A high-throughput screening method for cross-reactive antibodies and its application

Abstract

The present invention provides a high-throughput screening method for cross-reactive antibodies, comprising the following steps: step 1, constructing an antibody gene library, and obtaining a complex for displaying antibodies based on the antibody gene library; step 2, using at least two antigen display systems to screen the complexes in sequence, each antigen display system displaying different target antigen molecules on its surface, so as to screen out target complexes that can bind to different target antigen molecules at the same time; step 3, obtaining the gene information of the target antibody according to the target complex. The present invention displays a variety of target antigen molecules on the surface of artificial lipid droplets as an antigen display system for multiple rounds of antibody screening, which helps to efficiently, quickly and economically screen out broad-spectrum antibodies with cross-reactivity.

Description

Technical Field

[0001] The invention relates to a high-throughput screening method for cross-reactive antibodies and application thereof, and relates to the field of biotechnology. Background Art

[0002] Clinical intervention with genetically engineered antibodies is very effective in preventing and treating viral infections. Since there are many types of viruses and they have extremely high mutation rates, they will produce a variety of mutants. It is very challenging to screen antibodies for all viruses and mutants one by one, and the speed of screening specific antibodies is often slower than the speed of virus mutation. Therefore, there is an urgent need in this field to develop broad-spectrum antibodies with high affinity that can well target various viruses and their mutants. Summary of the invention

[0003] The present invention provides a high-throughput screening method for cross-reactive antibodies, which is used for screening high-affinity broad-spectrum antibodies targeting various viruses and their mutants.

[0004] The present invention also provides the application of the above-mentioned screening method in the screening of broad-spectrum antibodies targeting the new coronavirus and its mutant strains.

[0005] The first aspect of the present invention provides a high-throughput screening method for cross-reactive antibodies, comprising the following steps:

[0006] Step 1, constructing an antibody gene library, and obtaining a complex for displaying antibodies based on the antibody gene library;

[0007] Step 2, using at least two antigen display systems to screen the complexes in sequence, each antigen display system displaying different target antigen molecules on its surface, so as to screen out target complexes that can bind to different target antigen molecules at the same time;

[0008] Step 3, obtaining the gene information of the target antibody according to the target complex;

[0009] The antigen display system comprises artificial lipid droplets and amphiphilic target antigen molecules; the artificial lipid droplets are assembled into particles having a hydrophilic surface and a hydrophobic core in a hydrophilic medium, the amphiphilic target antigen molecules are target antigen molecules modified with hydrophobic groups, and the target antigen molecules are displayed on the hydrophilic surface of the artificial lipid droplets.

[0010] Based on the specificity of antibody and antigen binding, the present invention first establishes an antibody gene library, and uses conventional antibody library screening technology in the art to construct a complex for displaying antibodies; secondly, the complex displaying antibodies is incubated with an antigen display system displaying different target antigen molecules to screen out antibodies with cross-activity to multiple target antigen molecules; finally, the gene information of cross-active antibodies can be obtained according to conventional technical means in the art. It is understandable that different target antigen molecules can be derived from at least two of the original strains of the virus and its mutants, specifically the receptor binding domain (RBD) on the surface of the virus and its mutants.

[0011] Streptavidin magnetic beads are commonly used in the art for antigen display. However, streptavidin magnetic beads have strong adsorption and streptavidin is fixed on the surface, so non-specific binding will occur during the interaction between the antibody displayed on the surface of the ribosome and the target antigen bound to the surface of the magnetic beads. This non-specific binding will result in false positives, which will seriously affect the accuracy of antibody screening, causing some antibodies that do not interact with the target protein to be mistakenly screened out, thereby affecting the screening efficiency and antibody specificity. Therefore, the present invention provides a method such as Figure 1 The antigen display system shown includes an artificial lipid droplet 1 and an amphiphilic target antigen molecule 2; wherein the artificial lipid droplet 1 is formed by a surfactant in a hydrophilic medium. Since the surfactant has a hydrophilic end and a hydrophobic end, it can form particles with a hydrophilic surface and a hydrophobic core in the hydrophilic medium; the amphiphilic target antigen molecule 2 is formed by hydrophobic modification of the target antigen molecule 21. The hydrophilic target antigen molecule 21 is displayed on the hydrophilic surface of the artificial lipid droplet 1 to facilitate the binding with the antibody during the antibody screening process. The hydrophobic end is located inside the artificial lipid droplet 1, so that the target antigen molecule 21 is closely displayed on the surface of the artificial lipid droplet 1. In the scheme of the present invention, the hydrophilic medium can be pure water.

