Nanometer self-assembly anti-A beta antibody as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of nano drug delivery and antibody preparation, and particularly relates to a nano self-assembly anti-Abeta antibody as well as a preparation method and application thereof. A first protein tag is connected to a site which does not influence protein self-assembly after a self-assembly nanoparticle protein is exposed to the outer surface and an exogenous sequence is inserted, a second protein tag is connected to the N end of an anti-Abeta antibody, and a polypeptide penetrating through a blood brain barrier is connected to the C end of the anti-Abeta antibody; the first protein tag and the second protein tag form a covalent bond through spontaneous reaction, and the anti-Abeta antibody is directionally loaded on the surface of the self-assembled nanoparticle protein to form the nano self-assembled anti-Abeta antibody, so that the stability of the anti-Abeta antibody is improved, and the reduction of curative effect caused by degradation of the anti-Abeta antibody is effectively avoided; moreover, the polypeptide which is connected to the C end of the anti-Abeta antibody and passes through the blood brain barrier can enable the anti-Abeta antibody to break through the blood brain barrier, so that the permeability of the antibody to the brain and the accurate delivery of the antibody to a target site are enhanced, and the treatment effect of the Alzheimer's disease is enhanced.

Description

Technical Field

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

[0002] Alzheimer's disease (AD) is a degenerative disease of the central nervous system that is closely related to age. The disease has an insidious onset, and the most typical manifestation is progressive memory loss or cognitive dysfunction, which may be accompanied by symptoms such as decreased language, executive function, visual-spatial function, and abnormal behavior. It is the most common type of dementia.

[0003] β-amyloid protein (Aβ) accumulates and aggregates in specific brain regions, such as the hippocampus and cerebral cortex (so-called neuritic plaques), and is one of the important pathogenic factors of Alzheimer's disease. Therefore, the extracellular deposition of Aβ is a key target to be tackled in the treatment of AD. Passive immunotherapy against Aβ has important research value and application prospects in the field of AD treatment.

[0004] To date, the U.S. 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 cognitive decline and alleviate AD disease progression, due to the presence of the blood-brain barrier, the penetration rate of antibodies in the brain is low and they cannot reach sufficiently effective concentrations in the lesion area of ​​the brain. Therefore, the efficacy of passive antibody therapy is still greatly limited. Therefore, it is crucial to develop antibody delivery technology that can quickly break through the blood-brain barrier, transport sufficient antibodies to the brain and act on lesion targets to improve the passive immunotherapy system for AD. Summary of the invention

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

[0006] 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; The self-assembling nanoparticle protein functional element comprises a self-assembling nanoparticle protein; the self-assembling nanoparticle protein is exposed on the outer surface and is connected to a first protein tag at a site where the self-assembling protein does not affect the protein self-assembly after the insertion of an exogenous sequence; The anti-Aβ antibody functional element comprises an anti-Aβ antibody; the N-terminus of the anti-Aβ antibody is connected to a second protein tag, and the C-terminus of the anti-Aβ antibody is connected to a polypeptide that crosses the blood-brain barrier; The first protein tag and the second protein tag form a covalent bond through a spontaneous reaction.

[0007] Preferably, the self-assembling nanoparticle protein is exposed on the outer surface and the site where the insertion of the exogenous sequence does not affect the protein self-assembly is connected to the first protein tag through the first flexible amino acid; The N-terminus of the anti-Aβ antibody is connected to the second protein tag via a second flexible amino acid, and the C-terminus is connected to a polypeptide that crosses the blood-brain barrier via a third flexible amino acid; The amino acid sequence of the first flexible amino acid is GGGGS, GGGGSGGGGS, GGGGSGGGGSGGGGS or GGGGSGGGGSGGGGSGGGGS; The amino acid sequence of the second flexible amino acid is GGGGS, GGGGSGGGGS, GGGGSGGGGSGGGGS or GGGGSGGGGSGGGGSGGGGS; The amino acid sequence of the third flexible amino acid is GGGGS, GGGGSGGGGS, GGGGSGGGGSGGGGS or GGGGSGGGGSGGGGSGGGGS.

[0008] Preferably, the anti-Aβ antibody comprises one or more of aducanumab, lencanizumab and donetumab.

[0009] Preferably, the self-assembling nanoparticle protein includes Z105 protein; the accession number of the subunit of the Z105 protein in UniProtKB is A0A497XTC1.

[0010] Preferably, the polypeptide that crosses the blood-brain barrier includes one or more of angiopeptide-2, HIV-penetrating peptide, leptin, amylin and insulin polypeptide.

[0011] Preferably, when the first protein tag is SpyTag, the second protein tag is SpyCatcher; When the first protein tag is SnoopTag, the second protein tag is SnoopCatcher; When the first protein tag is DogTag, the second protein tag is DogCatcher.

[0012] Preferably, the N-terminus of the first protein tag and the N-terminus of the second protein tag are connected to purification tags respectively.

[0013] The present invention also provides a method for preparing the nano self-assembled anti-Aβ antibody described in the above technical solution, comprising the following steps: Prokaryotic expression and purification of the recombinant plasmid subcloned with the self-assembling nanoparticle protein functional element to obtain the purified self-assembling nanoparticle protein; Prokaryotic expression and purification of the recombinant plasmid subcloned with the anti-Aβ antibody functional element to obtain a purified anti-Aβ antibody; The purified self-assembled nanoparticle protein and the purified anti-Aβ antibody are coupled in a buffer to obtain a nano self-assembled anti-Aβ antibody; the pH value of the buffer is 7.0-7.4.

