A nano self-assembled anti-Aβ antibody, its preparation method and application

Abstract

The present invention belongs to the technical fields of nano-drug delivery and antibody preparation, and particularly relates to a nano-self-assembled anti-Aβ antibody and its preparation method and application. In the present invention, a first protein tag is linked to a site on the self-assembled nanoparticle protein where the protein is exposed on the outer surface and insertion of an exogenous sequence does not affect protein self-assembly. A second protein tag is linked to the N-terminus of the anti-Aβ antibody, and a polypeptide that crosses the blood-brain barrier is linked to the C-terminus. The first protein tag and the second protein tag form a covalent bond through a spontaneous reaction, and the anti-Aβ antibody is directionally loaded onto the surface of the self-assembled nanoparticle protein to form a nano-self-assembled anti-Aβ antibody, improving the stability of the anti-Aβ antibody and effectively avoiding the reduction in efficacy caused by the degradation of the anti-Aβ antibody. Moreover, the polypeptide that crosses the blood-brain barrier linked to the C-terminus of the anti-Aβ antibody enables the anti-Aβ antibody to cross the blood-brain barrier, enhancing the permeability of the antibody to the brain and the precise delivery to the target site, and enhancing the therapeutic effect of Alzheimer's disease.

Description

Technical Field

[0001] The present invention belongs to the technical fields of nano drug delivery and antibody preparation, and particularly relates to a nano self-assembled anti-Aβ antibody, a preparation method thereof, and an application thereof. Background Art

[0002] Alzheimer's Disease (AD) is a central nervous system degenerative disease closely related to age. The onset of the disease is insidious, and the most typical manifestation is progressive memory decline or cognitive dysfunction, which may be accompanied by symptoms such as language, executive function, visuospatial function decline, and behavioral abnormalities. It is the most common type of dementia.

[0003] The massive accumulation and aggregation of β-amyloid protein (Aβ) in specific regions of the brain, such as the hippocampus and cerebral cortex (i.e., the so-called neuritic plaques), is one of the important pathogenic factors of Alzheimer's disease. Therefore, the extracellular deposition of Aβ is a key target that needs to be urgently overcome in the treatment of AD. The passive immunotherapy targeting Aβ has important research value and application prospects in the field of AD treatment.

[0004] To date, the US Food and Drug Administration (FDA) has approved three anti-Aβ monoclonal antibodies, namely Aducanumab, Lecanemab, and Donanemab, for the treatment of mild cognitive impairment and mild AD. Although these three antibodies can reduce brain amyloid deposition to slow down cognitive decline and relieve the progression of AD, due to the presence of the blood-brain barrier, the penetration rate of antibodies in the brain is low and sufficient effective concentration cannot be achieved in the diseased areas of the brain. Therefore, the efficacy of the current antibody passive therapy is still greatly limited. Thus, developing an antibody delivery technology that can quickly break through the blood-brain barrier, transport a sufficient amount of antibodies to the brain and act on the diseased target is crucial for improving the passive immunotherapy system of AD. Summary of the Invention

[0005] The purpose of the present invention is to provide a nano self-assembled anti-Aβ antibody, a preparation method thereof, and an application thereof, so that the anti-Aβ antibody can quickly penetrate the blood-brain barrier, significantly enhance the penetration ability of the anti-Aβ antibody to the brain, be accurately delivered to the diseased target site, improve the stability of the anti-Aβ antibody, effectively avoid the degradation of the anti-Aβ antibody, and improve the treatment effect of Alzheimer's disease.

[0006] The present invention provides a nano self-assembled anti-Aβ antibody, which comprises a self-assembled nanoparticle protein functional element and an anti-Aβ antibody functional element;

[0007] The self-assembled nanoparticle protein functional element includes a self-assembled nanoparticle protein; a first protein tag is linked to a site on the outer surface of the self-assembled nanoparticle protein where exogenous sequences are inserted without affecting protein self-assembly.

[0008] The anti-Aβ antibody functional element includes an anti-Aβ antibody; a second protein tag is linked to the N-terminus of the anti-Aβ antibody, and a polypeptide that crosses the blood-brain barrier is linked to the C-terminus.

[0009] The first protein tag and the second protein tag form a covalent bond through a spontaneous reaction.

[0010] Preferably, a first flexible amino acid is used to link the first protein tag to a site on the outer surface of the self-assembled nanoparticle protein where exogenous sequences are inserted without affecting protein self-assembly.

[0011] The N-terminus of the anti-Aβ antibody is linked to the second protein tag through a second flexible amino acid, and the C-terminus is linked to the polypeptide that crosses the blood-brain barrier through a third flexible amino acid.

[0012] The amino acid sequence of the first flexible amino acid is GGGGS, GGGGSGGGGS, GGGGSGGGGSGGGGS, or GGGGSGGGGSGGGGSGGGGSGGGGS.

[0013] The amino acid sequence of the second flexible amino acid is GGGGS, GGGGSGGGGS, GGGGSGGGGSGGGGS, or GGGGSGGGGSGGGGSGGGGSGGGGS.

[0014] The amino acid sequence of the third flexible amino acid is GGGGS, GGGGSGGGGS, GGGGSGGGGSGGGGS, or GGGGSGGGGSGGGGSGGGGSGGGGS.

[0015] Preferably, the anti-Aβ antibody includes one or more of aducanumab, lecanemab, and donanemab.

[0016] Preferably, the self-assembled nanoparticle protein includes the Z105 protein; the subunit of the Z105 protein has the accession number A0A497XTC1 in UniProtKB.

[0017] Preferably, the polypeptide that crosses the blood-brain barrier includes one or more of angiopep-2, HIV trans-activator of transcription (TAT) peptide, leptin, islet amyloid polypeptide, and insulin polypeptide.

[0018] Preferably, when the first protein tag is SpyTag, the second protein tag is SpyCatcher.

[0019] When the first protein tag is SnoopTag, the second protein tag is SnoopCatcher;

[0020] When the first protein tag is DogTag, the second protein tag is DogCatcher.

