CN118085081A

CN118085081A - Nano antibody specifically binding VEGF and application thereof

Info

Publication number: CN118085081A
Application number: CN202410471608.6A
Authority: CN
Inventors: 陈大华; 章文信; 周一珉
Original assignee: Yunnan University YNU
Current assignee: Yunnan University YNU
Priority date: 2024-04-19
Filing date: 2024-04-19
Publication date: 2024-05-28
Anticipated expiration: 2044-04-19
Also published as: CN118085081B

Abstract

The invention discloses a nanometer antibody specifically binding VEGF and application thereof, wherein the nanometer antibody specifically binds with human or mouse VEGF to seal the activity of the VEGF, the amino acid sequence of the nanometer antibody is shown as SEQ ID NO.1 or has the sequence of substitution, deletion or addition of one or more amino acids compared with SEQ ID NO.1, the complementarity determining region of the nanometer antibody comprises CDR1, CDR2 and CDR3, wherein the amino acid sequence of the CDR1 is SEQ ID NO.2, the amino acid sequence of the CDR2 is SEQ ID NO.3, and the amino acid sequence of the CDR3 is SEQ ID NO.4; the nano antibody has small relative molecular mass (12-15 KDa), is specifically combined with human or mouse VEGF to block the activity of the VEGF, effectively inhibits abnormal vascular proliferation caused by increasing VEGF, and has great application potential in clinical treatment of wet age-related maculopathy by verifying through a mouse disease model.

Description

Nano antibody specifically binding VEGF and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a nano antibody specifically binding VEGF and application thereof.

Background

Vascular endothelial growth factor (Vascular endothelial growth factor, VEGF) is the main factor regulating angiogenesis, and has effects of promoting vascular permeability increase, extracellular matrix degeneration, vascular endothelial cell migration, proliferation and angiogenesis. The vascular endothelial growth factor family has Sub>A variety of subtypes, including VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E and Placental Growth Factor (PGF), in which VEGF-A is abundant and has Sub>A more intensive functional study, and VEGF refers generally to VEGF-A unless specified. In the process of angiogenesis, VEGF is combined with a receptor (Vascular Endothelial Growth Factor Receptors, VEGFR) thereof to activate intracellular tyrosine kinase, and downstream cell signals are started to promote angiogenesis, so that angiogenesis is an important cause for promoting malignant tumor development and inducing ophthalmic diseases, and therefore, blocking angiogenesis is a key for treating tumors and related ophthalmic diseases, and VEGF pathway is also an important target point for developing related medicines.

At present, research on pharmacodynamic molecular mechanisms for blocking VEGF is clear, and various monoclonal antibody medicines are applied to blocking treatment of targeting VEGF channels, wherein representative medicines comprise bevacizumab and ranibizumab, and clinical treatment of senile macular degeneration (age-related macular degeneration, AMD, common eye diseases causing vision loss of the elderly) is taken as an example, and due to the short half-life of the applied anti-VEGF monoclonal antibody medicine, intraocular injection of the monoclonal antibody medicine needs to be repeatedly carried out every 4-8 weeks to maintain curative effect, and frequent injection brings high administration cost and inconvenience to remarkably reduce compliance and treatment effect of patients. Moreover, conventional monoclonal antibodies are classical macromolecular drugs (molecular weight about 150 KDa), which have shortcomings in terms of binding antigen specificity, avidity, and tissue penetration capacity (drug effective absorption). Thus there remains a need for miniaturized engineering of antibodies.

Disclosure of Invention

The invention aims to solve the defects of the prior art and provides a preparation method and application of a heavy chain single domain VHH antibody, which has the characteristics of small relative molecular mass (12-15 KDa) and high antigen binding specificity and specifically binds VEGF.

The technical scheme adopted by the invention is as follows:

A nanobody that specifically binds to VEGF, which specifically binds to human or mouse VEGF to block the activity of the VEGF, the nanobody having an amino acid sequence as shown in SEQ ID No. 1 or a sequence having one or more amino acid substitutions, deletions or additions as compared to SEQ ID No. 1.