[0014] 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 subunits of the purified self-assembled nanoparticle protein; The coupling time is 8 to 16 h, and the buffer comprises TBS buffer or PBS buffer; The present invention also provides the use of the nano self-assembled anti-Aβ antibody described in the above technical solution or the nano self-assembled anti-Aβ antibody prepared by the preparation method described in the above technical solution in the preparation of products for treating Alzheimer's disease.

[0015] Beneficial effects: The present invention provides a nano self-assembling anti-Aβ antibody, comprising a self-assembling nanoparticle protein functional element and an anti-Aβ antibody functional element; the self-assembling nanoparticle protein functional element comprises a self-assembling nanoparticle protein; the self-assembling nanoparticle protein is exposed on the outer surface and is connected to a first protein tag at a site where the self-assembling nanoparticle protein is inserted without affecting the protein self-assembly; the anti-Aβ antibody functional element comprises an anti-Aβ antibody; the N-terminus of the anti-Aβ antibody is connected to a second protein tag, and the C-terminus is connected to a polypeptide that crosses the blood-brain barrier; the first protein tag and the second protein tag form a covalent bond through a spontaneous reaction. The present invention connects a polypeptide that crosses the blood-brain barrier 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; connects a second protein tag to the N-terminus of the anti-Aβ antibody, which can spontaneously react with the first protein tag exposed to the outer surface of the self-assembled nanoparticle protein and does not affect the site of protein self-assembly after the exogenous sequence is inserted to form a covalent bond, and the anti-Aβ antibody is geometrically enriched on the surface of the self-assembled nanoparticle protein to form a nano self-assembled anti-Aβ antibody. The anti-Aβ antibody is exposed on the surface of the self-assembled nanoparticle protein, thereby improving the stability of the anti-Aβ antibody, effectively avoiding the reduction in efficacy due to degradation of the anti-Aβ antibody, and enhancing the therapeutic effect of Alzheimer's disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0017] Figure 1 This is a schematic diagram of the structure of the anti-Aβ antibody functional element of Example 1; Figure 2 A_Z105 expression element and B_PP-Ab in Example 1 Aβ Schematic diagram of the structure of the expression element; Figure 3 This is a schematic diagram of the directed assembly of the monovalent nano self-assembled anti-Aβ antibody in Example 1; Figure 4 This is a schematic diagram of the directed assembly of multivalent nano self-assembled anti-Aβ antibodies in Example 2; Figure 5 Negative staining transmission electron microscopy images of A_Z105 and nano-self-assembled anti-Aβ antibody; Figure 6 A_Z105 and AD_PP-NanoAb Aβ Particle size detection chart; Figure 7 For free anti-Aβ antibody and AD_PP-NanoAb Aβ In vitro affinity assay diagram; Figure 8 Based on heat treatment of free anti-Aβ antibody and AD_PP-NanoAbAβ Affinity detection diagram of Fig. 9 Based on serum storage of free anti-Aβ antibody and AD_PP-NanoAb Aβ Particle size variation diagram; Fig.10 Intracellular free anti-Aβ antibody and AD_PP-NanoAb Aβ Relative fluorescence intensity detection result diagram; Fig.11 Intracellular free anti-Aβ antibody and AD_PP-NanoAb Aβ The Pearson correlation coefficient test result diagram; Fig.12 For different AD_PP-NanoAb Aβ The relative activity test results of cells at different concentrations; Fig.13 Inject free anti-Aβ antibody and AD_PP-NanoAb into the tail vein of mice Aβ Post-fluorescence intensity analysis result diagram; Fig.14 This is a graph showing the escape latency test results of mice in different treatment groups at different training durations; Fig.15 The target limited residence time detection results of mice in different treatment groups during the test period are shown in the figure; Fig.16 This is the result of the average synapse number detection in the brain tissue of mice in different treatment groups; Fig.17 The graph shows the results of relative Aβ content detection in brain tissue of mice in different treatment groups. DETAILED DESCRIPTION

[0018] 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; The self-assembling nanoparticle protein functional element comprises a self-assembling nanoparticle protein; the self-assembling nanoparticle protein is exposed on the outer surface and is connected to a first protein tag at a site where the self-assembling protein does not affect the protein self-assembly after the insertion of an exogenous sequence; The anti-Aβ antibody functional element comprises an anti-Aβ antibody; the N-terminus of the anti-Aβ antibody is connected to a second protein tag, and the C-terminus of the anti-Aβ antibody is connected to a polypeptide that crosses the blood-brain barrier; The first protein tag and the second protein tag form a covalent bond through a spontaneous reaction.

[0019] As an embodiment, the self-assembled nanoparticle protein of the present invention includes 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 nanoprotein particle formed by self-assembly of 60 identical subunits, and the N-terminus of each subunit is exposed on the surface of the particle, derived from 6,7-dimethyl-8-ribityllumazine synthase (UniProtK accession number A0A497XTC1), named after the 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 anti-Aβ antibodies to achieve efficient anti-Aβ antibody delivery.

