Multi-specific anti-gp120 artificial antibody as well as preparation method and application thereof

Abstract

The invention discloses a multispecific anti-gp120 artificial antibody as well as a preparation method and application thereof. The multispecific anti-gp120 artificial antibody is prepared from gold nanoparticles and polypeptide, the two ends of the polypeptide are fixedly connected to the surfaces of the gold nanoparticles through Au-S bonds. According to the invention, the SPR (Surface Plasmon Resonance) technology is utilized to prove that the multispecific anti-gp120 artificial antibody has strong binding force and high affinity to gp120. The cost of the novel multispecific anti-gp120 artificial antibody is the same as that of a single monospecific gold antibody, but the affinity of the novel multispecific anti-gp120 artificial antibody with gp120 is higher, and the novel multispecific anti-gp120 artificial antibody is expected to effectively neutralize various gp120 mutants and inhibit immune escape of HIV viruses. Gp120 of HIV can be prevented from being combined with host cells CD4, and the application prospect in the aspect of HIV prevention and treatment is achieved.

Description

Technical Field

[0001] The present invention relates to the fields of biological antibody technology and nanotechnology, and in particular to a multi-specific anti-gp120 artificial antibody and a preparation method and application thereof. Background Art

[0002] Antibodies can recognize and bind to specific antigenic determinants on pathogens to prevent them from entering healthy cells, making broadly neutralizing antibodies (bNAbs) potential therapeutics against these pathogens. However, immune evasion through mutation is a common survival strategy used by pathogens to evade bNAbs, which is the main reason why there is still no effective vaccine against human immunodeficiency virus (HIV) despite decades of research.

[0003] As a potential alternative or supplement to antiretroviral therapy (ART), preclinical and clinical studies have demonstrated the safety of antibodies and their ability to reduce viral load in HIV-infected individuals. The primary targets of anti-HIV antibodies are relatively conserved epitopes on the HIV-1 envelope glycoprotein (Env), with the glycoprotein gp120 being the most extensively studied. Essentially, HIV infection is initiated by binding of its gp120 to CD4 on host CD4+ T cells. This subsequently triggers a conformational rearrangement of gp120 to expose coreceptor (CCR5 / CXCR4) binding sites, ultimately leading to viral envelope-cell membrane fusion. Based on this mechanism, in addition to anti-gp120 antibodies, a number of CD4 mimetics have been developed as potential inhibitors to prevent gp120 binding to CD4, similar to the effects of anti-gp120 antibodies. Alternatively, antibodies can also target host CD4 and act as post-attachment inhibitors to block HIV entry; such an antibody (ibalizumab) has been approved for the treatment of HIV-1 infection.

[0004] Due to the large size of conventional IgG antibodies, some potential conserved antigenic determinants of HIV Envs are inaccessible to conventional antibodies. Fortunately, there is a class of minimized antibodies, called nanobodies, which are much smaller in size (~15kD) than conventional IgG antibodies (~150kD). Nanobodies are a type of single variable domain antibody that is naturally produced in some animals, such as camels, llamas, and sharks. Its small size enables it to reach hidden antigenic determinants on HIV Envs. Several anti-HIV antibodies identified as bnAbs, such as A12, D7, and J3, were produced by immunizing camels with HIV-1 Envs. Therefore, nanobodies provide an effective solution to overcome the limitations of traditional antibodies.

[0005] The inventor's previous patent, CN108794626B, disclosed a polypeptide and a gold nanobody that can target the HIV envelope protein gp120. This artificial antibody is composed of gold nanoparticles and a designed polypeptide sequence. The resulting gold nanobody can enhance the binding strength between the drug and the target, maintaining a high binding strength even when the concentration of viral gp120 in the body is very low. However, these artificial antibodies are only monospecific and can only target a single target, the HIV envelope protein gp120. This limits their effectiveness and application range when faced with the complex and diverse escape mechanisms and immune evasion strategies of the virus.