Further, the complementarity determining regions of the nanobody comprise CDR1, CDR2 and CDR3, wherein the amino acid sequence of the CDR1 is SEQ ID NO. 2, the amino acid sequence of the CDR2 is SEQ ID NO. 3, and the amino acid sequence of the CDR3 is SEQ ID NO. 4.

Further, the framework region of the nano antibody comprises FR1, FR2, FR3 and FR4, wherein the amino acid sequence of the FR1 is shown as SEQ ID NO. 5, the amino acid sequence of the FR2 is shown as SEQ ID NO. 6, the amino acid sequence of the FR3 is shown as SEQ ID NO. 7, and the amino acid sequence of the FR4 is shown as SEQ ID NO. 8.

Furthermore, n nanobodies are directly connected or connected through a connecting sequence to form fusion protein, and the fusion protein still has the capability of binding VEGF, wherein n is more than or equal to 2.

The invention provides a pharmaceutical composition comprising the nanobody and a pharmaceutically acceptable excipient, carrier or diluent.

The invention provides an application of the nano antibody in preparing a medicament for treating diseases related to blocking VEGF signals.

Further, the disease associated with blocking VEGF signals by the treatment is age related macular degeneration disease.

The invention has the beneficial effects that:

the nanobody of the invention has a relative molecular mass of about (12-15 KDa), and specifically binds to human or mouse VEGF to block the activity of the VEGF, so that the abnormal proliferation of blood vessels caused by the increase of VEGF can be effectively inhibited. The mouse disease model proves that the nano antibody can effectively inhibit the formation of choroidal neovascularization induced by laser, and has great application potential in clinical treatment of wet age-related maculopathy.

Drawings

FIG. 1 is a chart showing the serum antibody titer assay performed after 4-immunization of alpaca with vascular endothelial growth factor A antigen protein of example 1;

FIG. 2 is a diagram showing the detection of binding between candidate nanobody molecules and vascular endothelial growth factor A antigen protein by ELISA method in example 2;

FIG. 3 is a diagram showing the detection of the binding between candidate nanobody molecules and vascular endothelial growth factor A antigen protein by co-immunoprecipitation in example 3;

FIG. 4 is a graph showing the affinity of the candidate nanobody of example 4 with vascular endothelial growth factor A antigen protein;

FIG. 5 is a statistical graph showing the effect of nanobody VE12 and fusion protein 2VE12 of example 5 on inhibiting choroidal neovascularization in a laser induced mouse.

Detailed Description

The term "antibody" is used in its broadest sense to include a variety of antibody structures including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies, bispecific antibodies, and antibody fragments so long as they exhibit a particular antigen-binding activity. The binding specificity and avidity of antibodies are determined primarily by the CDR sequences, and amino acid sequences of non-CDR regions can be readily altered according to well-established and well-known techniques to obtain variants with similar biological activities. As is known in the art, an antigen binding domain refers to a region that specifically interacts with a target molecule, such as an antigen, with a high degree of selectivity in its action, and a sequence that recognizes one target molecule is generally unable to recognize other molecular sequences.

The term "binding" according to the present invention refers to a non-covalent interaction between a single binding site of a molecule (e.g. an antibody) and its binding partner (e.g. an antigen). Unless otherwise indicated, "binding force" as used herein refers to the inherent binding affinity of a 1:1 interaction between members of a binding pair (e.g., antibodies and antigens). The affinity of a molecule X for its partner Y can generally be expressed by a dissociation constant (KD), which can be measured by common methods known in the art.

The present invention will be described in further detail below for the purpose of making the objects, technical solutions and advantages of the present invention more apparent.