[0020] As an embodiment, the site where the self-assembling nanoparticle protein of the present invention is exposed on the outer surface and does not affect the self-assembly of the protein after the insertion of an exogenous sequence is connected to the first protein tag through a first flexible amino acid. As an embodiment, the site where the self-assembling nanoparticle protein of the present invention is exposed on the outer surface and does not affect the self-assembly of the protein after the insertion of an exogenous sequence includes the N-terminus of the subunit of the self-assembling nanoparticle protein.

[0021] As an embodiment, the C-terminus of the first protein tag of the present invention is connected to the N-terminus of the monomer of the self-assembling 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).

[0022] As an embodiment, the N-terminus of the first protein tag of the present invention is connected to a purification tag. As an embodiment, the purification tag of the present invention includes a His tag. The present invention connects 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, which is convenient for subsequent protein purification.

[0023] As an embodiment, the anti-Aβ antibody of the present invention includes one or more of aducanumab, lencanezumab and donenetumab; as another embodiment, the anti-Aβ antibody of the present invention is lencanezumab, or a combination of aducanumab, lencanezumab and donenetumab. In the specific embodiments of the present invention, lencanezumab and the combination of aducanumab, lencanezumab and donenetumab are used as examples for illustration, but they cannot be understood as the entire protection scope of the present invention. Since different anti-Aβ antibodies target different links in the pathogenesis of Alzheimer's disease, compared with single antibody treatment, the combination of aducanumab, lencanezumab and donenetumab has multivalency, can synergistically act from multiple targets, intervene in the pathological process of Alzheimer's disease more comprehensively, and further enhance the therapeutic effect of Alzheimer's disease. As an embodiment, the CAS numbers of aducanumab, lencanezumab and donetumab described in the present invention are 1384260-65-41260393-98-3 and 1931944-80-7, respectively.

[0024] As an embodiment, the N-terminus of the anti-Aβ antibody of the present invention is connected to the second protein tag via the second flexible amino acid, and the C-terminus is connected to the polypeptide that crosses the blood-brain barrier via the third flexible amino acid.

[0025] As an embodiment, the amino acid sequence of the second 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). The present invention connects the second protein tag to the N-terminus of the anti-Aβ antibody, which has the advantage of improving the purification efficiency compared with the C-terminus connection, and the use of the second flexible amino acid connection can protect the functional independence of the protein domains on both sides. The amino acid sequence of the third 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). 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.

[0026] As an embodiment, the N-terminus of the second protein tag of the present invention is connected to a purification tag. As an embodiment, the purification tag of the present invention includes a His tag. The present invention has the advantage of improving the purification efficiency by connecting a purification tag to the N-terminus of the second protein tag.

[0027] As an embodiment, the polypeptide that crosses the blood-brain barrier of the present invention includes one or more of vascular peptide-2, HIV penetrating peptide, leptin, islet amylin and insulin peptide. As an embodiment, the polypeptide that crosses the blood-brain barrier of the present invention is vascular peptide-2. As an embodiment, the amino acid sequence of vascular peptide-2 of 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 HIV penetrating peptide of the present invention is GRKKRRQRRRPQ (SEQ ID NO: 6), which relies on the cationic properties rich in arginine and penetrates the cell membrane and BBB through adsorption-mediated endocytosis (AMT). As an embodiment, the leptin of the present invention binds to the leptin receptor (LepR) and crosses the BBB by receptor-mediated transport. The core sequence of leptin that binds to the leptin receptor is LPYFDLA (SEQ ID NO: 7). As an embodiment, the amino acid sequence of the amylin of the present invention is KCNTATCATQRLANFLVHSSNNFGPILPPTNVGSNTY (SEQ ID NO: 8), which penetrates the BBB through adsorption-mediated transcellular action, relying on its amphipathic structure. As an embodiment, the insulin polypeptide of the present invention binds to the insulin receptor of brain endothelial cells, triggering receptor-mediated transport, and the sequence of the insulin α chain receptor binding domain in the insulin polypeptide is GIVEQCCTSICSLYQLENYCN (SEQID NO: 9).

[0028] The polypeptide that crosses the blood-brain barrier of the present invention has the significant advantages of small molecular weight and low toxic side effects, and is easy to modify on the anti-Aβ antibody to enhance the permeability of the anti-Aβ antibody to the brain; and the polypeptide that crosses the blood-brain barrier of 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, enhance the permeability of the anti-Aβ antibody to the brain and the precise delivery to the target site. In the specific embodiments of the present invention, vascular peptide-2 is used as an example for explanation, but it cannot be understood as the entire protection scope of the present invention.

[0029] The first protein tag and the second protein tag of 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 of the present invention is SpyTag, the second protein tag is SpyCatcher; as another embodiment, when the first protein tag of the present invention is SnoopTag, the second protein tag is SnoopCatcher; as another embodiment, when the first protein tag of the present invention is DogTag, the second protein tag is DogCatcher.