[0021] Preferably, purification tags are respectively connected to the N-terminus of the first protein tag and the N-terminus of the second protein tag.

[0022] The present invention also provides a method for preparing the nano self-assembled anti-Aβ antibody according to the above technical solution, including the following steps:

[0023] Prokaryotically express and purify the recombinant plasmid subcloning the self-assembled nanoparticle protein functional element to obtain the purified self-assembled nanoparticle protein;

[0024] Prokaryotically express and purify the recombinant plasmid subcloning the anti-Aβ antibody functional element to obtain the purified anti-Aβ antibody;

[0025] Couple the purified self-assembled nanoparticle protein and the purified anti-Aβ antibody in a buffer solution to obtain the nano self-assembled anti-Aβ antibody; the pH value of the buffer solution is 7.0 - 7.4.

[0026] Preferably, the molar ratio of the purified self-assembled nanoparticle protein to the purified anti-Aβ antibody is 1:(1.2 - 3); the molar number of the purified self-assembled nanoparticle protein is calculated based on the total molar number of the subunits of the purified self-assembled nanoparticle protein;

[0027] The coupling time is 8 - 16 h, and the buffer solution includes TBS buffer solution or PBS buffer solution;

[0028] The present invention also provides the application of the nano self-assembled anti-Aβ antibody according to the above technical solution or the nano self-assembled anti-Aβ antibody prepared by the preparation method according to the above technical solution in the preparation of products for treating Alzheimer's disease.

[0029] Beneficial effects:

[0030] The present invention provides a nano self-assembled anti-Aβ antibody, which comprises a self-assembled nanoparticle protein functional element and an anti-Aβ antibody functional element; the self-assembled nanoparticle protein functional element comprises a self-assembled nanoparticle protein; a first protein tag is connected to a site on the outer surface of the self-assembled nanoparticle protein where insertion of an exogenous sequence does not affect protein self-assembly; the anti-Aβ antibody functional element comprises an anti-Aβ antibody; a second protein tag is connected to the N-terminus of the anti-Aβ antibody, and a polypeptide that crosses the blood-brain barrier is connected to the C-terminus; the first protein tag and the second protein tag form a covalent bond through a spontaneous reaction. In the present invention, a polypeptide that crosses the blood-brain barrier is connected to the C-terminus of the anti-Aβ antibody, which can break through the blood-brain barrier, enhance the permeability of the antibody to the brain and the precise delivery to the target site; a second protein tag is connected to the N-terminus of the anti-Aβ antibody, which can spontaneously react with the first protein tag at a site on the outer surface of the self-assembled nanoparticle protein where insertion of an exogenous sequence does not affect protein self-assembly to form a covalent bond, enrich the anti-Aβ antibody on the surface of the self-assembled nanoparticle protein in a geometric form, form a nano self-assembled anti-Aβ antibody, expose the anti-Aβ antibody on the surface of the self-assembled nanoparticle protein, improve the stability of the anti-Aβ antibody, effectively avoid the reduction of therapeutic effect caused by the degradation of the anti-Aβ antibody, and enhance the therapeutic effect of Alzheimer's disease. Description of the Drawings

[0031] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments.

[0032] Figure 1 It is a schematic structural diagram of the anti-Aβ antibody functional element in Example 1;

[0033] Figure 2 It is a schematic structural diagram of the A_Z105 expression element and the B_PP-Ab Aβ expression element in Example 1;

[0034] Figure 3 It is a schematic diagram of the directional assembly of the monovalent nano self-assembled anti-Aβ antibody in Example 1;

[0035] Figure 4 It is a schematic diagram of the directional assembly of the multivalent nano self-assembled anti-Aβ antibody in Example 2;

[0036] Figure 5 It is a negative staining transmission electron micrograph of A_Z105 and the nano self-assembled anti-Aβ antibody;

[0037] Figure 6 It is of A_Z105 and AD_PP-NanoAb Aβ particle size detection chart;

[0038] Figure 7For free anti-Aβ antibody and AD_PP-NanoAb Aβ In vitro affinity detection chart;

[0039] Figure 8 For free anti-Aβ antibody and AD_PP-NanoAb based on heat treatment Aβ Affinity detection chart;

[0040] Figure 9 For free anti-Aβ antibody and AD_PP-NanoAb based on serum storage Aβ Particle size change chart;

[0041] Figure 10 For intracellular free anti-Aβ antibody and AD_PP-NanoAb Aβ Relative fluorescence intensity detection result chart;

[0042] Figure 11 For intracellular free anti-Aβ antibody and AD_PP-NanoAb Aβ Pearson correlation coefficient detection result chart;

[0043] Figure 12 For different AD_PP-NanoAb Aβ Cell relative activity detection result chart at different concentrations;

[0044] Figure 13 For fluorescence intensity analysis results after intravenous injection of free anti-Aβ antibody and AD_PP-NanoAb into mouse tail veins Aβ Chart;

[0045] Figure 14 For escape latency detection results of mice in different treatment groups at different training durations;

[0046] Figure 15 For targeted limited residence time detection results of mice in different treatment groups during the test period;

[0047] Figure 16 For average synaptic number detection results in the brain tissues of mice in different treatment groups;

[0048] Figure 17 For relative Aβ content detection results in the brain tissues of mice in different treatment groups. Specific implementation mode

[0049] The present invention provides a nano self-assembled anti-Aβ antibody, comprising a self-assembled nanoparticle protein functional element and an anti-Aβ antibody functional element;

[0050] The self-assembled nanoparticle protein functional element includes a self-assembled nanoparticle protein; a first protein tag is linked to a site on the self-assembled nanoparticle protein that is exposed on the outer surface and does not affect protein self-assembly after insertion of an exogenous sequence.

[0051] The anti-Aβ antibody functional element includes an anti-Aβ antibody; a second protein tag is linked to the N-terminus of the anti-Aβ antibody, and a polypeptide for crossing the blood-brain barrier is linked to the C-terminus.

[0052] The first protein tag and the second protein tag form a covalent bond through a spontaneous reaction.

