IgA antibodies targeting neurotrophins bdnf and ngf and uses thereof

Abstract

The application discloses an IgA antibody targeting neurotrophic factors BDNF and NGF and application thereof. The IgA antibody targeting BDNF and NGF is obtained by expression and purification of expression plasmids thereof in HEK-293T cells, and the two IgA antibodies can specifically recognize and combine with BDNF and NGF respectively and have high affinity. The results of examples show that the IgA antibody targeting NGF and BDNF provided by the application can inhibit the growth, proliferation and survival of glioma cells, induce apoptosis of the glioma cells, and reduce the migration and invasion abilities of the cells, and the IgA antibody can also penetrate the blood-brain barrier and reach the glioma tissue to exert a significantly stronger anti-tumor effect compared with IgG antibodies.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceuticals and relates to an IgA antibody targeting neurotrophic factors BDNF and NGF and its application, specifically to an IgA antibody targeting neurotrophic factors BDNF and NGF, its preparation method, and its application in the preparation of drugs for treating glioma. Background Technology

[0002] Gliomas are the most aggressive malignant brain tumors, accounting for approximately 40% of central nervous system tumors, with a mortality rate as high as 61.4% in China. However, there are currently no effective treatments. The main obstacle is the diffuse infiltration of tumor cells throughout the brain, which allows the tumor to evade complete surgical resection and radiotherapy / chemotherapy. Gliomas grow in a "root-like" pattern, intertwining with neurons and glial cells. Studies have shown that multiple neurotrophic factors, such as brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF), can promote the proliferation and invasion of gliomas (Cameron M et al. Neurons promote glioma proliferation through NLGN3 and BDNF. Neuro Oncol, 2019. 18(10): p. 1192-1203; Demir, IE et al. Nerve growth factor & TrkA as novel therapeutic targets in cancer. Biochim Biophys Acta, 2016. 1866(1): p. 37-50).

[0003] Currently, most antibodies used in glioma immunotherapy are monoclonal antibodies, and all are IgG isotypes. For example, one of them is bevacizumab, which targets vascular endothelial growth factor (VEGF). It is a humanized IgG1 monoclonal antibody that inhibits tumor angiogenesis and reduces tumor blood supply and edema by binding to VEGF and preventing its interaction with the receptor. Bevacizumab has been approved by the US FDA for the treatment of recurrent glioblastoma (GBM). Studies have shown that bevacizumab can prolong progression-free survival (PFS), but has limited impact on overall survival (OS). Nevertheless, for some patients, it can significantly improve quality of life and control symptoms (Chinot OL et al. Bevacizumab plus radiotherapy-temozolomide for newly diagnosed glioblastoma. N Engl J Med. 2014; 370(8):709-722. doi:10.1056 / NEJMoa1308573). Another treatment is nivolumab, which targets programmed death receptor-1 (PD-1). Nivolumab is a humanized IgG4 monoclonal antibody that enhances the T-cell immune response against tumor cells by blocking the interaction between PD-1 and its ligands PD-L1 and PD-L2. While nivolumab is not yet widely approved for first-line treatment of gliomas, it has been approved for the treatment of other types of cancer in some countries and regions and has shown potential in clinical trials for recurrent glioblastoma. Clinical trial results indicate that the efficacy of nivolumab alone or in combination with other therapies is inconsistent; for newly diagnosed GBM patients, overall survival is not significantly improved. However, objective response and disease stabilization have been observed in some patients with recurrent GBM (Reardon DA et al. Nivolumab in patients with recurrent glioblastoma and cediranib plus nivolumab in patients with recurrent glioblastoma or other solid tumors (CheckMate 143 and CheckMate 040): early safety and efficacy data from two open-label, phase 2 trials. Lancet Oncol. 2016; 17(10):1373-1385. doi:10.1016 / S1470-2045(16)30348-5).

[0004] Although these IgG monoclonal antibodies have shown some potential in the treatment of glioma, they also have some significant limitations and challenges.

[0005] 1. Limited ability to penetrate the blood-brain barrier (BBB).

[0006] The blood-brain barrier is a protective mechanism of the brain that prevents many large molecules from entering brain tissue, including most IgG monoclonal antibodies. This limits the effective concentration of IgG antibodies reaching the tumor site, thus affecting treatment efficacy. Even if IgG antibodies can specifically bind to the target site, their therapeutic effect may be greatly limited due to their difficulty in penetrating the blood-brain barrier.

[0007] 2. Tumor heterogeneity and drug resistance

[0008] Glioma cells exhibit high genetic and phenotypic heterogeneity, meaning that not all tumor cells express the same targets, and single-target IgG antibodies may not cover all tumor cell variations. Furthermore, as treatment progresses, tumors may develop resistance to IgG antibody therapy. This heterogeneity and resistance may lead to some patients being unresponsive to or developing resistance to IgG monoclonal antibody therapy, resulting in poor long-term efficacy.

[0009] 3. Unsatisfactory treatment window

[0010] IgG monoclonal antibodies may require higher doses to achieve effective therapeutic effects, but this also means that high doses of IgG antibodies may be widely distributed throughout the body, increasing the risk of side effects. Furthermore, high doses may lead to more serious toxic reactions, such as immune-related adverse events (irAEs), limiting the safe use of the drug.