[0030] The present invention also provides a method for preparing the nano self-assembled anti-Aβ antibody described in the above technical solution, comprising the following steps: Prokaryotic expression and purification of the recombinant plasmid subcloned with the self-assembling nanoparticle protein functional element to obtain the purified self-assembling nanoparticle protein; Prokaryotic expression and purification of the recombinant plasmid subcloned with the anti-Aβ antibody functional element to obtain a purified anti-Aβ antibody; The purified self-assembled nanoparticle protein and the purified anti-Aβ antibody are coupled in a buffer to obtain a nano self-assembled anti-Aβ antibody; the pH value of the buffer is 7.0-7.4.

[0031] The present invention performs prokaryotic expression and purification on a recombinant plasmid subcloned with the self-assembling nanoparticle protein functional element to obtain the purified self-assembling nanoparticle protein. The present invention has no strict requirements on the construction of the recombinant plasmid, the prokaryotic expression step and the purification step, and conventional operations in the art can be used. As an embodiment, the basic plasmid of the recombinant plasmid of the present invention includes a pet28a plasmid. As an embodiment, the prokaryotic expression of the present invention includes Escherichia coli BL21DE3 As an embodiment, the purification of the present invention comprises Ni-NTA purification and size exclusion chromatography purification performed sequentially.

[0032] The present invention performs prokaryotic expression and purification on a recombinant plasmid subcloned with the anti-Aβ antibody functional element to obtain the purified anti-Aβ antibody. The present invention has no strict requirements on the construction of the recombinant plasmid, the prokaryotic expression step and the purification step, and conventional operations in the art can be used. As an embodiment, the basic plasmid of the recombinant plasmid of the present invention includes a pet28a plasmid. As an embodiment, the prokaryotic expression of the present invention includes Escherichia coli BL21DE3 As an embodiment, the purification of the present invention comprises Ni-NTA purification.

[0033] 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 coupled product; the coupled product contains nano self-assembled anti-Aβ antibody; the pH value of the buffer is 7.0-7.4.

[0034] As one embodiment, the molar ratio of the purified self-assembled nanoparticle protein of the present invention 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 molar number of subunits of the purified self-assembled nanoparticle protein; as another embodiment, the molar ratio of the purified self-assembled nanoparticle protein of the present invention to the purified anti-Aβ antibody is 1:(1.5~2).

[0035] As an embodiment, the coupling time of the present invention is 8 to 16 hours; as another embodiment, the coupling time of the present invention is 10 to 15 hours; as another embodiment, the coupling time of the present invention is 12 hours. As an embodiment, the buffer of the present invention includes TBS buffer or PBS buffer. The buffer of the present invention can prevent protein precipitation.

[0036] As an embodiment, after obtaining the coupled product, the present invention removes uncoupled free antibodies in the coupled product by size exclusion chromatography to obtain nano self-assembled anti-Aβ antibodies.

[0037] The preparation method of the present invention is simple to operate, and the obtained nano self-assembled anti-Aβ antibody can quickly 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 to the brain, and accurately delivers it to the target site of the lesion, providing a new strategy with great potential for the treatment of Alzheimer's disease.

[0038] In view of the advantages of the nano self-assembled anti-Aβ antibody described in the present invention, the use 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 falls within the protection scope of the present invention.

[0039] The present invention utilizes an Alzheimer's disease mouse model and finds that after administration of nano self-assembled anti-Aβ antibodies, it can significantly improve animal behavior, improve brain tissue nerve damage, and reduce brain tissue Aβ deposition. It is a highly potential new strategy for passive antibody treatment of Alzheimer's disease.

[0040] To further illustrate the present invention, a nanometer self-assembled anti-Aβ antibody and its preparation method and application provided by the present invention are described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0041] Example 1 Design of monovalent nano-self-assembled anti-Aβ antibodies 1. Modification of anti-Aβ antibody functional elements The polypeptide molecule (PP) that crosses the blood-brain barrier is fused to the anti-Aβ antibody (Ab) through a flexible amino acid (SEQ ID NO: 2). Aβ ) at the C-terminus; the His tag was fused to the anti-Aβ antibody (Ab) through a flexible amino acid (SEQ ID NO: 1) Aβ ) to construct an anti-Aβ antibody (PP-Ab) expressing blood-brain barrier crossing function. Aβ ) components, as shown in the schematic diagram Figure 1 As shown. Among them, Figure 1 PP in the middle is Angiopep-2, Ab Aβ It is Lecanemab (CAS No. 1260393-98-3).

[0042] 2. PP-Ab Aβ Directional assembly with Z105 (1) The covalent bond A is fused to a site of the Z105 protein that is exposed to the outer surface and does not affect the self-assembly of the protein after the insertion of the exogenous sequence through a flexible amino acid (amino acid sequence as shown in SEQ ID NO: 4). In this step, the covalent bond A is fused to the N-terminus of the Z105 protein subunit; The His tag was connected to the N-terminus of the covalent bond A via GGGGS (SEQ ID NO: 1) to construct an element expressing A_Z105, as shown in the schematic diagram. Figure 2 As shown. Among them, Figure 2 A is a covalent bond A, specifically SpyTag, and its amino acid sequence is RGVPHIVMVDAYKRYK (SEQ ID NO: 10).