[0006] Although both conventional IgG antibodies and nanobodies can be used to treat HIV, the rapid emergence of HIV escape mutants and the coexistence of genetically diverse viruses in the host are major obstacles to the clinical application of anti-HIV antibodies. An effective strategy to improve antibody efficacy and limit viral escape is to use a combination of multiple antibodies, each targeting a variant. Of course, antibody combinations are much more expensive than single antibodies. A more effective strategy to improve antibody efficacy and limit viral escape is to develop multispecific antibodies targeting multiple epitopes on HIV Envs (some may also target an epitope on the host). Due to increased affinity, multispecific antibodies generally have higher efficacy than any single or combination of monospecific antibodies. For nanobodies, due to their small size and limited number of complementary determining regions (CDRs), it is still impossible to develop multispecific nanobodies on the same single domain. Summary of the Invention

[0007] In order to solve the problem of "lack of effective multispecific antibodies against HIV virus" in the prior art, improve the therapeutic effect of anti-HIV antibodies, and achieve more efficient, economical and stable treatment of HIV infection, the purpose of the present invention is to provide a multispecific anti-gp120 artificial antibody and its preparation method and application.

[0008] Complementarity-determining regions (CDRs) are found in the variable regions of antibody molecules. The variable region of an antibody consists of multiple regions, among which the CDRs are the highly variable amino acid sequences within the variable region and are the key sites for specific binding between the antibody and the antigen. The amino acid sequence of the CDRs determines the specificity of the antibody, with different CDR sequences capable of recognizing and binding to different antigenic epitopes. Due to the diversity of CDRs, the immune system can produce a wide variety of antibodies to respond to a variety of pathogens.

[0009] The inventors have demonstrated that the CDR3 of an antibody can also be transplanted onto gold nanoparticles (AuNPs). (Reference Yan G H, Wang K, Shao Z X. Artificial antibody created by conformational reconstruction of the complementary-determining region on gold nanoparticles [J]. Proceedings of the National Academy of Sciences of the United States of America, 2018, 115 (1): E34–E43.) Restoring the native conformation and activity of the antibody CDR on AuNPs can produce a new class of artificial antibodies, called gold antibodies. On the surface, goldification is similar to humanization. Both technologies involve grafting the CDR of an antibody onto another scaffold, both of which can eliminate possible immunogenicity. However, the biggest difference between goldification and humanization lies in how to refold the grafted CDR into its original active conformation. Since animal and human antibodies are highly uniform in sequence and very similar in structure, refolding the transplanted CDR has never been a problem in humanization. However, before the invention of gold antibody technology, it was almost unimaginable that the highly flexible CDR would refold into its original conformation on the surface of AuNPs. Gold antibody technology involves grafting CDR to the surface of AuNPs through two (or more) Au-S bonds, rather than the traditional single Au-S bond conjugation method. It is these two (or more) Au-S bonds that mimic the key long-range interactions in the original antibody, refolding the CDR into its active binding conformation. And the size of gold antibodies is comparable to that of nanobodies, so they may be able to enter the hidden epitopes of HIV Envs like nanobodies. The large surface area of ​​AuNPs can accommodate dozens of CDRs on a small particle, which makes it easier to produce multi-specific gold bodies than traditional antibodies and is not limited to a maximum of four epitopes.

[0010] This study leverages the large surface area and ease of coupling of AuNPs to graft the gp120-binding fragment of the CD4 protein and the CDR3 of three anti-gp120 nanoantibodies onto AuNPs, creating four monospecific anti-HIV gold antibodies. Furthermore, a potential tetraspecific anti-gp120 gold antibody was prepared by grafting four different fragments onto the same AuNP. This antibody exhibits strong specificity, high accuracy, high affinity, and high binding capacity.

[0011] Based on the above principles and objectives, the present invention adopts the following technical solutions:

[0012] One of the technical solutions of the present invention provides a multispecific anti-gp120 artificial antibody, which is composed of gold nanoparticles and a polypeptide; both ends of the polypeptide are cysteine, and the cysteine ​​has a sulfhydryl residue; the sulfhydryl residue forms an Au-S bond with the gold nanoparticle; the polypeptide is fixedly connected to the surface of the gold nanoparticle through the Au-S bond to form a specific cyclic conformation; the polypeptide serves as the antibody determinant.

[0013] Furthermore, the polypeptide includes at least any one of pJ as shown in SEQ ID NO.1, pD as shown in SEQ ID NO.2, pC as shown in SEQ ID NO.3 or pA as shown in SEQ ID NO.4, preferably including four polypeptides.