[0053] As an embodiment, the self-assembled nanoparticle protein of the present invention includes the Z105 protein; the accession number of the Z105 protein in UniProtKB is A0A497XTC1. As an embodiment, the Z105 protein of the present invention is a spherical nanoparticle protein formed by self-assembly of 60 identical subunits, and the N-terminus of each subunit is exposed on the particle surface. It is derived from 6,7-dimethyl-8-ribityllumazine synthase (UniProtK accession number A0A497XTC1) and is named Z105 protein. For specific information, see: https: / / www.uniprot.org / uniprotkb / A0A497XTC1 / entry. The amino acid sequence of the subunit of the Z105 protein of the present invention is specifically MKKYEGELRAEGIRFAIVAARFNHLLVDRLVEGAIDCILRHGGSEDNIELARVPGSWEIPVAVKKFLEREDIDAVIALGVLVRGSTPHFDYIAAEVSKGIANLSIDTGKPVSFGVVTADTLEQAIERAGTKMGNKGWEAALSAIEMANLFKKLG (SEQ ID NO:12). The self-assembled nanoparticle protein of the present invention has the characteristics of high biocompatibility and long biological half-life, and its outer surface can carry an anti-Aβ antibody to achieve efficient delivery of the anti-Aβ antibody.

[0054] As an embodiment, a first flexible amino acid links the first protein tag to a site on the self-assembled nanoparticle protein that is exposed on the outer surface and does not affect protein self-assembly after insertion of an exogenous sequence. As an embodiment, the site on the self-assembled nanoparticle protein that is exposed on the outer surface and does not affect protein self-assembly after insertion of an exogenous sequence includes the N-terminus of the subunit of the self-assembled nanoparticle protein.

[0055] As an embodiment, the C-terminus of the first protein tag of the present invention is linked to the N-terminus of the monomer of the self-assembled nanoparticle protein through the first flexible amino acid. As an embodiment, the amino acid sequence of the first flexible amino acid of the present invention is GGGGS (SEQ ID NO:1), GGGGSGGGGS (SEQ ID NO:2), GGGGSGGGGSGGGGS (SEQ ID NO:3) or GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:4).

[0056] As an embodiment, the N-terminus of the first protein tag of the present invention is linked to a purification tag. As an embodiment, the purification tag of the present invention includes a His tag. The present invention links a purification tag to the N-terminus of the first protein tag, so that the purification tag is fully exposed on the surface of the self-assembled nanoparticle protein, facilitating subsequent protein purification.

[0057] As an embodiment, the anti-Aβ antibody of the present invention includes one or more of aducanumab, lecanemab, and donanemab; as another embodiment, the anti-Aβ antibody of the present invention is lecanemab, or a combination of aducanumab, lecanemab, and donanemab. In specific embodiments of the present invention, lecanemab, and the combination of aducanumab, lecanemab, and donanemab are taken as examples for illustration, but it should not be understood as the entire scope of protection of the present invention. Since different anti-Aβ antibodies target different links in the pathogenesis of Alzheimer's disease, compared with single antibody therapy, the combination of aducanumab, lecanemab, and donanemab has multivalence and can act synergistically from multiple action targets to more comprehensively intervene in the pathological process of Alzheimer's disease and further enhance the therapeutic effect of Alzheimer's disease. As an embodiment, the CAS numbers of aducanumab, lecanemab, and donanemab of the present invention are 1384260-65-4, 1260393-98-3, and 1931944-80-7 in sequence.

[0058] As an embodiment, the N-terminus of the anti-Aβ antibody of the present invention is linked to a second protein tag through a second flexible amino acid, and the C-terminus is linked to a polypeptide that crosses the blood-brain barrier through a third flexible amino acid.

[0059] As an embodiment, the amino acid sequence of the second flexible amino acid in the present invention is GGGGS (SEQ ID NO: 1), GGGGSGGGGS (SEQ ID NO: 2), GGGGSGGGGSGGGGS (SEQ ID NO: 3), or GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 4). Connecting the second protein tag to the N-terminus of the anti-Aβ antibody in the present invention has the advantage of improving the purification efficiency compared to connecting it to the C-terminus, and the use of the second flexible amino acid for connection can protect the functional independence of the protein domains on both sides. The amino acid sequence of the third flexible amino acid in the present invention is GGGGS (SEQ ID NO: 1), GGGGSGGGGS (SEQ ID NO: 2), GGGGSGGGGSGGGGS (SEQ ID NO: 3), or GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 4). The present invention uses the third flexible amino acid to connect the polypeptide that crosses the blood-brain barrier, which can protect the functional independence of the protein domains on both sides.

[0060] As an embodiment, a purification tag is connected to the N-terminus of the second protein tag in the present invention. As an embodiment, the purification tag in the present invention includes a His tag. Connecting the purification tag to the N-terminus of the second protein tag in the present invention has the advantage of improving the purification efficiency.

[0061] As an embodiment, the polypeptide for crossing the blood-brain barrier in the present invention includes one or more of angiotrophin-2, HIV trans-activator of transcription (TAT) peptide, leptin, islet amyloid polypeptide, and insulin polypeptide. As an embodiment, the polypeptide for crossing the blood-brain barrier in the present invention is angiotrophin-2. As an embodiment, the amino acid sequence of the angiotrophin-2 in the present invention is TFFYGGSRGKRNNFKTEEY (SEQ ID NO:5), which can target low density lipoprotein receptor-related protein 1 (LRP1) and cross the blood-brain barrier (BBB) through receptor-mediated endocytosis. As an embodiment, the amino acid sequence of the HIV TAT peptide in the present invention is GRKKRRQRRRPQ (SEQ ID NO:6), which penetrates cell membranes and the BBB through adsorption-mediated endocytosis (AMT) relying on the cationic property rich in arginine. As an embodiment, the leptin in the present invention binds to the leptin receptor (LepR) and crosses the BBB by receptor-mediated transport, and the core sequence binding to the leptin receptor in the leptin is LPYFDLA (SEQ ID NO:7). As an embodiment, the amino acid sequence of the islet amyloid polypeptide in the present invention is KCNTATCATQRLANFLVHSSNNFGPILPPTNVGSNTY (SEQ ID NO:8), which penetrates the BBB through adsorption-mediated transcytosis relying on its amphiphilic structure. As an embodiment, the insulin polypeptide in the present invention binds to the insulin receptor on brain endothelial cells and triggers receptor-mediated transport, and the sequence of the insulin α-chain receptor-binding domain in the insulin polypeptide is GIVEQCCTSICSLYQLENYCN (SEQ ID NO:9).

