and then 15-25 times with PBS; the phage adsorbed in the wells were eluted with 100. Mu.L of eluent (glycine-hydrochloric acid, pH 2.2), the eluate was neutralized with 35. Mu.L of Tris-HCl (1 mol/L, pH 8.0), 10. Mu.L of the eluate was taken for titer determination, and the remaining 125. Mu.L of the eluate was amplified for the next round of panning. After four rounds of panning, randomly picked monoclonals are rescued by using the auxiliary phage KM13 to respectively obtain phage particles displaying antibody variable regions, and the binding activity and specificity of the phage particles are measured by using indirect phage-ELISA. The ELISA positive clones were sent to biotechnology service for sequencing to obtain the DNA sequence of the insert, which encodes a single domain heavy chain antibody against PCSK 9. The sequence of the monovalent nano antibody P2-11D (SEQ ID NO. 8) is:

DVQLQESGGGLVQPGGSLRLSCAASGFTFRAYDMGWYRQAPGKQRDLVAVISSSGGTPNYADSVKDRFTISRDNDKNTVYLQMNSLKSEDTALYYCNARWESSAYERDYWGRGTQVTVSS wherein the 1 st to 25 th amino acid sequence is FR1, the 26 th to 33 th amino acid sequence is CDR1, the 34 th to 50 th amino acid sequence is FR2, the 51 th to 57 th amino acid sequence is CDR2, the 58 th to 95 th amino acid sequence is FR3, the 96 th to 111 th amino acid sequence is CDR3, and the 112 th to 122 th amino acid sequence is FR4.

Preparation of anti-PCSK 9 single-domain heavy-chain antibody

An anti-PCSK 9 single-domain heavy chain antibody gene fragment (SEQ ID NO. 9)

atgtgcagctgcaagagtccgggggcggcctggtccaacccgggggcagcctgagactgagctgcgccgctagcggcttcaccttcagagcctacgacatgggctggtacagacaagcccccggcaagcagagagacctggtggccgtgatcagcagcagcggcggcacccccaactacgccgacagcgtgaaggacagattcaccatcagcagagacaacgacaagaacaccgtgtacctgcagatgaacagcctgaagagcgaggacaccgccctgtactactgcaacgctagatgggagagcagcgcctacgagagagactactggggcagaggcacccaagtgaccgtgagcag is cloned to an expression vector pET22b, PCR and enzyme digestion identification are carried out, the expression vector of the PCSK9 single-domain heavy chain antibody is constructed and converted to escherichia coli BL21, monoclonal bacteria are picked and dropped into 5ml of LB culture medium containing antibiotics, and bacteria are shaken for 5h at 37 ℃;5ml of bacterial liquid is added into 500ml of LB culture medium containing antibiotics for amplification culture; when the OD value of the bacterial liquid is 0.6-0.8, 0.5mM IPTG is added, and the overnight induction is carried out at 18 ℃. Centrifuging at 4000rpm for 5min and collecting bacterial precipitate; the pellet was resuspended in a solution containing 20mM Tris-HCl,150mM NaCl,1mM PMSF and 5mM imidazole, sonicated 300W,20min; centrifuging at 18000rpm for 15min, and collecting supernatant; mixing and incubating the supernatant with 1ml of Ni resin for 30min; centrifuging at 1000rpm for 2min, discarding the supernatant, and suspending the buffer solution containing 20mM imidazole into a gravity column; sequentially eluting with 20mM,40mM and 60mM imidazole solutions to remove foreign proteins, eluting with 6ml solution containing 500mM imidazole, directly flowing into a concentration tube, sampling, running gel for detection, and concentrating to 1ml at 3000rpm and 10min/time; and (4) passing through a molecular sieve, and freezing and storing after glue running detection is not problematic.

Affinity assay for anti-PCSK 9 nanobodies

The affinity of the anti-PCSK 9 single-domain heavy-chain antibody prepared above was determined using an intermolecular interaction test method SPR (surface plasma resonance).

Affinity KD (M) = kdis (1/s)/kon (1/Ms). The detection results show that: kdis (1/s) =0.009213; kon (1/Ms) =129500; KD (M) = kdis (1/s)/kon (1/Ms) =7 × 10 ^-8 M。

Referring to fig. 1, fig. 1 is a graph illustrating the detection of the potency of PCSK9 immune alpaca according to an embodiment of the present invention; it can be seen from the figure that the antigen immune alpaca of the invention is effective, which is the key for obtaining the high-affinity and specificity anti-PCSK 9 nano antibody and provides a basis for obtaining the effective anti-PCSK 9 nano antibody.

FIG. 2 is a SDS-PAGE picture of expression and purification of the nanobody P2-11D; as can be seen from fig. 2, the nanobody has a small molecular weight.

Fig. 3 is a graph of the nanobody P2-11D affinity test (KD =7 × 10-8M). The figure shows that the nano antibody has high affinity and good specificity.

In conclusion, the invention provides a VHH chain of a PCSK 9-resistant nano antibody and an application thereof, and the VHH chain has the following beneficial effects:

(1) Small molecule, drug delivery. The nanometer antibody molecule for resisting PCSK9 is small, when the nanometer antibody molecule is used as a targeting molecule, the conformational influence and the steric hindrance on an active site (effector molecule) are small, the activity of the effector molecule is high, and the nanometer antibody molecule can be applied to a drug delivery system.

(2) Easy to manufacture and express: the anti-PCSK 9 nano antibody can be efficiently expressed by utilizing escherichia coli.

(3) High affinity and strong specificity: the anti-PCSK 9 nanobody has a VHH chain, and obtains high affinity to PCSK9 protein.

(4) The performance is stable, and the plasticity is good: the anti-PCSK 9 nanobody can be coupled with other molecules, and can keep the stable binding capacity against PCSK 9.

(5) Low immunogenicity and good metabolic characteristics: the nanometer antibody of PCSK9 of the invention retains the ability of VVH chain antibody, avoids the introduction of non-human exogenous protein as much as possible, and combines the functions of effectively binding to PCSK9 and the heterogeneity as low as possible.

The present invention is not limited to the above preferred embodiments, and any modifications, equivalent substitutions and improvements made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (10)

1. A VHH chain of a nanobody against PCSK9 comprising a framework region FR and a complementarity determining region CDR, wherein the framework region FR comprises FR1, FR2, FR3 and FR4, and the complementarity determining region CDR comprises: 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, FR4 is SEQ ID NO. 7.

2. The VHH chain of the nanobody against PCSK9 of claim 1, having the amino acid sequence shown in SEQ ID NO. 8.

3. An anti-PCSK 9 nanobody, comprising: the VHH chain of the anti-PCSK 9 nanobody of claim 1 or 2.

4. A gene sequence encoding a VHH chain of the anti-PCSK 9 nanobody of claim 1 or 2 or encoding an anti-PCSK 9 nanobody of claim 3.

5. A gene sequence according to claim 4, which has the nucleotide sequence shown in SEQ ID NO. 9.

6. A nucleotide construct comprising the gene sequence of claim 4 or 5.

7. A recombinant expression vector comprising the nucleotide construct of claim 6.

8. A recombinant host cell comprising the nucleotide construct of claim 6 or the recombinant expression vector of claim 7.

9. A method for producing nanobodies against PCSK9, characterized in that it is prepared by culturing the recombinant host cell of claim 8, inducing the recombinant host cell to express the form of nanobodies against PCSK 9.

10. Use of a diagnostic reagent for the detection of PCSK9, wherein the VHH chain of the nanobody against PCSK9 of claim 1 or 2 or the nanobody against PCSK9 of claim 3 is used.

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