[0043] (2) Using flexible amino acids (amino acid sequence as shown in SEQ ID NO: 4), the PP-Ab obtained in step 1 Aβ His tag and Ab Aβ Insert a covalent bond B between them to construct the expression B_PP-Ab Aβ The components are shown in the schematic diagram Figure 2 As shown. Among them, Figure 2Wherein B is a covalent bond B, specifically SpyCatcher, and its amino acid sequence is VTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATMELRDSSGKTISTWISDGHVKDFYLYPGKYTFVETAAPDGYEVATPIEFTVNEDGQVTVDGEATEGDAHT (SEQ ID NO: 11).

[0044] Due to the covalent bond A in A_Z105 obtained in step (1) and B_PP-Ab obtained in step (2) Aβ An irreversible amide bond is formed between the covalent bonds B to realize A_Z105 and B_PP-Ab Aβ The monovalent nano self-assembled anti-Aβ antibody (AD_PP-NanoAb Aβ ), the N-terminus of each subunit of Z105 protein is exposed on the particle surface, and the self-assembly diagram is shown in Figure 3 shown.

[0045] Example 2 Design of multivalent nano-assembly anti-Aβ antibodies 1. Modification of anti-Aβ antibody functional elements (1) The polypeptide molecule (PP) that crosses the blood-brain barrier is fused to the anti-Aβ antibody 1 (Ab1) through a flexible amino acid (SEQ ID NO: 2). Aβ ) at the C-terminus; the His tag was fused to the anti-Aβ antibody 1 (Ab1) via a flexible amino acid (SEQ ID NO: 1) Aβ ) to construct an anti-Aβ antibody 1 (PP-Ab1) expressing blood-brain barrier-crossing function. Aβ ) elements. Among them, PP is Angiopep-2, Ab1 Aβ Aducanumab, CAS: 1384260-65-4; (2) The polypeptide molecule (PP) that crosses the blood-brain barrier is fused to the anti-Aβ antibody 2 (Ab2) through a flexible amino acid (SEQ ID NO: 2). Aβ ) at the C-terminus; the His tag was fused to the anti-Aβ antibody 2 (Ab2) via a flexible amino acid (SEQ ID NO: 1) Aβ ) to construct an anti-Aβ antibody 2 (PP-Ab2) with blood-brain barrier crossing function. Aβ ) elements. Among them, PP is Angiopep-2, Ab2 Aβ It is Lecanemab (CAS No. 1260393-98-3).

[0046] (3) The polypeptide molecule (PP) that crosses the blood-brain barrier is fused to the anti-Aβ antibody 3 (Ab3 Aβ ) at the C-terminus; the His tag was fused to the anti-Aβ antibody 3 (Ab3 Aβ ) to construct an anti-Aβ antibody 3 (PP-Ab3) with blood-brain barrier crossing function. Aβ ) elements. Among them, PP is Angiopep-2, Ab3 Aβ Donanemab, CAS: 1931944-80-7 2. PP-Ab1 Aβ PP-Ab2 Aβ PP-Ab3 Aβ Directional assembly with A_Z105 (1) The covalent bond A is fused to a site of the Z105 protein that is exposed on the outer surface and does not affect the self-assembly of the protein after the insertion of the exogenous sequence through a flexible amino acid (SEQ ID NO: 4). In this step, the covalent bond A is fused to the N-terminus of the Z105 protein subunit; The His tag was connected to the N-terminus of the covalent bond A via GGGGS (SEQ ID NO: 1) to construct an element expressing A_Z105, as shown in the schematic diagram. Figure 2 As shown. Among them, Figure 2 A in the sequence is a covalent bond A, specifically SpyTag (SEQ ID NO: 10).

[0047] (2) Using the flexible amino acid (SEQ ID NO: 4), the PP-Ab1 obtained in step 1 was Aβ PP-Ab2 Aβ and PP-Ab3 Aβ His tag and Ab Aβ Insert a covalent bond B between them to obtain the expression B_PP-Ab1 Aβ Elements, expression of B_PP-Ab2 Aβ Elements and expression of B_PP-Ab3 Aβ wherein the covalent bond B is specifically SpyCatcher (SEQ ID NO: 11).

[0048] Since the covalent bond A in A_Z105 obtained in step (1) is respectively with B_PP-Ab1 obtained in step (2) Aβ , B_PP-Ab2 Aβ and B_PP-Ab3 AβThe covalent bond B forms an irreversible amide bond to achieve A_Z105, B_PP-Ab1 Aβ , B_PP-Ab2 Aβ and B_PP-Ab3 Aβ The multivalent nano-assembled anti-Aβ antibody (AD_MultiPP-NanoAb) was obtained by coupling and self-assembly. Aβ ), the N-terminus of each subunit of Z105 protein is exposed on the particle surface, and the self-assembly diagram is shown in Figure 4 shown.

[0049] Example 3 Preparation of Nano-Self-Assembled Anti-Aβ Antibodies 1. The pet28a plasmid subcloned with the element expressing A_Z105 in Example 1 was transformed into E. coli BL21 DE3, and after amplification, 0.1-1 mM IPTG was used for induction for 3-16 hours at a temperature of 16-37° C. A_Z105 was purified by Ni-NTA, and the particle protein was further separated by size exclusion chromatography (SEC) to obtain purified A_Z105.