[0062] The polypeptide for crossing the blood-brain barrier in the present invention has the significant advantages of small molecular weight and low toxicity and side effects, and is easy to be modified on the anti-Aβ antibody to enhance the permeability of the anti-Aβ antibody to the brain; and the polypeptide for crossing the blood-brain barrier in the present invention can specifically interact with the transport protein on the blood-brain barrier, mediate the anti-Aβ antibody to cross the blood-brain barrier and enter the brain, and enhance the permeability of the anti-Aβ antibody to the brain and the precise delivery to the target site. In the specific embodiment of the present invention, angiotrophin-2 is taken as an example for illustration, but it should not be understood as the entire protection scope of the present invention only.

[0063] The first protein tag and the second protein tag in the present invention form a covalent bond through a spontaneous reaction, thereby realizing the coupling of the self-assembled nanoparticle protein and the anti-Aβ antibody. As an embodiment, when the first protein tag in the present invention is SpyTag, the second protein tag is SpyCatcher; as another embodiment, when the first protein tag in the present invention is SnoopTag, the second protein tag is SnoopCatcher; as another embodiment, when the first protein tag in the present invention is DogTag, the second protein tag is DogCatcher.

[0064] The present invention also provides a method for preparing the nano self-assembled anti-Aβ antibody described in the above technical solution, including the following steps:

[0065] Prokaryotically express and purify the recombinant plasmid subcloning the functional element of the self-assembled nanoparticle protein to obtain the purified self-assembled nanoparticle protein;

[0066] Prokaryotically express and purify the recombinant plasmid subcloning the functional element of the anti-Aβ antibody to obtain the purified anti-Aβ antibody;

[0067] Couple the purified self-assembled nanoparticle protein and the purified anti-Aβ antibody in a buffer solution to obtain the nano self-assembled anti-Aβ antibody; the pH value of the buffer solution is 7.0-7.4.

[0068] The present invention prokaryotically expresses and purifies the recombinant plasmid subcloning the functional element of the self-assembled nanoparticle protein to obtain the purified self-assembled nanoparticle protein. The present invention has no strict requirements for the construction, prokaryotic expression step and purification step of the recombinant plasmid, and conventional operations in the art can be used. As an embodiment, the basic plasmid of the recombinant plasmid in the present invention includes the pet28a plasmid. As an embodiment, the prokaryotic expression in the present invention includes Escherichia coli BL21 DE3 transformation. As an embodiment, the purification in the present invention includes Ni-NTA purification and size exclusion chromatography purification carried out in sequence.

[0069] The present invention prokaryotically expresses and purifies the recombinant plasmid subcloning the functional element of the anti-Aβ antibody to obtain the purified anti-Aβ antibody. The present invention has no strict requirements for the construction, prokaryotic expression step and purification step of the recombinant plasmid, and conventional operations in the art can be used. As an embodiment, the basic plasmid of the recombinant plasmid in the present invention includes the pet28a plasmid. As an embodiment, the prokaryotic expression in the present invention includes Escherichia coli BL21 DE3 transformation. As an embodiment, the purification in the present invention includes Ni-NTA purification.

[0070] After obtaining the purified self-assembled nanoparticle protein and the purified anti-Aβ antibody, the present invention couples the purified self-assembled nanoparticle protein and the purified anti-Aβ antibody in a buffer to obtain a conjugated product; the conjugated product contains a nano self-assembled anti-Aβ antibody; the pH value of the buffer is 7.0-7.4.

[0071] As an embodiment, the molar ratio of the purified self-assembled nanoparticle protein to the purified anti-Aβ antibody in the present invention is 1:(1.2-3); the molar number of the purified self-assembled nanoparticle protein is calculated based on the molar number of the subunit number of the purified self-assembled nanoparticle protein; as another embodiment, the molar ratio of the purified self-assembled nanoparticle protein to the purified anti-Aβ antibody in the present invention is 1:(1.5-2).

[0072] As an embodiment, the coupling time in the present invention is 8-16 h; as another embodiment, the coupling time in the present invention is 10-15 h; as another embodiment, the coupling time in the present invention is 12 h. As an embodiment, the buffer in the present invention includes TBS buffer or PBS buffer. The buffer in the present invention can avoid protein precipitation.

[0073] As an embodiment, after obtaining the conjugated product, the present invention removes the uncoupled free antibody in the conjugated product by size exclusion chromatography to obtain a nano self-assembled anti-Aβ antibody.

[0074] The preparation method of the present invention is simple to operate, and the obtained nano self-assembled anti-Aβ antibody can rapidly penetrate the blood-brain barrier, has good stability, effectively avoids the degradation of the anti-Aβ antibody, significantly enhances the penetration ability of the anti-Aβ antibody into the brain, and is accurately delivered to the diseased target site, providing a highly potential new strategy for the treatment of Alzheimer's disease.

[0075] In view of the advantages of the nano self-assembled anti-Aβ antibody described in the present invention, the application of the nano self-assembled anti-Aβ antibody described in the present invention or the nano self-assembled anti-Aβ antibody prepared by the preparation method in the preparation of products for treating Alzheimer's disease also belongs to the protection scope of the present invention.

[0076] The present invention uses a mouse model of Alzheimer's disease and finds that after administration of the nano self-assembled anti-Aβ antibody, it can significantly improve animal behavior, improve nerve damage in the brain tissue, and reduce Aβ deposition in the brain tissue, which is a highly potential new strategy for passive treatment of Alzheimer's disease with antibodies.