[0014] Furthermore, the diameter of the gold nanoparticles is (1-100) nm, preferably 3.6 nm.

[0015] Furthermore, the number of peptides attached to each gold nanoparticle is 10 to 100. When only one type of peptide is attached, the number is preferably 5 to 40, more preferably 10 to 20. In particular, the optimal peptide densities for the four peptides pC, pA, pJ, and pD are 20, 15, 15, and 10 peptides per AuNP, respectively. When four types of peptides are attached, the number is preferably 12 to 100 (i.e., 4n, where n is a positive integer from 3 to 25); more preferably, 16, with four peptides attached to each type of peptide.

[0016] A second technical solution of the present invention provides a method for preparing a multispecific anti-gp120 artificial antibody, comprising the following steps: dropwise adding a polypeptide solution to a gold nanoparticle solution, and after the dropwise addition is completed, stirring the solution to react, thereby preparing a multispecific anti-gp120 artificial antibody.

[0017] In some specific embodiments of the present invention, the specific steps are:

[0018] (1) preparing gold nanoparticles to obtain a gold nanoparticle solution;

[0019] In this step, the solvent for the gold nanoparticle solution is water. The preparation method for the gold nanoparticles is not a distinguishing technical feature of the present invention and will not be described in detail. The gold nanoparticle solution exhibits excellent dispersibility; in some specific embodiments, it is filtered through a 2 μm membrane before use and trisodium citrate solution is added dropwise to prevent agglomeration.

[0020] (2) adding the polypeptide solution dropwise to the gold nanoparticle solution obtained in step (1), stirring the mixture to react, and preparing a multispecific anti-gp120 artificial antibody;

[0021] In this step, the polypeptide solution is dissolved using any one of an alkaline solution, DMSO, or acetonitrile; the alkaline solution is preferably a sodium hydroxide solution, and the concentration of the sodium hydroxide solution is 1 to 3 mM, preferably 2 mM. The stirring reaction temperature is room temperature, and the reaction time is 1 to 1.5 hours.

[0022] Furthermore, the specificity of the multispecific anti-gp120 artificial antibody was detected by SPR technology (Biacore T200, Cytiva), and the method was specifically as follows:

[0023] gp120 was immobilized on the first channel of a CM5 chip using a standard amine coupling method at 25°C to characterize the specific interaction of the anti-gp120 gold antibody with gp120. BSA and IgG were coupled to the second and third channels of the CM5 chip, respectively, as controls. The anti-gp120 gold antibody was simultaneously injected into the first to third channels to allow antigen-antibody binding to occur, thus verifying the specificity of the anti-gp120 artificial antibody.

[0024] Furthermore, the diluent used in the standard amine coupling method is Acetate 4.0, Acetate 4.5 or Acetate 5.0, preferably Acetate 5.0. The binding time of the antigen-antibody specific binding is 90-120 s, and the dissociation time is 800-1200 s.

[0025] The third technical solution of the present invention provides an application of a multi-specific anti-gp120 artificial antibody, which is used to prepare an anti-HIV drug and binds to the antigenic determinant of the HIV virus and exerts its effect.

[0026] A fourth technical solution of the present invention provides an anti-HIV drug containing a multi-specific anti-gp120 artificial antibody.

[0027] A fifth technical solution of the present invention provides a polypeptide, which is the polypeptide of technical solution 1. The polypeptide is selected from any one of pJ as shown in SEQ ID NO. 1, pD as shown in SEQ ID NO. 2, pC as shown in SEQ ID NO. 3, or pA as shown in SEQ ID NO. 4.

[0028] Compared with the prior art, the present invention has the following obvious outstanding substantial features and significant advantages:

[0029] (1) The present invention reconstructs the gp120-binding fragment of CD4 and the CDR3 loops of three anti-gp120 antibodies to obtain four monospecific anti-gp120 gold antibodies on 3.6 nm AuNPs. All four monospecific anti-gp120 gold bodies exhibit strong specific interactions with gp120, with high binding affinity and high affinity.

[0030] (2) The present invention demonstrates that all four different protein fragments (including three CDR3s) can be reconstructed on the same AuNPs to produce a potential tetraspecific anti-gp120 gold body GbMix, which can strongly and specifically bind to gp120 and has more sensitive recognition ability, preventing HIV virus mutation and escape.