EXAMPLE 1 preparation of nanobody VE12

Step 1: expression and purification of human VEGFa antigen. Inquiring human VEGFA protein and coding mRNA sequence information in NCBI database, constructing pcDNA3.1-VEGFA-6 XHis plasmid and transferring HEK293F cells, collecting cell supernatant after 72 hours of transfection, performing affinity purification on the supernatant by using AKTA, concentrating the eluate, and further purifying by using a molecular sieve to obtain human VEGFA protein as an antigen molecule;

Step 2: antigen immunized alpaca. Mixing the antigen molecules in the step 1 with an equal volume of Freund's adjuvant, vibrating and uniformly mixing, performing multipoint subcutaneous injection on the neck of the alpaca, performing immunization on the alpaca for five times repeatedly, performing immunization for 1 time every 14 days, detecting antibody titer after four times of immunization, and forming a good immunization effect after antigen injection as shown in a figure 1;

Step 3: and (3) extracting and reverse transcribing RNA of alpaca peripheral blood lymphocyte. Collecting blood of leg veins of alpaca in the step 2 by adopting a heparin sodium vacuum blood collection tube, separating lymphocytes, extracting total RNA, and then carrying out reverse transcription to synthesize cDNA for constructing a ribosome display library;

Step 4: a ribosome display library was constructed. Designing primers (primer sequences: alpVh-L2: CTGARCTKGGTGGTCCTGGCTGC; finger-R: TGGRCATTTGGGACACTTGGA) for amplifying antibody fragments by taking the cDNA in the step 3 as a template according to the heavy chain antibody structure of alpaca, wherein the amplified fragments are about 500-600bp in size, and adding necessary elements for ribosome display at two ends of the antibody fragments by an overlap extension PCR (SOE-PCR) method, wherein phage P3 protein fragments are used for ribosome surface display of translated proteins; polyP and PolyA structures are used to trigger ribosome arrest, terminal no stop codon;

Step 5: ribosome display screening. Coating antigen proteins in a 96-well plate for later use, performing in-vitro translation reaction on the ribosome display library in the step 4, incubating reaction products with the coated antigen proteins, washing off a non-specific binding, eluting an incubation mixture, recovering RNA in the mixture, and performing reverse transcription by using the finger-R primer to obtain cDNA, namely an antibody fragment template of the ribosome display library in the next round; adding necessary elements for ribosome display at two ends of the antibody fragment by SOE-PCR to construct a new ribosome display library, repeating the three times of screening to finish the next step (step 6) of sequence confirmation of the screened cDNA library;

Step 6: bacterial ELISA detection. Bovine serum albumin (Bovine Serum Albumin, BSA) (negative control) and antigen protein were coated separately in 96 well plates for use; taking cDNA obtained by screening in the step 5, designing a primer (a primer sequence ：RDLT28A-F：agcaaatgggtcgcggatccGtggtcctggctgctcttctac;HA-28A-R：gtggtggtggtggtggtgctcgagTTAAGCGTAATCTGGTACGTCGTATGGGTA）, is introduced with an HA tag at the tail end of the primer sequence and is connected with a prokaryotic expression plasmid pET-28a, a recombinant product is transformed into escherichia coli BL21 (DE 3) competent cells, adding IPTG to induce target protein expression after picking up monoclonal amplification culture, finishing induced escherichia coli lysis, respectively co-incubating the product and BSA or antigen protein, washing off nonspecific binding, using an anti-HA tag antibody to perform ELISA detection, and removing repeated sequences and antibody structural incomplete sequences in 50 antibody sequences with the highest ratio of the antigen protein to the corresponding BSA, taking the first 5 candidate nanobody sequences (sequences with frames and bold fonts) according to the ratio, and performing further experiments on the candidate A, the candidate B, the candidate C, the candidate D and the candidate E;

TABLE 1 preliminary screening of candidate nanobody sequences by bacterial ELISA

Step 7: antibody-antigen affinity detection. Constructing a prokaryotic expression plasmid of the candidate antibody sequence in the step 6, expressing and purifying the candidate nano-antibody, and detecting antigen-antibody affinity by adopting a surface plasmon resonance (Surface plasmon resonance, SPR) technology;

Optimization based on nanobody in step 7: the two VE12 antibody sequences are serially connected by a linker (linker sequence: GGGGS), the formed fusion protein is named as 2VE12, and compared with the original nano antibody, the capability of the fusion protein 2VE12 for binding antigen is obviously improved.