[0012] The present invention uses artificial lipid droplets to replace streptavidin magnetic beads, which helps to reduce the nonspecific binding to the target antigen molecules during the antibody screening process and improve the specificity and screening efficiency of the target antibody; and the display system provided by the present invention simulates the spatial structure of the virus to a great extent, but does not contain genetic material, which is safer and more effective; in addition, those skilled in the art can accurately control the parameters of its structure, function, etc. by controlling the size of the artificial lipid droplets, the number and type of antigens displayed on the surface, and thus have higher controllability. The present invention displays a variety of target antigen molecules on the surface of artificial lipid droplets as an antigen display system for multiple rounds of antibody screening, which helps to efficiently, quickly and economically screen out broad-spectrum antibodies with cross-activity.

[0013] In a specific embodiment, the preparation method of the antigen display system provided by the present invention comprises:

[0014] Modifying the target antigen molecule with hydrophobic groups to obtain an amphiphilic target antigen molecule;

[0015] The surfactant and the amphiphilic target antigen molecules are mixed and dispersed in a hydrophilic medium to form the antigen display system.

[0016] First, the present invention provides two methods for modifying target antigen molecules with hydrophobic groups. In a specific embodiment: poly(N-isopropylacrylamide) is activated using mercaptothiazoline or N-hydroxysuccinimide, the activated poly(N-isopropylacrylamide) is mixed with the target antigen molecule and reacted, and lysine is added to terminate the reaction to obtain an amphiphilic target antigen molecule.

[0017] Furthermore, the molar ratio of the activated poly(N-isopropylacrylamide) to the target antigen molecule was 2:1, the pH of the reaction system was controlled to be 8.0, and after reacting at 4°C for 12 hours, lysine was added to terminate the reaction.

[0018] Furthermore, the number average molecular weight of poly(N-isopropylacrylamide) is 8000-15000 g / mol. Controlling the molecular weight of the polymer helps to balance the hydrophilicity and hydrophobicity of the antigen-polymer, thereby allowing the antigen molecules to be better displayed on the surface of the artificial lipid droplets.

[0019] In another specific embodiment, the target antigen molecule is prenylated to obtain an amphiphilic target antigen molecule; specifically, the target antigen molecule can be modified using farnesyltransferase (FTase) or geranylgeranyltransferase (GGTase) to form a hydrophobic group at the C-terminus of the target antigen molecule; this can be done specifically according to conventional technical means in the art.

[0020] Secondly, the surfactant is mixed with the prepared amphiphilic target antigen molecules and dispersed in a hydrophilic medium to form the antigen surface display system. Furthermore, the artificial lipid droplets are assembled by tributyrin in a hydrophilic medium; the density of tributyrin is greater than that of water, which is conducive to the artificial lipid droplets formed by the assembly sinking to the bottom of the water medium, which is conducive to the enrichment of artificial lipid droplets and prevents the artificial lipid droplets from being suspended at the interface between air and water to inactivate the antigen molecules.

[0021] In a specific embodiment, in order to facilitate the recovery of the magnetic enrichment and display system, the particles assembled by the artificial lipid droplets in the hydrophilic medium are wrapped with magnetic nanoparticles 3. The magnetic nanoparticles are highly hydrophobic and can be dispersed in the hydrophobic interior of the artificial lipid droplets formed by tributyrin.

[0022] Furthermore, the magnetic nanoparticles may be Fe3O4 nanoparticles, and the mass of the magnetic nanoparticles is 1%-2% of the mass of tributyrin.