[0031] (3) The multispecific anti-gp120 artificial antibody of the present invention, which is completely based on gold antibodies, has obvious advantages in the strategy of developing multispecific gold bodies, including ease of production and no valency restriction, indicating the great potential of this simple method.

[0032] (4) Compared with the monospecific artificial antibodies in the prior art, the multispecific artificial antibodies provided by the present invention can simultaneously target multiple key sites of HIV, effectively curbing the drug resistance of the virus caused by mutations in a single target site, and showing stronger inhibitory power against different subtypes and mutants. By synergistically activating multispecific immune responses, the recognition and killing of infected cells are enhanced, opening up new paths for conquering HIV infection and treating complex viral diseases. At the same time, the development of multispecific antibodies also provides new ideas for the targeted treatment of other diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Schematic diagram of the principle of a multispecific anti-gp120 artificial antibody in a preferred embodiment of the present invention;

[0034] Figure 2 This is the SPR graph of the gp120 dilution screening in Example 1 of the present invention and Comparative Examples 1-2;

[0035] Figure 3 This is an SPR graph for screening the number of peptides binding to the gp120 protein by the monospecific anti-gp120 artificial antibody in Example 1 of the present invention; the labels are as follows: (A) screening for the number of peptides pC, (B) screening for the number of peptides pA, (C) screening for the number of peptides pJ, and (D) screening for the number of peptides pD;

[0036] Figure 4Figures 1 and 2 show the SPR patterns of specific binding of artificial antibodies (monoclonal anti-gp120 artificial antibody, multispecific anti-gp120 artificial antibody) to proteins (gp120, BSA, IgG) in Example 1 and Comparative Examples 3-4 of the present invention. Reference symbols: (A) Binding of anti-gp120 artificial antibodies (GbC, GbA, GbJ, GbD, GbMix) after restoration of their native conformation to proteins (gp120, BSA, IgG); (B) Binding of pure peptides (pC, pA, pJ, pD, pMix) to proteins (gp120, BSA, IgG).

[0037] Figure 5 These are SPR plots of the binding kinetics of the monoclonal anti-gp120 artificial antibody and the multispecific anti-gp120 artificial antibody to gp120 in Example 2 of the present invention; legend of the numbers: (A) kinetic curve of GbC, (B) kinetic curve of GbA, (C) kinetic curve of GbJ, (D) kinetic curve of GbD, and (E) kinetic curve of GbMix. DETAILED DESCRIPTION

[0038] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention is described in detail below with reference to specific embodiments. It should be noted that the following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present invention. These all fall within the scope of protection of the present invention.

[0039] All raw materials of the present invention are not particularly limited in their sources and can be purchased commercially or prepared according to conventional methods known to those skilled in the art. The glassware and polytetrafluoroethylene rotor used in the synthesis of the present invention were cleaned by immersion in aqua regia for 2 hours. After immersion, they were rinsed once with deionized water and then soaked in ultrapure water for 3 days. After the ultrapure water soaking, they were rinsed again with ultrapure water, dried, and sealed with a film before use.

[0040] The materials and instruments used in the examples are shown in Tables 1 and 2.

[0041] Table 1 Specifications and manufacturers of experimental reagents

[0042]

[0043]

[0044] Table 2 Experimental instruments

[0045]

[0046]

[0047] The anti-gp120 gold antibody in the preferred embodiment of the present invention obtains four monospecific anti-gp120 gold bodies on 3.6nm AuNPs by reconstructing the gp120 binding fragment of CD4 and the CDR3 loop of three anti-gp120 antibodies. All four different protein fragments (including three CDR3s) can be reconstructed on the same AuNPs to produce a potential four-specific anti-gp120 gold body GbMix. The polypeptide sequences of the gp120 binding fragment of CD4 and the CDR3 loop of the three anti-gp120 antibodies are named pC, pA, pJ, and pD, respectively. The sulfhydryl group in the cysteine ​​(Cys) at both ends of the pC, pA, pJ, and pD sequences can form an Au-S bond with the nanogold surface, thereby fixing the two ends of the polypeptide Pep2 on the surface of the gold nanoparticles to form an anti-gp120 gold antibody that can specifically bind to gp120.

