An anti-RBD single-chain antibody No. 35 and its use in preparing antibody-drug conjugates

Abstract

The present invention provides an anti-RBD single-chain antibody No.35 and the use of preparing an antibody-drug conjugate, wherein the DNA sequence of the anti-RBD single-chain antibody No.35 is shown in SEQ ID No.1, and the anti-RBD single-chain antibody No.35 comprises complete heavy chain and light chain variable region sequences, and can specifically bind to RBD. The single-chain antibody No.35 is connected to the N-glycosylation inhibitor tunicamycin (TM) through glutaraldehyde, binds to the RBD region of the new coronavirus S protein, is internalized by a cell line expressing ACE2, and the release of the small molecule drug tunicamycin can inhibit the glycosylation of the S protein and ACE2, block the binding of the new coronavirus to the receptor, and inhibit the further invasion of the coronavirus into the cell, thereby affecting the infection and proliferation of the progeny virus. It can be used for the development of coronavirus-targeted therapeutic antibody drugs, especially for the preparation of antibody-drug conjugates that inhibit new coronavirus infection.

Description

Technical Field

[0001] The present invention belongs to gene and antibody engineering, and relates to an anti-RBD single-chain antibody No. 35 and its use in preparing antibody-drug conjugates. It includes the synthesis of an anti-receptor-binding domain (RBD) single-chain antibody No. 35 and a tunicamycin drug conjugate and analysis of its antiviral effect, demonstrating the application prospect of ADC-35 as a targeted anti-coronavirus therapeutic drug. Background Art

[0002] Coronaviruses are a large family of viruses found widely in nature. They possess an envelope structure and are classified into four genera: alpha, beta, gamma, and delta coronaviruses. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is composed of four major structural proteins: the spike protein (S protein), the nucleocapsid protein (N protein), the membrane protein (M protein), and the envelope protein (E protein). The S protein mediates fusion between the virus and the cell membrane, participating in viral invasion and binding, and is a popular target for studying the pathogenicity of SARS-CoV-2. The S protein consists of the S1 and S2 subunits. The S1 subunit comprises the receptor-binding domain (RBD) and the N-terminal domain (NTD). The RBD interacts with host cell ACE2. The S2 subunit primarily mediates the fusion of the virus with the host cell membrane and releases viral RNA into the cytoplasm for replication. Currently, therapeutic antibodies and vaccines targeting the S protein or RBD protein have demonstrated good safety and protective properties.

[0003] Glycoproteomics is of great significance in virus identification, immunology, and drug discovery, and is an emerging frontier in the search for biomarkers. Glycosylated proteins are involved in viral invasion, the hydrolysis of virus-associated cellular receptor proteins, and viral recognition and neutralization by the host immune system. Studies have reported that pathogens use glycosylation to evade recognition by the host immune system, potentially interfering with host adaptive immunity and even enhancing viral infectivity. Glycosylation can be primarily categorized as N-glycosylation and O-glycosylation. SARS-CoV-2 invades cells via a highly glycosylated spike protein. The S protein has 22 N-glycosylation sites, and the loss of double glycosylation at sites N331 and N343 can dramatically reduce viral infectivity by 1200-fold. Seven sites on the hACE2 molecule are N-glycosylated, with N-glycosylation at residues N90, N322, and N546 playing a crucial role in ACE2 binding to the RBD. These findings demonstrate the crucial role of N-glycosylation in viral infection. Intervening in the N-glycosylation modification of ACE2 or S protein can block the binding of the virus to the receptor, inhibit the synthesis and release of progeny viruses, and is an effective antiviral solution. Summary of the Invention

[0004] One of the objectives of the present invention is to provide an anti-RBD single-chain antibody No. 35, which is screened from a murine phage single-chain antibody library and is capable of specifically binding to RBD. The DNA sequence of the anti-RBD single-chain antibody No. 35 is shown in SEQ ID No. 1:

[0005] CAGGTGCAACTGCAGGAGTCTGGGGCTGAACTGGCAAGACCTGGGGCCTCAGTGAAGA

[0006] TGTCCTGCAAGGCTTCTGGCTACACCTTTACTAGGTACACGATGCACTGGGTAAAACAG

[0007] AGGCCTGGACAGGGTCTGGAATGGATTGGATACATTAATCCTAGCCGTGGTTATAACTAAT

[0008] TACAATCAGAAGTTCAAGGACAAGGCCACATTGACTACAGACAAATCCTCCAGCACAG

[0009] CCTACATGCAACTGAGCAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAGAT

[0010] ATTATGATGATCATTACTGCCTTGACTACTGGGGCCAAGGGACCACGGTCACCGTCTCCT

[0011] CAGGTGGAGGCGGTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCGGACATTGAGCT

[0012] CACCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAGAAGGTCACCATGACCTGCA

[0013] GTGCCAGCTCAAGTGTAAGTTACATGTACTGGTACCAGCAGAAGTCAGATGCCTCCCCC

[0014] AAACTATGGATTTATTACACATCCAACCTGGCTCCTGGAGTCCCAGCTCGCTTCAGTGGC

[0015] AGTGGGTCTGGGAACTCTTATTCTCTCACAATCAGCAGCATGGAGGGTGAAGATGCTGC

[0016] CACTTATTACTGCCAGCAGTTTACTAGTTCCCCATTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAA。

[0017] The amino acid sequence of the anti-RBD single-chain antibody No. 35 is shown in SEQ ID No. 2: QVQLQESGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIELTQSPAIMSASPGEKVTMTCSASSSVSYMYWYQQKSDASPKLWIYYTSNLAPGVPARFSGSGSGNSYSLTISSMEGEDAATYYCQQFTSSPFTFGSGTKLEIK.

[0018] The anti-RBD single-chain antibody No. 35 contains a complete antibody heavy chain variable region VH and light chain variable region VL, wherein the amino acid sequence of the heavy chain variable region VH CDR1 is GYTFTRYTMH (SEQ ID No. 3), the amino acid sequence of the heavy chain variable region VH CDR2 is YINPSRGYTNYNQKFKD (SEQ ID No. 4), and the amino acid sequence of the heavy chain variable region VH CDR3 is YYDDHYCLDY (SEQ ID No. 5); the amino acid sequence of the light chain variable region VL CDR1 is SASSVSYMY (SEQ ID No. 6), the amino acid sequence of the light chain variable region VL CDR2 is YTSNLAP (SEQ ID No. 7), and the amino acid sequence of the light chain variable region VL CDR3 is QQFTSSPFT (SEQ ID No. 8).

[0019] In addition, another object of the present invention is to provide the use of the anti-RBD single-chain antibody No. 35 in the preparation of an antibody-drug conjugate for inhibiting novel coronavirus infection. The drug is an antibody-drug conjugate that can target coronaviruses by synthesizing an anti-RBD single-chain antibody No. 35 and an N-glycosylation inhibitor tunicamycin (TM) through a glutaraldehyde linker under certain conditions. This antibody-drug conjugate can specifically target coronaviruses. After the virus binds to ACE2 on the cell surface and enters the host cell, the antibody-drug conjugate releases tunicamycin, inhibiting the action of dolichyl-phosphate N-acetylglucosamine phosphotransferase 1 (DPAGT1) in the endoplasmic reticulum, blocking the glycosylation of coronavirus S protein and ACE2, thereby inhibiting the infection of N-glycosylated coronaviruses.

[0020] The antibody-drug conjugate includes the anti-RBD single-chain antibody No. 35 itself and its variable region sequence, the variable region sequence including the amino acid sequence of the heavy chain variable region VH CDR1, CDR2, and CDR3, and the amino acid sequence of the light chain variable region VL CDR1, CDR2, and CDR3; it also includes the small molecule drug tunicamycin and its antibody-drug conjugate ADC-35 synthesized under certain conditions.