[0023] Taking the screening of antibodies with cross-reactivity to the new coronavirus SARS-CoV-2 and its mutants as an example, the screening method specifically includes the following steps:

[0024] Step 1: construct an antibody gene library, and obtain a complex for displaying antibodies based on the antibody gene library.

[0025] In the scheme provided by the present invention, the antibody can be a ScFv antibody. According to the formation mechanism of the antibody in vivo, mutations are introduced into the hypermutation region to construct an antibody gene library. Further, the hypermutation region includes CDR3.

[0026] Taking ribosome display technology as an example, the antibody gene also includes a TonB group, which is located at the 3' end of the antibody gene to improve the binding stability of the complex formed during mRNA translation.

[0027] In the solution provided by the present invention, the 5' and 3' ends of the antibody gene are also connected to a stem-loop structure, and the 5' end is also connected to a promoter and a ribosome binding site RBS.

[0028] The created antibody gene is transcribed into mRNA in vitro, and the mRNA is translated in an in vitro expression system rich in ribosome cell extract fluid to form an "mRNA-ribosome-polypeptide" complex.

[0029] Step 2: Use at least two antigen display systems to screen the complexes in sequence, with each antigen display system displaying different target antigen molecules on its surface, so as to screen out target complexes that can bind to different target antigen molecules simultaneously.

[0030] Taking the new coronavirus SARS-CoV-2 and its mutants Omicron, Gamma, and Alpha as examples, first, the RBD on the surface of the virus and its mutants was used as the target antigen molecule, and four antigen display systems were prepared after modification with hydrophobic groups; secondly, the first antigen display system displaying SARS-CoV-2-RBD was used to perform the first round of screening on the antibody gene library. After the screening, the mRNA on the complex that could bind to the first antigen display system was released, and cDNA was obtained by reverse transcription. After connecting the promoter, the antibody gene library for the second round of screening was obtained; then, the Omicron -RBD's second antigen display system was used to perform a second round of screening on the antibody gene library obtained in the first round of screening, following the same method as after the first round of screening; then, the third antigen display system displaying Gamma-RBD was used to perform a third round of screening on the antibody gene library obtained in the second round of screening; then, the fourth target antigen artificial lipid droplet display system displaying Alpha-RBD was used to perform a fourth round of screening on the antibody genes obtained in the third round of screening, and finally, the target complex that could bind to the four target antigens SARS-CoV-2-RBD, Omicron-RBD, Gamma-RBD, and Alpha-RBD was obtained.

[0031] It is understandable that the target antigen molecules and their sequence during the four rounds of screening can be adjusted according to conventional techniques in the art.

[0032] Step 3: obtaining the gene information of the target antibody according to the target complex.

[0033] Finally, the gene information of the target antibody can be obtained according to the target complex, and the target antibody can be analyzed and identified by methods such as enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR).

[0034] The present invention provides a method for high-throughput screening of broad-spectrum antibodies with cross-activity, wherein an antibody gene library is transcribed and translated in vitro to form an "mRNA-ribosome-antibody" complex, and the complex is incubated with an artificial lipid droplet display system displaying different target antigen molecules, and at least two rounds of screening are performed to obtain broad-spectrum antibodies with cross-activity, thereby solving the problem that the current cross-activity antibody screening method cannot perform fast, simple, and high-throughput screening, thereby causing high screening costs.

[0035] It is understandable that in addition to being used for broad-spectrum antibody screening of the new coronavirus and its mutant strains, the present invention can also be used for screening other types of viruses, cancers, bacteria, etc.