[0077] To further illustrate the present invention, a nano self-assembled anti-Aβ antibody provided by the present invention, its preparation method and application will be described in detail below with reference to the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0078] Example 1

[0079] Design of monovalent nano self-assembled anti-Aβ antibody

[0080] 1. Modification of anti-Aβ antibody functional elements

[0081] The polypeptide molecule (PP) that crosses the blood-brain barrier is fused to the C-terminus of the anti-Aβ antibody (Ab Aβ ) through a flexible amino acid (SEQ ID NO: 2); the His tag is fused to the N-terminus of the anti-Aβ antibody (Ab Aβ ) through a flexible amino acid (SEQ ID NO: 1) to construct an element expressing an anti-Aβ antibody (PP-Ab Aβ ) with blood-brain barrier crossing function, and the schematic diagram is as shown in Figure 1 . Among them, Figure 1 PP is Angiopep-2, and Ab Aβ is Lecanemab (CAS No. 1260393-98-3).

[0082] 2. Directed assembly of PP-Ab Aβ with Z105

[0083] (1) The covalent bond A is fused through a flexible amino acid (amino acid sequence as shown in SEQ ID NO: 4) to a site on the outer surface of the Z105 protein that does not affect protein self-assembly after inserting the exogenous sequence. Specifically, in this step, the covalent bond A is fused to the N-terminus of the Z105 protein subunit;

[0084] The His tag is connected to the N-terminus of the covalent bond A through GGGGS (SEQ ID NO: 1) to construct an element expressing A_Z105, and the schematic diagram is as shown in Figure 2 . Among them, Figure 2 A is the covalent bond A, specifically SpyTag, and its amino acid sequence is RGVPHIVMVDAYKRYK (SEQ ID NO: 10).

[0085] (2) Using a flexible amino acid (amino acid sequence as shown in SEQ ID NO: 4), the covalent bond B is inserted between the His tag of the PP-Ab Aβ obtained in step 1 and Ab Aβ to construct an element expressing B_PP-Ab Aβ , and the schematic diagram is as shown in Figure 2 . Among them,Figure 2 In B, B is a covalent bond B, specifically SpyCatcher, and its amino acid sequence is VTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATMELRDSSGKTISTWISDGHVKDFYLYPGKYTFVETAAPDGYEVATPIEFTVNEDGQVTVDGEATEGDAHT (SEQ ID NO: 11).

[0086] Due to the formation of an irreversible amide bond between the covalent bond A in A_Z105 obtained in step (1) and the covalent bond B in B_PP-Ab obtained in step (2), Aβ the coupling and binding of A_Z105 and B_PP-Ab are achieved, Aβ resulting in a monovalent nano self-assembled anti-Aβ antibody (AD_PP-NanoAb Aβ ). The N-terminus of each subunit of the Z105 protein is exposed on the particle surface, and the self-assembly schematic diagram is as Figure 3 shown.

[0087] Example 2

[0088] Design of multivalent nano self-assembled anti-Aβ antibody

[0089] 1. Modification of anti-Aβ antibody functional elements

[0090] (1) The polypeptide molecule (PP) that crosses the blood-brain barrier is fused to the C-terminus of anti-Aβ antibody 1 (Ab1 Aβ ) through a flexible amino acid (SEQ ID NO: 2); the His tag is fused to the N-terminus of anti-Aβ antibody 1 (Ab1 Aβ ) through a flexible amino acid (SEQ ID NO: 1), constructing an element that expresses anti-Aβ antibody 1 (PP-Ab1 Aβ ) with blood-brain barrier crossing function. Among them, PP is Angiopep-2, and Ab1 Aβ is aducanumab, CAS: 1384260-65-4;

[0091] (2) The polypeptide molecule (PP) that crosses the blood-brain barrier is fused to the C-terminus of anti-Aβ antibody 2 (Ab2 Aβ ) through a flexible amino acid (SEQ ID NO: 2); the His tag is fused to the N-terminus of anti-Aβ antibody 2 (Ab2 Aβ ) through a flexible amino acid (SEQ ID NO: 1), constructing an element that expresses anti-Aβ antibody 2 (PP-Ab2 Aβ ) with blood-brain barrier crossing function. Among them, PP is Angiopep-2, and Ab2Aβ It is lecanemab (CAS No.: 1260393-98-3).

[0092] (3) Fuse the polypeptide molecule (PP) that can cross the blood-brain barrier to the C-terminus of anti-Aβ antibody 3 (Ab3 Aβ ) through a flexible amino acid (SEQ ID NO: 2); fuse the His tag to the N-terminus of anti-Aβ antibody 3 (Ab3 Aβ ) through a flexible amino acid (SEQ ID NO: 1), and construct an element that expresses anti-Aβ antibody 3 (PP-Ab3 Aβ ) with the function of crossing the blood-brain barrier. Among them, PP is angiopep-2, and Ab3 Aβ is donanemab, CAS: 1931944-80-7

[0093] 2. PP-Ab1 Aβ , PP-Ab2 Aβ , PP-Ab3 Aβ and A_Z105 are assembled directionally

[0094] (1) Fuse covalent bond A to the site on the outer surface of Z105 protein that is exposed and does not affect the self-assembly of the protein after inserting the exogenous sequence through a flexible amino acid (SEQ ID NO: 4). Specifically, in this step, the covalent bond A is fused to the N-terminus of the Z105 protein subunit;

[0095] Connect the His tag to the N-terminus of covalent bond A through GGGGS (SEQ ID NO: 1) to construct an element that expresses A_Z105, as shown in the schematic diagram Figure 2 . Among them, Figure 2 A is covalent bond A, specifically SpyTag (SEQ ID NO: 10).

[0096] (2) Using a flexible amino acid (SEQ ID NO: 4), insert covalent bond B between the His tag and Ab Aβ of PP-Ab1 Aβ , PP-Ab2 Aβ and PP-Ab3 Aβ obtained in step 1 respectively, to obtain an element that expresses B_PP-Ab1 Aβ , an element that expresses B_PP-Ab2 Aβ and an element that expresses B_PP-Ab3 Aβ . Among them, covalent bond B is specifically SpyCatcher (SEQ ID NO: 11).