[0048] The anti-gp120 antibody GbC, used in a preferred embodiment of the present invention, has a surface-linked peptide pC. The peptide pC (CGYLGNQGSFLTYDGC) was designed and synthesized based on the CD4 CDR2 peptide fragment NQIKILGNQGSFLTKGPS. The anti-gp120 antibodies GbA, GbJ, and GbD, used in this embodiment of the present invention, have surface-linked peptides pA, pJ, and pD. The peptides pA, pJ, and pD (CAAKWRPLAYSDYPSNSDYYDWG, SKSTYISYNSNGYD, and WRPLRYSDNPSNSDYN) were designed and synthesized based on the CDR3 peptide fragments CAAKWRPLAYSDYPSNSDYYDC, CSKSTYISYNSNGYDC, and CRPLAYSDNPSNSDYNC, respectively. (The GbA gold antibody in the preferred embodiment of the present invention uses the anti-gp120 gold nanoparticle artificial antibody disclosed in the patent document with publication number CN108794626B.) The polypeptides pC, pA, pJ, and pD sequences have cysteine ​​(Cys) residues at both ends. This allows the polypeptide fragments to be fixed to the surface of the gold nanoparticles via Au-S bonds at both ends. Through conformational regulation, their native conformation is restored to form anti-gp120 gold antibodies that can specifically bind to gp120. The gold nanoparticles have a diameter of approximately 3.6 nm, and their surfaces are modified with polypeptides pC, pA, pJ, and pD to form four monospecific antibodies. The polypeptides form Au-S bonds with the gold nanoparticles, which are then fixed to the surface of the gold nanoparticles.

[0049] The preferred embodiment of the present invention uses the above-mentioned anti-HIV gold antibody and uses surface plasmon resonance (SPR) technology to test the binding of anti-HIV antibodies to gp120 protein. Figure 1As shown, in this experiment, the inventors selected gp120 as a target for investigation. Since gp120 requires CD4 binding, they mimicked a CD4 binding region, allowing HIV gp120 to initially bind to the gold antibody designed by the present invention, thereby occupying the gp120 binding site and protecting the CD4 target. The inventors extracted the CDR3 region from the protein sequence context of 2NY1 (reference: Zhou T, Xu L, Dey B, et al. Structural definition of a conserved neutralization epitope on HIV-1 gp120 [J]. Nature, 2007, 445(7129):732–737) and designed the peptide pC. In addition to directly mimicking the CD4-gp120 binding site, the inventors also considered natural antibodies against gp120. Camelids possess a variety of antibodies, which are part of the animal's natural immune response, used to identify and defend against foreign pathogens, including viruses and bacteria. A special type of antibody found in camelids, these antibodies naturally lack light chains and consist of only two heavy chains. Heavy chain antibodies (VHH) have structural characteristics different from traditional antibodies, such as larger variable regions and richer CDR3 regions. These characteristics enable them to recognize and bind to more diverse antigenic epitopes. VHH can recognize the specific antigenic epitope gp120 on the surface of HIV virus. Compared with traditional antibodies, VHH has higher specificity and affinity, and can more accurately lock onto key targets on the surface of the virus. Based on the CDR3 region A12 of VHH, CD5 was designed, and pA was also designed. The inventors retained alanine and mutated a positively charged arginine in the middle of the sequence to alanine to reduce the effect of electrostatic adsorption on structural recovery. At the same time, the inventors designed pJ and pD, which were designed from the CDR3 regions of different antibodies produced by camels. The polypeptide design of pJ retained the original sequence, only adding a cysteine ​​at both ends. The positively charged arginine in the middle of the pD sequence was mutated to alanine (Reference: Zhou T, Chen L, Gorman J, et al. Structural basis for llamananobody recognition and neutralization of HIV-1 at the CD4-binding site [J]. Structure, 2022, 30 (6): 862–875.).