[0036] The present invention displays a variety of target antigen molecules on the surface of artificial lipid droplets as an antigen display system for multiple rounds of antibody screening, which helps to efficiently, quickly and economically screen out broad-spectrum antibodies with cross-activity; at the same time, the artificial lipid droplet antigen display system provided by the present invention helps to reduce nonspecific binding; the screening efficiency is faster and more efficient; it can be reused, is more economical and environmentally friendly; the artificial lipid droplet antigen display system can display different types of antigens; and high-affinity antibodies can be obtained through high-throughput screening. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 A schematic diagram of the structure of the target antigen artificial lipid droplet surface display system provided by the present invention;

[0038] Figure 2 This is a bright field microscope photograph of the target antigen artificial lipid droplet surface display system provided in Example 1 of the present invention;

[0039] Figure 3 This is a fluorescence microscope image of the target antigen artificial lipid droplet surface display system provided in Example 1 of the present invention, with a scale of 50 micrometers;

[0040] Figure 4 is an agarose gel electrophoresis diagram of mRNA in the embodiment of the present invention, M is RNA marker;

[0041] Figure 5 is an agarose gel electrophoresis diagram of the DNA of the antibody gene library recovered after cross-screening in an embodiment of the present invention, M is a DNA marker, 1, 2, 3 and 4 represent the DNA bands of the antibody gene library recovered by the first, second, third and fourth rounds of cross-screening, respectively, using SARS-CoV-2-RBD, Omicron-RBD, Gamma-RBD and Alpha-RBD artificial cells as antigens, respectively;

[0042] Figure 6 Screening of antibodies targeting SARS-CoV-2-RBD, Omicron-RBD, Gamma-RBD and Alpha-RBD with cross-activity by ELISA in the embodiments of the present invention;

[0043] Figure 7 The figure is the SDS-PAGE electrophoresis diagram of the recombinant cross-reactive antibody G8 purified by Ni2+ column in the embodiment of the present invention, M is a protein marker, FT is a flow-through solution, HS is a high salt washing solution, W1, W2, and W3 are three column washings with a buffer solution, E1, E2, E3, and E4 are eluents of different concentrations, and Pooled is the collection of E1, E2, E3, and E4 for the next step of purification;

[0044] Figure 8The SDS-PAGE electrophoresis diagram of the antibody G8 further purified by ion exchange chromatography in the embodiment of the present invention, M is a protein marker, and Pooled means the eluate is collected and subjected to the next step of purification;

[0045] Fig. 9 The affinity chromatography SDS-PAGE electrophoresis diagram of the antibody G8 in the embodiment of the present invention, Pooled is the collected eluate;

[0046] Fig.10 This is the kinetic curve of the antibody G8 with cross-targeting SARS-CoV-2-RBD, Omicron-RBD, Gamma-RBD and Alpha-RBD in the embodiments of the present invention.

[0047] Description of reference numerals:

[0048] 1-artificial lipid droplets;

[0049] 2-amphiphilic target antigen molecule;

[0050] 21-target antigen molecule;

[0051] 3- Magnetic nanoparticles. DETAILED DESCRIPTION

[0052] In order to make the purpose, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0053] The primers used in the examples of the present invention are shown in Table 1:

[0054] Table 1 Nucleotide sequences of primers used in the examples of the present invention

[0055]

[0056]

[0057] Example 1: Amplification of antibody gene library

[0058] The ScFv antibody gene library for screening was amplified by PCR using primers (upstream primer: T7B-F and downstream primer: tonB-ScFv-R). The polymerase used was high-fidelity Q5 DNA enzyme, and the components of PCR are shown in Table 2. The primers used are shown in Table 1.

[0059] Table 2 PCR components

[0060] Components Volume (μL) concentration template 1 0.02-2 ng / μL Upstream primer (10 μM) 0.5 0.5μM Downstream primer (10 μM) 0.5 0.5μM GC enhancer (optional) 5 1x Q5 PCR mix 12.5 - <![CDATA[ddH2O]]> adjust to 25 -

[0061] The PCR program is: 98°C pre-denaturation for 30 seconds, 30 cycles: 98°C denaturation for 10 seconds, 72°C annealing for 20 seconds, 72°C extension for 30 seconds, and final extension for 2 minutes at 72°C. The antibody gene library is usually recovered to 200 ng / μl.

[0062] Example 2: Transcription and translation of antibody gene library

[0063] like Figure 4 As shown, 1 μg of ScFv antibody gene library was in vitro transcribed and purified to obtain mRNA. Then, 10 μg of mRNA was in vitro translated into "mRNA-ribosome-antibody" complex in ribosome-rich Escherichia coli cell extract and stabilized at 4°C.