[0097] Since the covalent bond A in A_Z105 obtained in step (1) respectively forms irreversible amide bonds with the covalent bond B in B_PP-Ab1 Aβ 、B_PP-Ab2 Aβ and B_PP-Ab3 Aβ obtained in step (2), the coupling combination of A_Z105, B_PP-Ab1 Aβ 、B_PP-Ab2 Aβ and B_PP-Ab3 Aβ is realized, and a multivalent nano self-assembled anti-Aβ antibody (AD_MultiPP-NanoAb Aβ ) is self-assembled. The N-terminus of each subunit of Z105 protein is exposed on the particle surface, and the self-assembly schematic diagram is as shown in Figure 4 .

[0098] Example 3

[0099] Preparation of Nano Self-Assembled Anti-Aβ Antibody

[0100] 1. Transform the pet28a plasmid subcloning the element expressing A_Z105 in Example 1 into Escherichia coli BL21 DE3. After amplification, induce with 0.1 - 1 mM IPTG for 3 - 16 h at an induction temperature of 16 - 37 °C. Purify A_Z105 using Ni-NTA, and further separate the particulate protein by size exclusion chromatography (SEC) to obtain purified A_Z105.

[0101] 2. Transform the pet28a plasmid subcloning the element expressing B_PP-Ab Aβ in Example 1 into Escherichia coli BL21 DE3. After amplification, induce with 0.1 - 1 mM IPTG for 3 - 16 h at an induction temperature of 16 - 37 °C. Purify using Ni-NTA to obtain purified B_PP-Ab Aβ .

[0102] 3. Couple the purified A_Z105 and the purified B_PP-Ab Aβ overnight in Tris buffer at pH 7.4 in a molar ratio of 1:(1.2 - 3); among them, the molar number of the purified A_Z105 is calculated based on the total molar number of the subunit number of A_Z105. Remove the uncoupled free antibody by size exclusion chromatography (SEC) to obtain a nano self-assembled anti-Aβ antibody (AD_PP-NanoAb Aβ ) with B_PP-Ab Aβ surface-oriented.

[0103] Test Example 1

[0104] Preliminary Characterization of AD_PP-NanoAb Aβ ​

[0105] 1. Morphological and appearance identification

[0106] The morphological and appearance of A_Z105 and AD_PP-NanoAb obtained in Example 3 were observed by negative staining transmission electron microscopy Aβ The results are as Figure 5 shown. Among them Figure 5 A_Z105 is on the left, and the arrow indicates a single A_Z105; AD_PP-NanoAb is on the right Aβ and the arrow indicates a single AD_PP-NanoAb Aβ . According to Figure 5 it can be seen that both A_Z105 and AD_PP-NanoAb Aβ formed spherical particles with relatively uniform particle sizes. Compared with A_Z105, AD_PP-NanoAb Aβ showed a more inflated spherical structure

[0107] 2. Particle size analysis

[0108] The particle sizes of A_Z105 and AD_PP-NanoAb obtained in Example 3 were analyzed Aβ . The results showed that compared with A_Z105 without conjugated antibody, the particle size of AD_PP-NanoAb Aβ was larger, increased by about 3 - 5 nm ( Figure 6 ). This data indicates that the self-assembled nanoparticle protein (A_Z105) was successfully conjugated with the antibody (Ab Aβ ), and the antibody carried on the surface increased the particle size of the self-assembled nanoparticle protein

[0109] Test Example 2

[0110] Functional evaluation of AD_PP-NanoAb Aβ

[0111]

[0112] 1. In vitro affinity evaluation

[0112] The free antibody Ab Aβ (lecanemab) and AD_PP-NanoAb obtained in Example 3 Aβ were respectively incubated with β-amyloid protein (Aβ) in an in vitro simulated physiological environment for 0.5 - 3 h, and then the affinity effect was quantitatively detected by ELISA. The results showed that compared with the free antibody Ab Aβ , the binding ability of AD_PP-NanoAb Aβ to Aβ was significantly enhanced ( Figure 7 ), indicating that the nano self-assembled anti-Aβ antibody AD_PP-NanoAb obtained in Example 3 AβCan effectively improve the antigen-binding ability of free antibodies.

[0113] 2. Stability assessment

[0114] (1) Thermal stability

[0115] To evaluate the effect of the nano self-assembled anti-Aβ antibody structure on the thermal stability of the antibody, the free antibody Ab Aβ (lecanemab) and AD_PP-NanoAb obtained in Example 3 Aβ were respectively incubated in a constant temperature environment of 37 °C and 65 °C for 48 h. The free antibody Ab Aβ and AD_PP-NanoAb Aβ before and after treatment were respectively incubated with β-amyloid (Aβ) in an in vitro simulated physiological environment, and then ELISA was used to quantitatively detect the affinity effect. The results showed that after high-temperature treatment, the binding ability of AD_PP-NanoAb Aβ against Aβ did not change significantly compared with that before treatment. However, the free antibody Ab Aβ almost completely lost its binding ability to Aβ under the same high-temperature treatment conditions ( Figure 8 ). This data indicates that the nano self-assembled anti-Aβ antibody AD_PP-NanoAb obtained in Example 3 Aβ can effectively improve the thermal stability of the free antibody, enabling it to maintain the integrity and biological activity of the antibody in a high-temperature environment.

[0116] (2) Serum storage stability

[0117] To evaluate the effect of the nano self-assembled anti-Aβ antibody structure on the serum storage stability of the antibody, the free antibody Ab Aβ (lecanemab) and AD_PP-NanoAb obtained in Example 3 Aβ were respectively placed in serum simulating the physiological environment, and the storage duration was set to 1 week. Dynamic light scattering technology was used to detect the change in the particle size of the samples before and after storage. The results showed that after 1 week of storage, the particle size of AD_PP-NanoAb Aβ did not change significantly compared with that before storage. However, the free antibody Ab Aβ showed obvious fluctuations in particle size under the same storage conditions ( Figure 9 ). This data indicates that the nano self-assembled anti-Aβ antibody AD_PP-NanoAb obtained in Example 3 Aβ has good storage stability in serum, ensuring that it can maintain the complete structure of the antibody during long-term storage. This characteristic plays a key role in avoiding premature clearance and degradation during the long-term blood circulation of the antibody in the body.