[0021] The preparation method of the RBD-targeting antibody-drug conjugate ADC-35 described herein is achieved by the following steps: First, 2.4 mg of TM was dissolved in 1 mL of anhydrous pyridine, followed by the addition of 0.9 mg of glutaric anhydride. After dissolution, the solution was shaken at 37°C and 220 rpm for 24 hours. 4 mL of anhydrous ether was added to the solution, and the synthesized product precipitated. The solution was centrifuged at 5000 rpm for 3 minutes, the supernatant discarded, and the product was washed twice with anhydrous ether. The precipitate was dissolved in 100 μl of DMF, and 2.4 mg of NHS and 1.2 mg of DCC were added to the solution. The product precipitated as white crystals, centrifuged at 5000 rpm for 3 minutes, the supernatant discarded, and the product was washed twice with anhydrous ether. 2 mg of purified anti-RBD single-chain antibody was added, and the solution was reacted at 4°C for 16 hours. Unreacted small molecules were then removed by ultrafiltration using a 10 kDa ultrafiltration tube. The synthesis of ADC-35 was verified by SDS-PAGE and mass spectrometry.

[0022] The anti-RBD single-chain antibody No. 35 of the present invention contains complete heavy chain and light chain variable region sequences and can specifically bind to RBD. In the antibody-drug conjugate, the single-chain antibody No. 35 is connected to the N-glycosylation inhibitor tunicamycin (TM) through glutaraldehyde. The antibody-drug conjugate ADC-35 can bind to the RBD region of the new coronavirus S protein, and through endocytosis by cell lines expressing ACE2, the small molecule drug tunicamycin is released to inhibit the glycosylation of the S protein and ACE2, thereby affecting the infection and proliferation of progeny viruses. The antibody-drug conjugate ADC-35 provided by the present invention can specifically bind to RBD, enter cells to inhibit protein glycosylation, block the binding of the new coronavirus to the receptor, and inhibit the further invasion of the coronavirus into the cell, and can be used for the development of coronavirus-targeted therapeutic antibody drugs. The beneficial effects of the present invention are: (1) The reagents used in the present invention are simple and easy to obtain, low in price, and can quickly screen single-chain antibodies that specifically bind to the RBD protein, with strong binding ability and high affinity; (2) The antibody-drug conjugate obtained by the present invention binds to the new coronavirus and enters the receptor cell to exert a deglycosylation effect, reducing the toxicity of tunicamycin to normal cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the SDS-PAGE electrophoresis of ADC-35, an antibody-drug conjugate targeting RBD ( Figure 1 A) and mass spectrometry analysis ( Figure 1 B).

[0024] Figure 2 It is a binding analysis of the RBD-targeted antibody-drug conjugate ADC-35.

[0025] Figure 3 This is an analysis of the cellular endocytosis of the RBD-targeting antibody-drug conjugate ADC-35.

[0026] Figure 4 This is an analysis of the inhibitory effect of RBD-targeted antibody-drug conjugate ADC-35 on HEK 293A-ACE2 and HEK 293-Spike cell fusion.

[0027] Figure 5 Analysis of the killing effect of RBD-targeted antibody-drug conjugate ADC-35 on Vero E6 cells ( Figure 5 A) and analysis of anti-SARS-CoV-2 virus infection effect ( Figure 5 B).

[0028] Figure 6 This is an analysis of the inhibitory effect of RBD-targeted antibody-drug conjugate ADC-35 on the killing of SARS-CoV-2 virus. DETAILED DESCRIPTION

[0029] The present invention is further described with reference to the following embodiments and accompanying drawings.

[0030] Example 1: Synthesis of RBD-targeted antibody-drug conjugate ADC-35 and SDS-PAGE electrophoresis and mass spectrometry analysis

[0031] Experimental Methods: First, dissolve 2.4 mg of TM in 1 ml of anhydrous pyridine, followed by the addition of 0.9 mg of glutaric anhydride. After dissolution, incubate at 37°C, 220 rpm, and shake for 24 hours. Add 4 ml of anhydrous ether to the solution, precipitate the synthesized product, centrifuge at 5000 rpm for 3 minutes, discard the supernatant, and wash twice with anhydrous ether. Dissolve the precipitate in 100 μl of DMF, add 2.4 mg of NHS and 1.2 mg of DCC, and shake at 37°C, 220 rpm, for 24 hours. Add 4 ml of anhydrous ether to the solution, precipitate the synthesized product as white crystals, centrifuge at 5000 rpm for 3 minutes, discard the supernatant, and wash twice with anhydrous ether. Add 2 mg of purified antibody and react at 4°C for 16 hours. Ultrafiltration through a 10 kDa ultrafiltration tube removes unreacted small molecules. Synthesis was confirmed by SDS-PAGE and mass spectrometry.