EXAMPLE 2 nanobody VE12, fusion protein 2VE12 binding to VEGF antigen

The cDNA obtained by the third ribosome display screening in example 1 was taken, primers were designed based on the conserved leader sequence and hinge region (primer sequence ：RDLT28A-F：agcaaatgggtcgcggatccGtggtcctggctgctcttctac;HA-28A-R：gtggtggtggtggtggtgctcgagTTAAGCGTAATCTGGTACGTCGTATGGGTA）, was introduced with HA tag at its C-terminal end and ligated into prokaryotic expression plasmid pET-28a, recombinant product transformed E.coli BL21 (DE 3) competent cells, the next day was picked up and monoclonal into 2mL 96 well sterile deep well plates containing 300. Mu.L LB medium (kanamycin resistance) each, shake cultured at 37℃shaker at 220rpm for more than 4 hours, stored at 4℃and antigen protein was diluted to 2. Mu.g/mL with ELISA coating solution, 100. Mu.L/well was coated in 96 well plates, shaking at 4℃at 50rpm overnight while the same conditions were coated with the same amount of BSA protein as a control. A new deep-well plate was prepared, containing 300. Mu.L of LB medium per well (kanamycin resistance), 50. Mu.L of the preserved monoclonal bacterial liquid per well was inoculated into the new deep-well plate, shake-cultured in a shaker at 37℃for 220rpm hours, and then 50. Mu.L of LB medium containing 1.6mM IPTG (kanamycin resistance) was added to the culture medium to give a final concentration of 0.2mM IPTG in the bacterial liquid, and the bacterial liquid was induced to express at 16℃at 200rpm overnight. The induced expression bacterial liquid is taken out, centrifuged for 15 minutes at 4 ℃ and 4000 Xg, and the culture medium supernatant is discarded. 300. Mu.L of E.coli lysis buffer of protease inhibitor Cocktail and lysozyme was added to each well, and the cells were resuspended and lysed on ice for 30 minutes. The coating solution in the antigen protein coated 96-well plate was discarded, 200. Mu.L of PBS was added to each well and washed 3 times for 5 minutes, 3% PBSTA blocking solution was added, 200. Mu.L/well, and the wells were sealed at room temperature for 1 hour. The blocking solution was discarded, and 200. Mu.L of PBST was added to each well and washed 3 times for 5 minutes. 100. Mu.L of 3% PBSTA blocking solution was added to each well, and then the supernatant of the E.coli lysate was taken, 100. Mu.L of each well was added to the corresponding well, and the BSA coated wells were incubated at 4℃for 4 hours with shaking at 50 rpm. The remaining liquid in the wells was discarded, and 200. Mu.L of PBST was added to each well and washed 3 times for 10 minutes. anti-HA mouse antibody is diluted with 3% PBSTA at a ratio of 1:3000, 100 μl/well is added to 96-well ELISA plate, incubated at room temperature for 2 hours, the remaining liquid in the well is discarded, 200 μl PBST is added to each well and washed 6 times for 10 minutes each time. HRP-labeled murine secondary antibody was diluted 1:5000 with 3% PBSTA; 100. Mu.L/well 96-well ELISA plate was added, incubated at room temperature for 30 minutes, the remaining liquid in the well was discarded, and each well was washed 6 times with 300. Mu.L of PBST for 5 minutes. The remaining liquid in the wells was discarded, 100. Mu.L of TMB single-component color development solution was added to each well, incubated at room temperature in the dark for 5-10 minutes, 100. Mu.L of 1mol/L HCl stop solution was added to each well to terminate the reaction, and OD450 values were read by using a microplate reader, as shown in FIG. 2, and nanobody VE12 and fusion protein 2VE12 were both able to bind to antigen VEGF.

EXAMPLE 3 binding of nanobodies to VEGF antigen

The binding of 6 nanobodies, namely, the nanobody VE12, the candidate A, the candidate B, the candidate C, the candidate D and the candidate E, with the human VEGF and the mouse VEGF is detected by using an immune coprecipitation method.