[0050] Based on the above principles and design ideas, the specific implementation methods of the present invention are as follows:

[0051] Example 1

[0052] Preparation, Screening, and Testing of Anti-gp120 Gold Antibodies

[0053] (1) Preparation of CM5 chip for screening anti-gp120 gold antibodies

[0054] In this example, gp120 was diluted to a concentration of 4 μg / mL in Acetate 5.0 (10 mM acetate buffer, pH 5.0) and coupled to channel 2 of a CM5 chip (a dextran chip modified with carboxymethylated dextran) via amino coupling (ENC, NHS activation, Ethanolamine-HCL blocking, both conventional techniques well known to those skilled in the art). The resulting response unit (RU) value ranged from 100 to 150, resulting in a gp120-coupled CM5 chip for subsequent screening. The specific steps are as follows:

[0055] A. Immobilize gp120 protein on the surface of a CM5 chip. Use Acetate 5.0 as the diluent for gp120 protein (400 μg / mL in the pre-enrichment process and 4 μg / mL in the actual experiment). Enter 10 μL / min in the Flow rate. Select Flow path 2 in the Flow path and use channel 2 for the pre-enrichment experiment (protein gp120 is immobilized on channel 2 through electrostatic adsorption). Figure 2 As shown, compared with Comparative Examples 1-2, the electrostatic adsorption RU value of Acetate 5.0 as a diluent of protein gp120 in the experiment is the highest.

[0056] B. The pre-enrichment experiment in A has determined the optimal dilution and pH conditions for the coupling experiment. Using the amino coupling method, ligand coupling can be performed using the wizard function in the Biacore T200 control software. First, activate the dextran on the channel with EDC and NHS. The coupling wizard program sets the flow rate to 10 μL / min. The Target program automatically controls the coupling level, increasing the gp120 level to an RU increase of 100-150 (this RU value serves as the initial value). Finally, ethylamine-HCl is used as a blocking agent.

[0057] (2) Preparation of anti-gp120 gold antibody

[0058] The present invention adopts the classical synthesis method in the literature and slightly improves it to prepare gold nanoparticles of about 3.6 nm (References [1] Jana NR, Gearheart L, Murphy J. Seeding growth for size control of 5-40 nm diameter gold nanoparticles [J]. Langmuir, 2001, 17 (22): 6782–6786. [2] Ji X, Song X, Li J, et al. Size control of gold nanocrystals in citrate reduction: the third role of citrate [J]. Journal of the American Chemical Society, 2007, 129 (45): 13939–13948. [3] Haiss W, Thanh NTK, Aveyard J, et al. Determination of size and concentration of gold nanoparticles from UV-Vis spectra[J].AnalyticalChemistry,2007,79(11):4215–4221.), and obtain the AuNPs solution for later use.

[0059] To 6 mL of the AuNP solution that had been filtered through a 2 μm membrane, 10 μL of 0.2 M trisodium citrate solution was added to stabilize the AuNPs and prevent aggregation. Subsequently, 2 mL of peptide solutions (pC, pA, pJ, and pD) of varying concentrations, dissolved in 2 mM sodium hydroxide solution, were added dropwise and at a constant rate. After the addition, the AuNPs and peptides were allowed to react on a magnetic stirrer at room temperature for 1 hour. After the reaction was complete, the AuNPs were refrigerated at 4°C until ready for use. This yielded anti-gp120 gold antibodies with varying numbers of attached stripes (5 to 40).

[0060] (3) Detection and screening of anti-gp120 gold antibodies

[0061] Subsequently, the anti-gp120 gold antibody AuNP-pC (number of strips: 10, 15, 20, 25, 30), AuNP-pA (number of strips: 10, 15, 20, 30, 40), AuNP-pJ (10, 15, 20, 30, 40), and AuNP-pD (5, 10, 15, 20, 30) prepared in step (2) were diluted to 4 nM using a 10-fold dilution of 0.1 M HBS-EP + 10× buffer, pH 7.4 (containing 0.1 M HEPES, 1.5 M NaCL, 0.03 M EDTA, and 0.5% v / v Surfactant P20) as samples. The flow rate was set to 30 μL / min, the association time was 90-120 s, and the dissociation time was 2 s. The solution was passed through the gp120-coupled CM5 chip prepared in step (1) to measure the increase in test RU. The four anti-gp120 gold antibodies specifically bound to the antigenic determinants on gp120, forming complexes and exhibiting specific binding, which was reflected in the increase in RU.

[0062] The results are as follows Figure 3 As shown, based on the RU values, the present invention selected 20 pCs attached to AuNPs as GbC, 15 pAs attached to AuNPs as GbA, 15 pJs attached to AuNPs as GbJ, and 10 pDs attached to AuNPs as GbD. In subsequent examples, based on the detection results of these four monoclonal anti-HIV gold antibodies, the present invention selected 4 pCs, 4 pAs, 4 pJs, and 4 pDs attached to the same AuNP as the multispecific antibody GbMix.