[0032] Experimental results: SDS-PAGE protein gel results showed that the molecular weight of the antibody-drug conjugate was higher than that of the single-chain antibody, and TM was successfully connected to the antibody ( Figure 1 A). Mass spectrometry results showed that the antibody-drug conjugate had two peaks compared to the anti-RBD single-chain antibody No. 35, proving that the anti-RBD single-chain antibody No. 35 was successfully linked to the TM ( Figure 1 B).

[0033] Example 2: Binding Analysis of RBD-Targeted Antibody-Drug Conjugate ADC-35

[0034] Experimental Methods: Recombinant RBD-Fc protein was plated onto an ELISA plate at 1 μg / well and incubated at 4°C overnight. Antiviral drug conjugates and anti-RBD single-chain antibody No. 35 were diluted to varying concentrations and added to the ELISA plate at 100 μl / well. Primary and secondary antibodies were added to detect binding of the antiviral drug conjugates and single-chain antibody to the recombinant RBD-Fc protein.

[0035] Experimental results: There is no significant difference in the binding of anti-RBD single-chain antibody No. 35 and antibody-drug conjugate ADC-35 to RBD-Fc, indicating that the connection of anti-RBD single-chain antibody No. 35 to TM does not affect its binding to RBD ( Figure 2 ).

[0036] Example 3: Analysis of endocytosis of RBD-targeted antibody-drug conjugate ADC-35

[0037] Experimental method: HEK 293-Spike cells were plated in 12-well plates and cultured overnight. ADC-35 was added and incubated for different time points. Cells were trypsinized and centrifuged at 1,000 rpm for 5 minutes to collect the cell pellet. 3 x 10 5 Cells / tube were aliquoted into 1.5 ml centrifuge tubes, with two replicates for each group of cells; washing: cells were washed with 2% FBS-PBS, centrifuged at 1,000 rpm for 5 minutes, and this step was repeated three times; incubation with primary antibody: anti-mouse His monoclonal antibody diluted in PBS (1:400 dilution) was added and incubated on ice for 1 hour; washing: cells were washed with 2% FBS-PBS, centrifuged at 1,000 rpm for 5 minutes, and this step was repeated three times; incubation with fluorescent antibody: anti-mouse fluorescent secondary antibody labeled with Alexa Fluor 647 diluted in PBS was added (1:500 dilution) and incubated on ice in the dark for 1 hour; flow cytometer detection: cells were resuspended in 300 μl PBS, filtered through a 300-mesh cell sieve, and detected on an ACEA NovoCyte™ flow cytometer.

[0038] Experimental results: ADC-35 endocytosis was detected at 0, 2, 4 and 8 hours. ADC-35 was internalized at 2 hours and the fluorescence intensity decreased. The endocytosis at 8 hours was more significant than that at 0 hours ( Figure 3 ).

[0039] Example 4: Analysis of the Inhibition of HEK 293A-ACE2 and HEK 293-Spike Cell Fusion by RBD-Targeted Antibody-Drug Conjugate ADC-35

[0040] Experimental method: HEK 293-Spike cells were plated in confocal culture dishes with an inoculum size of 5 x 10 cells per well. 4After 12 hours, add 0.02 μM ADC-35 to the culture dish after the cells adhere to the wall and continue to culture for 24 hours. Replace the culture medium with fresh medium and add HEK 293A-ACE2 cells to each dish. The inoculation volume is 2 x 10 4 After 48 hours, cell fusion was observed under a laser confocal microscope. Using a 20x objective lens, five fields of view were randomly selected, and the number of fused nuclei in each field of view was counted. Graphpad Prime was used to generate the graph.

[0041] Experimental results: The control group observed a more obvious cell fusion phenomenon, and the ADC-35 group had a smaller number of cell fusions. Compared with the control group, the ADC-35 group inhibited the cell fusion rate by 75% ( Figure 4 ).