[0118] 3. Cell Uptake and Affinity Assessment

[0119] To evaluate the cell uptake ability of the AD_PP-NanoAb obtained in Example 3 Aβ and its binding affinity with β-amyloid (Aβ) in cells, mouse microglial cell line BV-2 was selected as the research object and incubated with AD_PP-NanoAb labeled with green fluorescent dye FITC Aβ , and Aβ labeled with red fluorescent dye Cy5. After incubation for 12 - 24 h, flow cytometry was used to detect the fluorescence intensities of different types of dyes in cells. A free antibody Ab Aβ (lecanemab) group was set as a parallel control. The results showed that the relative fluorescence intensity of the cells treated with AD_PP-NanoAb Aβ was significantly higher than that of the cells treated with the free antibody group Ab Aβ , indicating that more AD_PP-NanoAb Aβ was taken up by the cells ( Figure 10 ). Further measurement and analysis of the co-localization relationship between the red fluorescence signal emitted by Aβ and the green fluorescence signal emitted by AD_PP-NanoAb Aβ or Ab Aβ showed that the co-localization correlation between AD_PP-NanoAb Aβ and Aβ was significantly higher than that of AbAβ ( Figure 11 ). These data indicate that the AD_PP-NanoAb obtained in Example 3 Aβ can not only significantly improve the cell uptake ability of the free antibody, but also enhance the binding affinity with Aβ after entering the cells, providing a strong material basis and mechanism guarantee for subsequent intervention in the pathological process of Alzheimer's disease.

[0120] 4. Biocompatibility Assessment

[0121] The AD_PP-NanoAb obtained in Example 3 Aβ was prepared into solutions with different concentration gradients (10, 50, 100, and 200 μg / mL) and used to treat mouse microglial cell line BV-2. After incubation for 48 h, the Cell Counting Kit-8 reagent was used to detect the changes in cell viability and evaluate the potential toxic effects on cells. Cells without any treatment were set as the negative control group to ensure the accuracy and reliability of the experimental results. The results showed that the viability of the cells treated with AD_PP-NanoAb Aβ showed no obvious change compared with that of the negative control group. In addition, with the increase in the concentration of AD_PP-NanoAb Aβ , the cell viability was still not significantly affected ( Figure 12). This data indicates that the AD_PP-NanoAb obtained in Example 3 Aβ has high biocompatibility, providing important experimental evidence for its safe application in vivo.

[0122] 5. In vivo retention and brain accumulation evaluation

[0123] Healthy adult mice were intravenously injected with 50 μg / mouse of AD_PP-NanoAb carrying the same fluorescence signal Aβ and free antibody Ab Aβ (lecanemab) via the tail vein. At 12 h, 24 h, 48 h, 72 h, 96 h, and 120 h after injection, a small animal in vivo imaging system was used to detect the change in fluorescence intensity in the brain tissue. The results showed that at each set detection time point, the fluorescence intensity in the brain tissue of mice treated with AD_PP-NanoAb Aβ was significantly higher than that in the free antibody Ab Aβ treatment group. Over time, the fluorescence intensity of AD_PP-NanoAb Aβ in the brain tissue showed a gradually increasing trend and reached a peak at 72 h. While for the free antibody Ab Aβ , the fluorescence signal in the mice's bodies basically disappeared at 72 h after injection, indicating a relatively fast clearance rate in vivo ( Figure 13 ). This data indicates that the AD_PP-NanoAb obtained in Example 3 Aβ can significantly extend the retention time of the antibody in vivo and effectively break through the blood-brain barrier, accumulating efficiently in the brain tissue.

[0124] Test Example 3

[0125] Efficacy evaluation of AD_PP-NanoAb Aβ

[0126] 1. Administration to AD model mice

[0127] AD model mice (6 months old, body weight 25 - 30 g) were randomly divided into a negative control group, a free antibody Ab Aβ group, and an AD_PP-NanoAb Aβ group, with 5 mice in each group, and were treated as follows:

[0128] AD_PP-NanoAb Aβ group: The mice were intravenously injected with 50 μg / mouse of the AD_PP-NanoAb obtained in Example 3 Aβ , 2 - 3 times a week for 4 - 6 weeks;

[0129] Free antibody Ab Aβ ​Group: Mice were injected with lecanemab via the tail vein, and each injection maintained the same molar amount of antibody injection dosage as the AD_PP-NanoAb Aβ group, 2 - 3 times a week for 4 - 6 weeks;

[0130] Negative control group: Each injection was the same volume of PBS.

[0131] 2. Evaluation of behavioral improvement

[0132] After completing all the drug administration procedures, the Morris water maze experiment was carried out. First was the adaptation period, the mice were placed in a pool without a platform and allowed to swim freely to fully familiarize themselves with the water environment; then entered the training period, for 5 - 7 consecutive days, the mice were placed in the pool in different quadrants every day, and the escape latency of the mice to find the hidden platform was accurately recorded; finally entered the testing period, the platform was removed, and the residence time of the mice in the original platform quadrant within 60 s was recorded, and the proportion in the total swimming time was calculated. The results showed that during the training period, compared with the negative control group, the escape latency of the free antibody Ab Aβ group and the AD_PP-NanoAb Aβ group mice gradually shortened with the increase of training days, and the shortening trend of the AD_PP-NanoAb Aβ group mice was the most obvious ( Figure 14 ). During the testing period, the proportion of the residence time of the AD_PP-NanoAb Aβ group mice in the original platform quadrant was significantly higher than that of the other two groups ( Figure 15 ). These data indicate that the AD_PP-NanoAb Aβ obtained in Example 3 can significantly improve the spatial learning and memory ability of AD mice.