[0050] 2. Subclone the B_PP-Ab expressed in Example 1 Aβ The pet28a plasmid containing the elements was transformed into E. coli BL21 DE3, and after amplification, 0.1~1mM IPTG was used for induction for 3~16h, and the induction temperature was 16~37℃. Ni-NTA purification was used to obtain the purified B_PP-Ab Aβ .

[0051] 3. Purified A_Z105 and purified B_PP-Ab Aβ , coupled overnight in a Tris buffer at pH 7.4 at a molar ratio of 1: (1.2-3); wherein the molar number of purified A_Z105 is calculated based on the total molar number of subunits of A_Z105. Uncoupled free antibodies were removed by size exclusion chromatography (SEC) to obtain surface-oriented B_PP-Ab Aβ Nano-assembled anti-Aβ antibody (AD_PP-NanoAb Aβ ).

[0052] Test Example 1 AD_PP-NanoAb Aβ Preliminary characterization 1. Morphological and appearance identification Negative staining transmission electron microscopy observation of A_Z105 and AD_PP-NanoAb obtained in Example 3 Aβ The appearance of Figure 5 As shown. Figure 5The left side shows A_Z105, and the arrow indicates a single A_Z105; the right side shows AD_PP-NanoAb Aβ , the arrow indicates a single AD_PP-NanoAb Aβ .according to Figure 5 It can be seen that A_Z105 and AD_PP-NanoAb Aβ All of them formed spherical particles with relatively uniform particle size. Compared with A_Z105, AD_PP-NanoAb Aβ Shows a more expanded spherical structure.

[0053] 2. Particle size analysis A_Z105 and AD_PP-NanoAb obtained in Example 3 Aβ The particle size of AD_PP-NanoAb was analyzed. The results showed that compared with A_Z105 without antibody conjugation, AD_PP-NanoAb Aβ The particle size is larger, increasing by about 3~5 nm ( Figure 6 This data indicates that the self-assembled nanoparticle protein (A_Z105) and the antibody (Ab Aβ ) were successfully coupled, and the antibodies carried on the surface increased the particle size of the self-assembled nanoparticle protein.

[0054] Test Example 2 AD_PP-NanoAb Aβ Functional assessment 1. In vitro affinity assessment Free antibody Ab Aβ (Lencanetumab) and AD_PP-NanoAb obtained in Example 3 Aβ The samples were incubated with β-amyloid protein (Aβ) in a simulated physiological environment in vitro for 0.5-3 hours, and then the affinity was quantitatively detected by ELISA. The results showed that compared with free antibody Ab Aβ , AD_PP-NanoAb Aβ The binding ability to Aβ was significantly enhanced ( Figure 7 ), indicating that the nano self-assembled anti-Aβ antibody AD_PP-NanoAb obtained in Example 3 Aβ It can effectively enhance the antigen binding ability of free antibodies.

[0055] 2. Stability Assessment (1) Thermal stability To evaluate the effect of nano-self-assembled anti-Aβ antibody structure on the thermal stability of antibodies, free antibodies Ab Aβ (Lencanetumab) and AD_PP-NanoAb obtained in Example 3 AβIncubate at 37°C and 65°C for 48h. Aβ and AD_PP-NanoAb Aβ They were incubated with β-amyloid protein (Aβ) in a simulated physiological environment in vitro, and then the affinity effect was quantitatively detected by ELISA. The results showed that after high temperature treatment, AD_PP-NanoAb Aβ The binding capacity to Aβ did not change significantly compared to before treatment. Aβ Under the same high temperature treatment conditions, the binding ability to Aβ was almost completely lost ( Figure 8 ). This data shows that the nano self-assembled anti-Aβ antibody AD_PP-NanoAb obtained in Example 3 Aβ It can effectively improve the thermal stability of free antibodies, so that they can still maintain the integrity and biological activity of antibodies in high temperature environments.

[0056] (2) Serum storage stability In order to evaluate the effect of nano-self-assembled anti-Aβ antibody structure on the storage stability of antibody serum, free antibody Ab Aβ (Lencanetumab) and AD_PP-NanoAb obtained in Example 3 Aβ The samples were placed in serum simulating a physiological environment and stored for one week. Dynamic light scattering technology was used to detect the changes in sample particle size before and after storage. The results showed that after one week of storage, AD_PP-NanoAb Aβ The particle size of the free antibody Ab Aβ Under the same storage conditions, the particle size fluctuates significantly ( Fig. 9 ). This data shows that the nano self-assembled anti-Aβ antibody AD_PP-NanoAb obtained in Example 3 Aβ Storage stability in serum ensures that the antibody can maintain its intact structure during long-term storage. This property plays a key role in avoiding premature clearance and degradation during the long-term blood circulation of antibodies in the body.