Constructing prokaryotic expression plasmid with HA tag, expressing prokaryotic expression in colibacillus, expressing Flag-hVEGF and Myc-mVegf in HEK293T, combining transfected cell lysate with Flag beads and Myc beads, incubating antigen-containing Flag or Myc beads with antibody-containing bacterial lysate at room temperature for 1 hr, washing off nonspecific binding with 500mM NaCl IP buffer, and performing Western Blot detection; as shown in fig. 3, all 6 nanobodies bound to VEGF antigen, but the degree of binding was greatly different; wherein VE12 and candidate E molecules bind most strongly to human VEGF antigen, but candidate E does not bind to mouse VEGF, and subsequent animal model tests cannot be performed, VE12 is the best nanobody in the invention.

Example 4 detection of nanobody VE12 based on surface plasmon resonance technique, affinity of fusion protein 2VE12 with VEGF antigen

The kinetic constants and affinity parameters of nanobody VE12 and fusion protein 2VE12 and VEGF were determined using Biacore X100. VEGF is used as a ligand and is immobilized on the surface of SERIES CHIP CM chips through amino coupling, HEB-EP+1mg/mL DSS (dextran sulfate sodium is used as a mobile phase); the nanobody is an analyte, and a response value (respond unit, RU) of the instrument is obtained through the surface of the chip; after analyte injection, the instrument response value rises, and the antibody binds to the equilibrium state, thereby obtaining a binding rate constant Kon; after the injection of the analyte, the mobile phase flows through the surface of the chip, and the antibody is dissociated to an equilibrium state, so that the dissociation rate constant Koff is obtained; koff/Kon gets the dissociation attempt KD of the antibody and antigen; in the multi-cycle mode, the combination of antibodies with different concentrations and chip surface antigens is detected, 0.5% SDS is used as a regeneration buffer solution, and after each round of detection is completed, the regeneration buffer solution flows through the chip surface for regeneration and then is subjected to the next round of analysis. As shown in FIG. 4, in HEB-EP+1mg/ml DSS solution, the affinity constant of the antibody VE12 and VEGFA-165 is 0.31 mu M, the affinity constant of the antibody 2VE12 and VEGFA-165 is 70.3 nM, and affinity detection shows that the candidate nano antibody molecules have stronger affinity with antigen protein molecules.

EXAMPLE 5 injection of nanobody VE12 and fusion protein 2VE12 for inhibiting laser-induced mouse choroidal neovascularization

Step 1: since Choroidal Neovascularization (CNV) is one of the important features of AMD, a laser induced CNV mouse model was first constructed:

a. selecting 6 to 8 weeks of healthy C57 mice, intraperitoneally injecting 0.3mL of anesthetic (i.e., 1.25% avermectin solution) and waiting for 3-5 minutes of anesthesia;

b. One drop of compound topiramate eye drops is dropped on the right eye of an anesthetized mouse to carry out pupil expansion treatment, and one drop of obucaine hydrochloride eye drops is dropped after about 1-3 minutes;

c. Setting laser photocoagulation instrument parameters:

pulse power: 150-200mw，duration: 50ms，interval: 300ms

e. Placing the mouse at a proper position in front of a microscope, adjusting the focal length of the microscope until eyes of the mouse are clearly seen, slightly attaching a fundus laser mirror to the eyes of the mouse, finely adjusting the eyes of the mouse to see the fundus, lasing for 3 times, and taking bubbles generated at the photocoagulation position as marks for breaking through Bruch membranes;

f. smearing tobramycin dexamethasone eye ointment on both eyes of the mice to prevent infection;

Step 2: OCT (A) detection

A. Preheating an OCT instrument of a Heidelberg ophthalmic diagnostic instrument in advance;

b. the mice were anesthetized and pupil expanded as above;

c. Placing the mouse in a suitable position in front of the instrument;

d. opening the Heidelberg software, and starting an OCT mode after creating a file;

e. adjusting the focal length, and obtaining an OCT image after fine tuning to see the fundus blood vessel clearly;

f. Switching to OCTA mode, setting a visual field frame, adjusting focal length, and obtaining OCTA images after fine tuning to see fundus blood vessels;

g. double-click images, namely selecting OCTA, adjusting the thickness until the new blood vessels at the laser spots are the clearest and other impurities are less, and storing the images and data;