[0064] Example 3: Construction of an artificial lipid droplet display system for target antigen molecules

[0065] SARS-CoV-2-RBD, Omicron-RBD, Gamma-RBD and Alpha-RBD proteins were modified with polymers to form water-oil amphiphilic molecules, and the antigen molecules were displayed on the surface of artificial lipid droplets.

[0066] Specifically, it includes: reacting SARS-CoV-2-RBD with poly (N-isopropylacrylamide) activated by mercaptothiazoline. Reaction conditions: protein concentration 2 mg / mL; polymer concentration 1 mg / mL; reaction pH = 8.0; reaction temperature 4 degrees Celsius; reaction time 12 hours. After the reaction is completed, a lysine solution with a final concentration of 60 mM is added to terminate the reaction. A two-phase solution is formed by mixing 6 microliters of tributyrin (1% w / w Fe3O4 nanoparticles) and 200 microliters of polymer-modified antigen solution. By manually shaking the mixture for 30 seconds, an antigen-polymer-stabilized artificial lipid droplet emulsion is obtained. Then add 1 mL of buffer for use. The bright field microscopy images and fluorescent images of artificial lipid droplets are shown as follows. Figure 2-3 As shown, according to Figure 3 As shown by fluorescence, the surface of the artificial lipid droplets displays the SARS-CoV-2 RBD protein.

[0067] Using the same method, Omicron-RBD, Gamma-RBD and Alpha-RBD were reacted with poly (N-isopropylacrylamide) activated by mercaptothiazoline, and artificial lipid droplets displaying different RBDs were constructed.

[0068] Example 4: Screening of broad-spectrum antibodies with cross-reactivity

[0069] In the first round of screening, the "mRNA-ribosome-antibody" complex was incubated with SARS-CoV-2-RBD artificial lipid droplets, and non-specific antibody binding was removed by 6 washes. The enriched antibody mRNA was eluted with EDTA solution and cDNA was obtained by reverse transcription. The enriched antibody gene library was recovered by PCR reaction using primers (upstream primer: SDA-ScFv-F and downstream primer: tonB-R). The polymerase used was Taq synthase, and the PCR components are shown in Table 3.

[0070] The T7 promoter was added to the N-terminus by primers (upstream primer: T7B-F and downstream primer: tonB-ScFv-R), and the PCR components and procedures used were the same as above. Then the second, third and fourth rounds of screening were carried out, and Omicron-RBD, Gamma-RBD and Alpha-RBD were used as antigens respectively. The primers used are shown in Table 1. The agarose gel electrophoresis of the screened DNA is shown in Figure 5 shown.

[0071] Table 3 PCR components

[0072] Components Volume (μL) concentration template 1 0.02-2 ng / μL Upstream primer (10 μM) 1 0.5μM Downstream primer (10 μM) 1 0.5μM GC enhancer (optional) 10 1x Platinum Taq PCR mix 25 - <![CDATA[ddH2O]]> adjust to 50 -

[0073] Reaction conditions: 94℃ pre-denaturation for 5 minutes, 30 cycles: 94℃ denaturation for 30 seconds, 66℃ annealing for 30 seconds, 72℃ extension for 1 minute, and final extension for 10 minutes at 72℃. 1% agarose gel electrophoresis was used to identify the amplified product and the target fragment was recovered.

[0074] Example 5: ELISA screening and nucleic acid sequencing to identify broad-spectrum antibodies with cross-reactivity

[0075] The antibodies screened were specifically amplified using primers (upstream primer: ScFv-transfer-F and downstream primer: ScFv-transfer-R). The polymerase used was high-fidelity Q5 DNA enzyme. The PCR components and procedures are shown in Table 3. The primers used are shown in Table 1. The antibodies were transferred to plasmids by Gibson DNA assembly and transformed into TG1 Escherichia coli cells by electroporation. The bacterial lysate of each clone was then used for ELISA detection (e.g. Figure 6 Specific antibodies with cross-reactivity to SARS-CoV-2-RBD, Omicron-RBD, Gamma-RBD and Alpha-RBD were screened out. The target antibodies were sequenced to obtain their gene and amino acid sequence information.