[0057] 3. Cellular Uptake and Affinity Assessment To evaluate the AD_PP-NanoAb obtained in Example 3 Aβ The cellular uptake ability of AD_PP-NanoAb and its binding affinity with β-amyloid protein (Aβ) in cells were investigated. Mouse microglial cells BV-2 were selected as the research object and were combined with AD_PP-NanoAb labeled with green fluorescent dye FITC. Aβ, and Aβ labeled with red fluorescent dye Cy5. After incubation for 12-24 hours, flow cytometry was used to detect the fluorescence intensity of different types of dyes in the cells. Aβ The results showed that AD_PP-NanoAb Aβ The relative fluorescence intensity of the treated cells was significantly higher than that of the free antibody group Ab Aβ Treated cells showed more AD_PP-NanoAb Aβ Uptake by cells ( Fig.10 ). The red fluorescence signal emitted by Aβ and AD_PP-NanoAb were further measured and analyzed. Aβ or Ab Aβ The co-localization relationship between the green fluorescence signals emitted by AD_PP-NanoAb Aβ The co-localization correlation with Aβ was significantly higher than that with AbAβ ( Fig.11 ). These data show that the AD_PP-NanoAb obtained in Example 3 Aβ It can not only significantly improve the cellular uptake ability of free antibodies, but also enhance the binding affinity with Aβ after entering the cells, providing a strong material basis and mechanism of action guarantee for subsequent intervention in the pathological process of Alzheimer's disease.

[0058] 4. Biocompatibility Assessment The AD_PP-NanoAb obtained in Example 3 Aβ The solution was prepared with different concentration gradients (10, 50, 100 and 200 μg / mL) and treated with mouse microglial BV-2 cells. After incubation for 48 hours, the Cell Counting Kit-8 reagent was used to detect the changes in cell activity and evaluate the potential toxicity to the cells. Cells without any treatment were set as negative control groups to ensure the accuracy and reliability of the experimental results. The results showed that AD_PP-NanoAb Aβ Compared with the negative control group, the activity of cells treated with AD_PP-NanoAb Aβ The cell activity was not significantly affected even with the increase of the concentration ( Fig.12 ). This data shows that the AD_PP-NanoAb obtained in Example 3 Aβ It has high biocompatibility, which provides important experimental basis for its safe application in vivo.

[0059] 5. Assessment of in vivo retention and brain accumulation Healthy adult mice were injected with 50 μg / mouse AD_PP-NanoAb carrying the same fluorescent signal into the tail vein Aβand free antibody Ab Aβ (Lencanetumab). At 12h, 24h, 48h, 72h, 96h, and 120h after injection, the small animal in vivo imaging system was used to detect the changes in fluorescence intensity in brain tissue. The results showed that at each set detection time point, AD_PP-NanoAb Aβ The fluorescence intensity in the brain tissue of treated mice was significantly higher than that of free antibody Ab Aβ Treatment group. Over time, AD_PP-NanoAb Aβ The fluorescence intensity in brain tissue showed a trend of gradual increase and reached a peak at 72 h. Aβ At 72 h after injection, the fluorescent signal in the mouse body basically disappeared, indicating that its clearance rate in the body was relatively fast ( Fig.13 ). This data shows that the AD_PP-NanoAb obtained in Example 3 Aβ It can significantly prolong the retention time of antibodies in the body, effectively break through the blood-brain barrier, and efficiently accumulate in brain tissue.

[0060] Test Example 3 AD_PP-NanoAb Aβ Evaluation of therapeutic efficacy 1. Drug administration to AD model mice AD model mice (6 months old, body weight 25-30 g) were randomly divided into negative control group and free antibody Ab group. Aβ Group and AD_PP-NanoAb Aβ There were 5 mice in each group, and they were treated as follows: AD_PP-NanoAb Aβ Group: 50 μg / mouse of AD_PP-NanoAb obtained in Example 3 was injected into the tail vein of mice Aβ , 2 to 3 times a week for 4 to 6 weeks; Free antibody Ab Aβ Group: Mice were injected with lencanezumab by tail vein, and each injection was kept constant with AD_PP-NanoAb Aβ The same molar amount of antibody was injected into each group, 2 to 3 times a week, for 4 to 6 weeks; Negative control group: the same volume of PBS was injected each time.

[0061] 2. Behavioral improvement evaluation After completing the entire dosing process, the Morris water maze experiment was carried out. The first is the adaptation period, in which the mice are placed in a pool without a platform, allowing them to swim freely and fully familiarize themselves with the water environment; then enter the training period, for 5 to 7 consecutive days, select different quadrants to place the mice in the pool every day, and accurately record the escape latency of the mice to find the hidden platform; finally enter the test period, remove the platform, record the time the mice stay in the original platform quadrant within 60 seconds, and calculate its proportion of the total swimming time. The results showed that during the training period, compared with the negative control group, the free antibody Ab Aβ Group and AD_PP-NanoAb Aβ The escape latency of mice in both groups gradually shortened with the increase of training days. Aβ The shortening trend was most obvious in the mice of group Fig.14 During the test period, AD_PP-NanoAb Aβ The proportion of time that mice in group A stayed in the original platform quadrant was significantly higher than that in the other two groups ( Fig.15 ). These data show that the AD_PP-NanoAb obtained in Example 3 Aβ It can significantly improve the spatial learning and memory ability of AD mice.