Step 3: preparation of injectable medicament

Construction of pLVX-VE 12 (or 2VE 12) -6 XHis plasmid transfected HEK293F cells, 60-72 hours after transfection, cell supernatants were harvested, purified on AKTA using a HisTrap excel 5mL affinity column, the eluate was concentrated by ultrafiltration, then further purified using a molecular sieve Superdex ™ 75 investment 10/300 GL equilibrated with PBS, single fractions were collected, antibody products were quantified using NanoDrop OneC (protein A205 mode was selected), antibody was diluted using PBS to final concentrations of 5mg/mL or 10mg/mL.

Step 4: glass cavity medicine injection

A. the mice were anesthetized and pupil expanded as above;

c. Placing the mouse at a proper position under a microscope, fixing the head of the mouse by the left hand to enable the eyes of the mouse to protrude, dipping iodophor with a cotton swab to wipe the eyes of the mouse, and observing the cornice limbus under the microscope;

d. Taking a 0.3mm syringe, sterilizing with alcohol, avoiding blood vessels 1mm below the corneosclera, vertically inserting a needle into a needle surface, punching, holding the microinjector to insert the needle from the hole, injecting 3 mu L of medicine, and observing the intraocular pressure under a microscope; the experimental group was injected with nanobody or fusion protein, and the control group was injected with Ranibizumab.

E. smearing tobramycin dexamethasone eye ointment on both eyes of a mouse to prevent infection, placing the mouse on a heating pad, and placing a mouse frame after waking;

step 5: the second OCT (A) detection is performed as above;

As shown in fig. 5, nanobody VE12 and double nanobody fusion protein 2VE12 were able to effectively inhibit laser-induced mouse choroidal neovascularization. Under the same injection dosage, the inhibition effect is equal to or better than that of the ranibizumab. Wherein, the grouping neovascular signal of the ranibizumab is reduced by about 16.2 percent on average, the grouping signal of the VE12 nano antibody is reduced by 15.1 percent (the effect is similar to that of the ranibizumab), and the grouping signal of the 2VE12 nano antibody is reduced by about 23.7 percent, and the effect is obviously better than that of the ranibizumab.

The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.

Claims

1. A nanobody that specifically binds to VEGF, wherein the nanobody specifically binds to human or mouse VEGF to block the activity of the VEGF, and wherein the nanobody has an amino acid sequence as shown in SEQ ID No. 1 or a sequence having one or more amino acid substitutions, deletions or additions as compared to SEQ ID No. 1.

2. A nanobody which specifically binds to VEGF according to claim 1 wherein the complementarity determining regions of said nanobody comprise CDR1, CDR2, CDR3, wherein CDR1 has the amino acid sequence of SEQ ID No. 2, CDR2 has the amino acid sequence of SEQ ID No. 3, and CDR3 has the amino acid sequence of SEQ ID No. 4.

3. The nanobody of claim 2, wherein the framework region of the nanobody comprises FR1, FR2, FR3, and FR4, wherein the amino acid sequence of FR1 is shown in SEQ ID No.5, the amino acid sequence of FR2 is shown in SEQ ID No.6, the amino acid sequence of FR3 is shown in SEQ ID No. 7, and the amino acid sequence of FR4 is shown in SEQ ID No. 8.

4. The nanobody of claim 1, wherein n nanobodies are linked directly or via a linker sequence to form a fusion protein, wherein n.gtoreq.2.

5. A pharmaceutical composition comprising the nanobody of any of claims 1-4 and a pharmaceutically acceptable excipient, carrier or diluent.

6. The use of the nanobody according to any one of claims 1 to 4 for the preparation of a medicament for the treatment of diseases related to blocking VEGF signaling.

7. The use according to claim 6, wherein the disease associated with blocking VEGF signaling is age-related macular degeneration.