[0076] Example 6: Expression and purification of antibodies and determination of binding kinetic constants

[0077] The positive clone G8 obtained in step 5 was cultured in BL21DE3 Escherichia coli in DYT medium containing ampicillin with shaking until OD 600 The final concentration of IPTG was 0.6-0.8, and the expression was induced overnight at 18°C. The cells were broken and the supernatant obtained by centrifugation was purified by Ni 2+ The protein was separated and purified by column, and the elution peaks were collected and subjected to SDS-PAGE after linear gradient elution. The elution peaks with a single band of the target protein were combined (such as Figure 7 In order to improve the purity of the protein, further ion exchange and gel filtration chromatography purification (as shown) Figure 8-9 The purified antibodies were coated on a streptavidin (SA) sensor chip, and the antigens SARS-CoV-2-RBD, Omicron-RBD, Gamma-RBD and Alpha-RBD were diluted in a gradient manner, and the binding affinity was measured on a surface plasmon resonance instrument. The results are shown in Fig.10 ,according to Fig.10 It can be seen that the screened antibodies have high binding affinity to the four antigens.

[0078] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A high-throughput screening method for cross-reactive antibodies, characterized in that: The steps include: Step 1, constructing an antibody gene library, and obtaining a complex for displaying antibodies based on the antibody gene library; Step 2, using at least two antigen display systems to screen the complexes in sequence, each antigen display system displaying different target antigen molecules on its surface, so as to screen out target complexes that can bind to different target antigen molecules at the same time; Step 3, obtaining the gene information of the target antibody according to the target complex; The antigen display system comprises artificial lipid droplets and amphiphilic target antigen molecules; the artificial lipid droplets are assembled from tributyrin in a hydrophilic medium and are encapsulated with magnetic nanoparticles; The artificial lipid droplets are formed by a surfactant in a hydrophilic medium; since the surfactant has a hydrophilic end and a hydrophobic end, the artificial lipid droplets are assembled into particles with a hydrophilic surface and a hydrophobic core in the hydrophilic medium; the amphiphilic target antigen molecule is formed after the target antigen molecule is hydrophobically modified; the hydrophilic target antigen molecule is displayed on the hydrophilic surface of the artificial lipid droplet to facilitate the binding with the antibody during the antibody screening process; the hydrophobic end is located inside the artificial lipid droplet, so that the target antigen molecule is closely displayed on the surface of the artificial lipid droplet.

2. The screening method according to claim 1, characterized in that Based on ribosome display technology, a complex including mRNA, ribosome and polypeptide was constructed.

3. The screening method according to claim 1 or 2, characterized in that The preparation method of the antigen display system comprises: Modifying the target antigen molecule with hydrophobic groups to obtain an amphiphilic target antigen molecule; The surfactant and the amphiphilic target antigen molecules are mixed and dispersed in a hydrophilic medium to form the antigen display system.

4. The screening method according to claim 3, characterized in that The target antigen molecule is modified with a hydrophobic group to obtain an amphiphilic target antigen molecule, which specifically includes the following steps: Poly(N-isopropylacrylamide) is activated using mercaptothiazoline or N-hydroxysuccinimide, the activated poly(N-isopropylacrylamide) is mixed with the target antigen molecule and reacted, and lysine is added to terminate the reaction to obtain an amphiphilic target antigen molecule.

5. The screening method according to claim 3, characterized in that The target antigen molecule is modified by isopentenylation to obtain an amphiphilic target antigen molecule.

6. The screening method according to any one of claims 1 to 5, characterized in that The target antigen molecule includes the receptor binding domain of the viral protein.

7. A method for screening antibodies with cross-reactivity to the novel coronavirus and its mutants, characterized in that: The method is carried out using the screening method according to any one of claims 1 to 5.

8. The screening method according to claim 7, characterized in that The target antigen molecules are selected from at least two of SARS-CoV-2-RBD, Omicron-RBD, Gamma-RBD, and Alpha-RBD.

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