[0063] Comparative Examples 1-2: Screening of dilution conditions for protein gp120

[0064] Comparative Examples 1 and 2 involve screening of dilution conditions in the coupling step of gp120 and CM5 chip, which are the same as the coupling step of gp120 and CM5 chip in Example 1 (1), except that the buffer was replaced with Acetate 4.0 and Acetate 4.5 respectively. Figure 2 shown.

[0065] Comparative Examples 3-4: Control Protein BSA and Control Protein IgG

[0066] The procedures of this example were essentially the same as those of Example 1, with the exception that a control protein, BSA, was coupled to channel 3 of the CM5 chip, and a control protein, IgG, was coupled to channel 4. In addition to specific binding experiments with the anti-gp120 gold antibody, peptides pC, pA, pJ, and pD were also used by themselves (i.e., not attached to gold nanoparticles) to bind to gp120 for comparison. The gold antibody concentration in Example 1 was 4 nM, so the corresponding peptide dosages were 80 nM for pC, 60 nM for pA, 60 nM for pJ, 40 nM for pD, and 64 nM for pMix.

[0067] from Figure 4 In B, it can be seen that pure peptides pC, pA, pJ, and pD show almost no binding signals with gp120, but after restoring their native conformations on AuNPs ( Figure 4 A), Four monospecific anti-gp120 gold antibodies showed strong binding signals to the target protein gp120, while the binding signals to two non-target proteins, bovine serum albumin (BSA) and IgG, were much weaker, indicating that the four anti-gp120 gold antibodies specifically bound to gp120, thereby successfully restoring the original native conformation of the grafted peptide.

[0068] Example 2 Kinetic Testing of Anti-gp120 Gold Antibodies

[0069] This embodiment is similar to Embodiment 1, but has some differences.

[0070] Same steps:

[0071] (1) Coupling of gp120 to CM5 chip;

[0072] (2) Preparation of anti-gp120 gold antibody

[0073] In this embodiment, 20 pCs are preferably connected to AuNP as GbC, 15 pA are connected to AuNP as GbA, 15 pJs are connected to AuNP as GbJ, and 10 pDs are connected to AuNP as GbD. 4 pCs, 4 pAs, 4 pJs, and 4 pDs are connected to the same AuNP as the multispecific antibody GbMix.

[0074] Different steps:

[0075] (3) Kinetic binding of anti-gp120 gold antibody to protein gp120

[0076] Anti-gp120 gold antibody was diluted with HBS-EP buffer to different concentration gradients (1nM, 2nM, 4nM, 8nM, 16nM or 2nM, 4nM, 8nM, 16nM, 32nM) and then injected into the gp120 immobilized channel. The binding time was 90-120s and the dissociation time was 800-1200s. The experimental data were fitted with a 1:1 binding model to obtain k on and k d , and calculated the dissociation constant (K D ).

[0077] To quantify the affinity between the synthetic anti-gp120 gold antibody and gp120, the binding kinetics of the gold antibody to gp120 were measured by SPR experiments. All kinetic curves could be well fitted by a 1:1 model (e.g. Figure 5 The apparent affinity K of GbC was obtained. D The value is 3.14×10 -10 M( Figure 5 A), Apparent affinity K of GbA D The value is 1.69×10 -10 M( Figure 5 B) Apparent affinity K of GbJ D The value is 4.14×10 -11 M( Figure 5 C), the apparent affinity K of GbD D The value is 7.27×10 -10 M( Figure 5 D), the apparent affinity K of GbMix D The value is 5.49×10 -11 M( Figure 5 E) These data quantitatively show that the gold antibodies strongly bind to gp120, and the K D The value is almost the highest affinity among all gold bodies, which proves the effective binding of multispecific antibodies.