[0133] 3. Evaluation of neuroprotective performance

[0134] After completing all the drug administration procedures, the mice were euthanized and brain tissue samples were quickly collected. The brain tissue was prepared into ultra-thin sections, and transmission electron microscopy was used to observe the morphology and quantity of synapses in the brain tissue sections. The results showed that compared with the negative control group, the brain tissue sections of the free antibody Ab Aβ group and the AD_PP-NanoAb Aβ group mice showed an increase in the number of synapses. The number of synapses in the AD_PP-NanoAb Aβ group was significantly higher than that in the Ab Aβ group ( Figure 16 ). In addition, in the AD_PP-NanoAb Aβ group, the synaptic structure was clearly distinguishable, the presynaptic expansion area was denser, and the number of vesicles was the largest. This data indicates that the AD_PP-NanoAb AβThe treated AD model mice showed healthy and active neurons, demonstrating the neuroprotective effect of AD_PP-NanoAb Aβ in AD model mice.

[0135] 4. Evaluation of reduction in Aβ deposition effect

[0136] After completing the entire drug administration process, the mice were euthanized and brain tissue samples were quickly collected. Immunoblotting was used to detect the Aβ content in the mouse brain tissue. The results showed that compared with the negative control group, the free antibody Ab Aβ group and the AD_PP-NanoAb Aβ group showed varying degrees of reduction in Aβ deposition in the mouse brain tissue. Among them, the Aβ content in the AD_PP-NanoAb Aβ group was significantly lower than that in other groups, indicating that AD_PP-NanoAb Aβ could effectively reduce Aβ deposition in the brains of AD mice and played a key role in the treatment of AD ( Figure 17 ).

[0137] It can be seen from the above that the nano self-assembled anti-Aβ antibody provided by the present invention can quickly penetrate the blood-brain barrier, significantly enhance the penetration ability of the anti-Aβ antibody into the brain, be accurately delivered to the diseased target site, improve the stability of the anti-Aβ antibody, effectively avoid the degradation of the anti-Aβ antibody, and improve the treatment effect of Alzheimer's disease.

[0138] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, rather than all embodiments. People can also obtain other embodiments without creative efforts based on these embodiments, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A nano self-assembled anti-Aβ antibody, characterized in that, It includes a self-assembled nanoparticle protein functional element and an anti-Aβ antibody functional element; the self-assembled nanoparticle protein functional element and the anti-Aβ antibody functional element are coupled by an amide bond; The self-assembled nanoparticle protein functional element consists of a self-assembled nanoparticle protein, a first flexible amino acid, and a first protein tag; the first protein tag is fused through the first flexible amino acid at a site on the outer surface of the self-assembled nanoparticle protein that is exposed and does not affect protein self-assembly after inserting an exogenous sequence; the self-assembled nanoparticle protein is the Z105 protein; the accession number of the Z105 protein in UniProtKB is A0A497XTC1; The anti-Aβ antibody functional element consists of a second flexible amino acid, a third flexible amino acid, an anti-Aβ antibody, a second protein tag, and a polypeptide for crossing the blood-brain barrier; the second protein tag is fused through the second flexible amino acid at the N-terminus of the anti-Aβ antibody; the polypeptide for crossing the blood-brain barrier is fused through the third flexible amino acid at the C-terminus of the anti-Aβ antibody; the anti-Aβ antibody includes one or more of aducanumab, lecanemab, and donanemab; the polypeptide for crossing the blood-brain barrier is angiopep-2; the amino acid sequence of angiopep-2 is as shown in SEQ ID NO:5; The first protein tag and the second protein tag form a covalent bond through a spontaneous reaction; the first protein tag includes SpyTag, and the second protein tag includes SpyCatcher; the amino acid sequence of SpyTag is as shown in SEQ ID NO:10; the amino acid sequence of SpyCatcher is as shown in SEQ ID NO:11; The amino acid sequence of the first flexible amino acid is GGGGS, GGGGSGGGGS, GGGGSGGGGSGGGGS, or GGGGSGGGGSGGGGSGGGGSGGGGS; The amino acid sequence of the second flexible amino acid is GGGGS, GGGGSGGGGS, GGGGSGGGGSGGGGS, or GGGGSGGGGSGGGGSGGGGSGGGGS; The amino acid sequence of the third flexible amino acid is GGGGS, GGGGSGGGGS, GGGGSGGGGSGGGGS, or GGGGSGGGGSGGGGSGGGGSGGGGS.

2. The nano self-assembled anti-Aβ antibody according to claim 1, characterized in that, The N-terminus of the first protein tag and the N-terminus of the second protein tag are respectively connected with purification tags.

3. The preparation method of the nano self-assembled anti-Aβ antibody according to claim 1 or 2, characterized in that, It includes the following steps: Prokaryotically express and purify the recombinant plasmid subcloning the self-assembled nanoparticle protein functional element to obtain the purified self-assembled nanoparticle protein; Prokaryotically express and purify the recombinant plasmid subcloning the anti-Aβ antibody functional element to obtain the purified anti-Aβ antibody; Couple the purified self-assembled nanoparticle protein and the purified anti-Aβ antibody in a buffer solution to obtain a nano self-assembled anti-Aβ antibody; the pH value of the buffer solution is 7.0 - 7.

4.

4. The preparation method according to claim 3, characterized in that, The molar ratio of the purified self-assembled nanoparticle protein to the purified anti-Aβ antibody is 1:1.2 to 3; the molar number of the purified self-assembled nanoparticle protein is calculated based on the total molar number of the subunit numbers of the purified self-assembled nanoparticle protein; The coupling time is 8 to 16 h, and the buffer solution includes TBS buffer solution or PBS buffer solution.

5. Use of the nano self-assembled anti-Aβ antibody according to claim 1 or 2 or the nano self-assembled anti-Aβ antibody obtained by the preparation method according to claim 3 or 4 in the preparation of a product for treating Alzheimer's disease.

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