[0062] 3. Evaluation of neuroprotective properties After the entire dosing process was completed, the mice were euthanized and brain tissue samples were quickly collected. The brain tissue was prepared into ultrathin sections, and the morphology and number of synapses in the brain tissue sections were observed using a transmission electron microscope. The results showed that compared with the negative control group, free antibody Ab Aβ Group and AD_PP-NanoAb Aβ The brain tissue sections of the mice in the AD_PP-NanoAb group all showed an increase in the number of synapses. Aβ The number of synapses in the group was significantly higher than that in the Ab Aβ Group( Fig.16 ). In addition, in AD_PP-NanoAb Aβ In the group, the synaptic structure is clearly discernible, the presynaptic expansion area is more dense, and the number of vesicles is the largest. This data shows that the AD_PP-NanoAb obtained in Example 3 Aβ The neurons of AD model mice treated with AD_PP-NanoAb showed healthy and active state, which proved that AD_PP-NanoAb Aβ It exerted a good neuroprotective effect in AD model mice.

[0063] 4. Evaluation of Aβ deposition reduction effect After the entire dosing process was completed, the mice were euthanized and brain tissue samples were quickly collected. The Aβ content of the mouse brain tissue was detected using immunoblotting technology. The results showed that compared with the negative control group, the free antibody AbAβ Group and AD_PP-NanoAb Aβ The Aβ deposition in the brain tissue of the mice in the two groups was reduced to varying degrees. Aβ The Aβ content in the group was significantly lower than that in the other groups, indicating that AD_PP-NanoAb Aβ It can effectively reduce Aβ deposition in the brain of AD mice and plays a key role in AD treatment ( Fig.17 ).

[0064] Based on the above content, it can be seen 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, accurately deliver it to the target site of the lesion, improve the stability of the anti-Aβ antibody, effectively avoid the degradation of the anti-Aβ antibody, and improve the therapeutic effect of Alzheimer's disease.

[0065] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, 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: Includes self-assembling nanoparticle protein functional elements and anti-Aβ antibody functional elements; The self-assembling nanoparticle protein functional element comprises a self-assembling nanoparticle protein; the self-assembling nanoparticle protein is exposed on the outer surface and is connected to a first protein tag at a site where the self-assembling protein does not affect the protein self-assembly after the insertion of an exogenous sequence; The anti-Aβ antibody functional element comprises an anti-Aβ antibody; the N-terminus of the anti-Aβ antibody is connected to a second protein tag, and the C-terminus of the anti-Aβ antibody is connected to a polypeptide that crosses the blood-brain barrier; The first protein tag and the second protein tag form a covalent bond through a spontaneous reaction.

2. The nano self-assembled anti-Aβ antibody according to claim 1, characterized in that: The self-assembling nanoparticle protein is exposed on the outer surface and the site where the insertion of the exogenous sequence does not affect the protein self-assembly is connected to the first protein tag through the first flexible amino acid; The N-terminus of the anti-Aβ antibody is connected to the second protein tag via a second flexible amino acid, and the C-terminus is connected to a polypeptide that crosses the blood-brain barrier via a third flexible amino acid; The amino acid sequence of the first flexible amino acid is GGGGS, GGGGSGGGGS, GGGGSGGGGSGGGGS or GGGGSGGGGSGGGGSGGGGS; The amino acid sequence of the second flexible amino acid is GGGGS, GGGGSGGGGS, GGGGSGGGGSGGGGS or GGGGSGGGGSGGGGSGGGGS; The amino acid sequence of the third flexible amino acid is GGGGS, GGGGSGGGGS, GGGGSGGGGSGGGGS or GGGGSGGGGSGGGGSGGGGS.

3. The nano self-assembled anti-Aβ antibody according to claim 1 or 2, characterized in that: The anti-Aβ antibody includes one or more of aducanumab, lencanizumab and donetumab.

4. The nano self-assembled anti-Aβ antibody according to claim 1 or 2, characterized in that: The self-assembling nanoparticle protein includes Z105 protein; the accession number of the Z105 protein in UniProtKB is A0A497XTC1.

5. The nano self-assembled anti-Aβ antibody according to claim 1 or 2, characterized in that: The polypeptides that cross the blood-brain barrier include one or more of angiopeptide-2, HIV transmembrane peptide, leptin, amylin and insulin polypeptide.

6. The nano self-assembled anti-Aβ antibody according to claim 1 or 2, characterized in that: When the first protein tag is SpyTag, the second protein tag is SpyCatcher; When the first protein tag is SnoopTag, the second protein tag is SnoopCatcher; When the first protein tag is DogTag, the second protein tag is DogCatcher.

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

8. The method for preparing the nano self-assembled anti-Aβ antibody according to any one of claims 1 to 7, characterized in that: The steps include: Prokaryotic expression and purification of the recombinant plasmid subcloned with the self-assembling nanoparticle protein functional element to obtain the purified self-assembling nanoparticle protein; Prokaryotic expression and purification of the recombinant plasmid subcloned with the anti-Aβ antibody functional element to obtain a purified anti-Aβ antibody; The purified self-assembled nanoparticle protein and the purified anti-Aβ antibody are coupled in a buffer to obtain a nano self-assembled anti-Aβ antibody; the pH value of the buffer is 7.0-7.

4.

9. The preparation method according to claim 8, characterized in that: 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 subunits of the purified self-assembled nanoparticle protein; The coupling time is 8 to 16 h, and the buffer includes TBS buffer or PBS buffer.

10. Use of the nanometer self-assembled anti-Aβ antibody according to any one of claims 1 to 7 or the nanometer self-assembled anti-Aβ antibody obtained by the preparation method according to claim 8 or 9 in the preparation of a product for treating Alzheimer's disease.

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