[0078] The above embodiments provide monoclonal anti-HIV gold antibodies and multispecific anti-gp120 gold antibodies. The present invention obtains four monospecific anti-gp120 gold bodies on 3.6nm AuNPs by reconstructing the gp120 binding fragment of CD4 and the CDR3 loop of three anti-gp120 antibodies. The four monospecific anti-gp120 gold bodies all show strong specific interactions with gp120. In addition, the present invention has demonstrated that all four different protein fragments (including three CDR3s) can be reconstructed on the same AuNPs to produce a potential four-specific anti-gp120 gold body GbMix, which can strongly and specifically bind to gp120. The strategy of developing multispecific gold bodies has obvious advantages, including ease of production and no valence restrictions, indicating the great potential of this simple method.

[0079] The above description of the embodiments is intended to facilitate understanding and use of the invention by those skilled in the art. It will be apparent that those skilled in the art can readily make various modifications to these embodiments and apply the general principles described herein to other embodiments without requiring inventive effort. Therefore, the present invention is not limited to the above-described embodiments. Improvements and modifications made by those skilled in the art based on the disclosure of the present invention, without departing from the scope of the present invention, should be within the scope of protection of the present invention.

[0080] The sequences involved in this application are as follows:

[0081] SEQ ID NO.1(pJ):CSKSTYISYNSNGYDC

[0082] SEQ ID NO.2(pD): CRPLAYSDNPSNSDYNC

[0083] SEQ ID NO.3(pC):CGYLGNQGSFLTYDGC

[0084] SEQ ID NO.4(pA):CAAKWRPLAYSDYPSNSDYYDC

[0085] The above description of the embodiments is intended to facilitate understanding and use of the invention by those skilled in the art. It will be apparent that those skilled in the art can readily make various modifications to these embodiments and apply the general principles described herein to other embodiments without requiring inventive effort. Therefore, the present invention is not limited to the above-described embodiments. Improvements and modifications made by those skilled in the art based on the disclosure of the present invention, without departing from the scope of the present invention, should be within the scope of protection of the present invention.

Claims

1. A multispecific anti-gp120 artificial antibody, characterized in that The artificial antibody is composed of gold nanoparticles and polypeptides; Both ends of the polypeptide have cysteines, and the cysteines have sulfhydryl residues; the sulfhydryl residues form Au-S bonds with gold nanoparticles; the polypeptide is fixedly connected to the surface of the gold nanoparticles through the Au-S bond to form a specific cyclic conformation; and the polypeptide serves as an antibody determinant.

2. A multispecific anti-gp120 artificial antibody according to claim 1, characterized in that: The polypeptide is selected from at least any one of pJ as shown in SEQ ID NO.1, pD as shown in SEQ ID NO.2, pC as shown in SEQ ID NO.3, and pA as shown in SEQ ID NO.

4.

3. The method for preparing a multispecific anti-gp120 artificial antibody according to claim 1, characterized in that: The diameter of the gold nanoparticles is (1-100) nm.

4. A multispecific anti-gp120 artificial antibody according to any one of claims 2 or 3, characterized in that: The number of polypeptides attached to each of the gold nanoparticles is 10 to 100; When only one polypeptide is connected, the number of polypeptides ranges from 5 to 40; When four polypeptides are connected, the number of polypeptides is 4n, where n is a positive integer ranging from 3 to 25.

5. The multispecific anti-gp120 artificial antibody according to claim 4, characterized in that: The polypeptide is a combination of pJ, pD, pC and pA, wherein the number of each polypeptide is 4.

6. A method for preparing the multispecific anti-gp120 artificial antibody according to claim 1, characterized in that: The steps include: The polypeptide solution is added dropwise to the gold nanoparticle solution. After the addition is completed, the mixture is stirred for reaction to prepare a multi-specific anti-gp120 artificial antibody.

7. The method for preparing a multispecific anti-gp120 artificial antibody according to claim 6, characterized in that: The stirring reaction temperature is room temperature, and the reaction time is 1 to 1.5 hours.

8. A use of the multispecific anti-gp120 artificial antibody according to claim 1, characterized in that: The artificial antibody is used for preparing medicine for resisting HIV virus.

9. An anti-HIV drug, characterized in that: The drug contains the multispecific anti-gp120 artificial antibody according to claim 1.

10. A polypeptide, characterized in that The polypeptide is selected from any one of: pJ as shown in SEQ ID NO.1, pD as shown in SEQ ID NO.2, pC as shown in SEQ ID NO.3 or pA as shown in SEQ ID NO.4.

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