[0042] Example 5: Analysis of the killing effect of RBD-targeted antibody-drug conjugate ADC-35 on Vero E6 cells and its anti-SARS-CoV-2 virus infection effect.

[0043] Experimental Methods: Different concentrations of antibody-drug conjugate ADC-35 were added to Vero E6 cells to test their cytotoxicity, and cell viability was determined by CCK8 assay. Vero E6 cells were pre-incubated with different concentrations of antibody-drug conjugate ADC-35 for 6 hours, followed by the addition of 100 TCID50 of SARS-CoV-2 and incubation at 37°C. After 72 hours, the cell supernatant was collected and the viral load was measured.

[0044] Experimental results: ADC-35 had no obvious toxic side effects on E6 cells at a concentration of 0-4nM, and CC 50 56.55nM( Figure 5 A); ADC-35 can significantly inhibit viral replication at a concentration of 4 nM, EC 50 0.05nM( Figure 5 B).

[0045] Example 6: Analysis of the inhibitory effect of the RBD-targeted antibody-drug conjugate ADC-35 on SARS-CoV-2 virus killing

[0046] Experimental method: Different concentrations of antibody-drug conjugate ADC-35 were added to Vero E6 cells and pre-incubated for 6 hours. Then 100 TCID50 of the new coronavirus was added and cultured at 37 degrees. After 72 hours, the cell field of view was selected for photography.

[0047] Experimental results: ADC-35 at a concentration of 4nM can significantly inhibit the killing effect of the virus on cells, and the cell viability suppressed by the virus is restored ( Figure 6 ).

[0048] For ordinary technicians in this field, the specific embodiments are only illustrative descriptions of the present invention. It is obvious that the specific implementation of the present invention is not limited to the above-mentioned methods. As long as various non-substantial improvements are made using the method concepts and technical solutions of the present invention, or the concepts and technical solutions of the present invention are directly applied to other occasions without improvement, they are all within the scope of protection of the present invention.

Claims

1. An anti-RBD single-chain antibody No. 35, characterized in that The DNA sequence of the anti-RBD single-chain antibody No. 35 is shown in SEQ ID No. 1, and the amino acid sequence thereof is shown in SEQ ID No.

2.

2. The anti-RBD single-chain antibody No. 35 according to claim 1, characterized in that: The anti-RBD single-chain antibody No. 35 contains a complete antibody heavy chain variable region VH and light chain variable region VL, the amino acid sequence of the heavy chain variable region VH CDR1 is shown in SEQ ID No. 3, the amino acid sequence of the heavy chain variable region VH CDR2 is shown in SEQ ID No. 4, and the amino acid sequence of the heavy chain variable region VHCDR3 is shown in SEQ ID No. 5; The amino acid sequence of the light chain variable region VL CDR1 is shown in SEQ ID No. 6, the amino acid sequence of the light chain variable region VL CDR2 is shown in SEQ ID No. 7, and the amino acid sequence of the light chain variable region VL CDR3 is shown in SEQ ID No.

8.

3. Use of the anti-RBD single-chain antibody No. 35 according to claim 1 or 2 in the preparation of an antibody-drug conjugate for inhibiting novel coronavirus infection.

4. The use according to claim 3, characterized in that The drug is an antibody-drug conjugate targeting the new coronavirus, synthesized by combining the anti-RBD single-chain antibody No. 35 and the N-glycosylation inhibitor tunicamycin through a glutaraldehyde linker.

5. The use according to claim 3, characterized in that The novel coronavirus is an N-glycosylated coronavirus.

6. The use according to claim 3, characterized in that The application is that the conjugated drug can specifically target the new coronavirus. After the virus binds to ACE2 on the cell surface, it enters the host cell. The conjugated drug releases tunicamycin, inhibiting the action of dolichol phosphate-N-acetylglucosamine phosphotransferase 1 in the endoplasmic reticulum, blocking the glycosylation of the new coronavirus S protein and ACE2, thereby inhibiting the infection of N-glycosylated new coronavirus.

Images