[0011] 4. Limited improvement in disease progression and survival.

[0012] While some IgG monoclonal antibodies can prolong progression-free survival (PFS), their impact on overall survival (OS) is usually limited. For most patients, IgG monoclonal antibodies may only provide temporary symptom relief or disease control, rather than significantly improving long-term survival.

[0013] 5. High cost

[0014] The development, production, and use of IgG monoclonal antibodies are extremely costly, increasing the burden on healthcare systems and potentially making treatment unaffordable for some patients. The high price may limit the accessibility of these therapies, especially in resource-constrained areas.

[0015] 6. Targeted nonspecific action

[0016] Some IgG monoclonal antibodies may not only target specific sites on tumor cells but may also bind to similar receptors in normal tissues, causing unwanted side effects. Non-specific binding can lead to damage to healthy tissues and increase treatment-related complications. First, IgG antibodies are mainly found in blood and tissue fluid and can activate immune responses through Fc receptors and the complement system. Due to their widespread distribution throughout the body, IgG antibodies may bind to similar receptors in normal tissues, triggering non-specific effects and side effects, especially at high doses. Second, IgG antibodies can activate various immune effects through Fc receptor-mediated mechanisms, including antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). This potent immune response, while enhancing anti-tumor effects, may also increase damage to normal cells.

[0017] Therefore, there is an urgent need in this field for new strategies and methods for treating tumors, especially gliomas. Summary of the Invention

[0018] In view of one or more problems existing in the prior art, one aspect of the present invention provides an IgA antibody targeting the neurotrophic factor BDNF, named BDNF Ab-IgA, which may contain or be composed of the amino acid sequence shown in SEQ ID NO:2.

[0019] Another aspect of the present invention provides an IgA antibody targeting the neurotrophic factor NGF, named NGF Ab-IgA, which may contain or be composed of the amino acid sequence shown in SEQ ID NO:1.

[0020] In some embodiments, the BDNF Ab-IgA or NGF Ab-IgA provided by the present invention may be in the form of a monomer or a polymer.

[0021] In some embodiments, the polymeric form includes a dimer or a tetramer.

[0022] In another aspect, the present invention provides an antibody composition comprising the above-described BDNF Ab-IgA and NGFAb-IgA.

[0023] In another aspect, the present invention provides a nucleic acid construct encoding the aforementioned BDNF Ab-IgA and / or the aforementioned NGF Ab-IgA.

[0024] The applications of BDNF Ab-IgA, NGF Ab-IgA, antibody compositions, or nucleic acid constructs provided by this invention in the preparation of drugs for treating gliomas, in the preparation of drugs for blocking nutrient delivery signals in gliomas, and in the preparation of drugs for triggering ADCC and ADCP effects in microglia are also within the scope of this invention.

[0025] In some embodiments, the glioma includes a brain glioma.

[0026] This study expressed two dimeric IgA antibodies targeting neurotrophic factors NGF or BDNF: NGF Ab-IgA and BDNF Ab-IgA. On one hand, by antagonizing the neurotrophic factor receptor pathway through its variable region, these antibodies directly block nutrient transport signals in gliomas, inhibiting glioma cell growth, proliferation, survival, and migration, and inducing apoptosis. In a glioma cell model with neurotrophic factor receptor knockout, the absence of TRKA, TRKB, or NGFR signaling directly inhibited glioma growth. On the other hand, the IgA antibodies trigger microglia ADCC and ADCP effects through Fc-linked FcαRI, ultimately leading to the lysis and phagocytosis of glioma cells. Simultaneously, the IgA antibodies may cross the blood-brain barrier or hematoma barrier via pIgR-mediated transcytosis, reaching the tumor tissue and binding to pIgR on the tumor cell surface, thereby promoting tumor cell lysis. These results indicate that IgA antibodies are more effective than IgG antibodies in crossing the blood-brain barrier and in demonstrating superior antitumor efficacy. Therefore, BDNF and NGF neurotrophic factors are expected to become new targets for the treatment of gliomas, and the properties of IgA antibodies are also expected to provide a new approach for glioma immunotherapy.

[0027] In addition, compared with existing IgG antibodies, the IgA antibody provided by this invention has significant advantages in terms of tumor heterogeneity and drug resistance, therapeutic window, cost, and targeted nonspecificity.

[0028] In terms of tumor heterogeneity and drug resistance, IgA antibodies can more effectively activate neutrophils and macrophages, which have broader targeting capabilities and may have an advantage in addressing tumor heterogeneity. Furthermore, IgA antibodies can act at mucosal sites, which may have unique advantages for certain types of cancer. Additionally, IgA antibodies exert their effects through different mechanisms, and in some cases can circumvent the mechanisms of IgG antibody resistance. For example, IgA antibodies can more effectively mediate neutrophil cytotoxicity, which could provide a new approach to overcoming certain drug resistances.

[0029] Regarding the therapeutic window, IgA antibodies offer several advantages over IgG antibodies in addressing less-than-ideal therapeutic windows and the risk of side effects. These advantages primarily stem from their limited distribution, their mechanism of specifically activating immune cells, and the potential for lower doses. First, their pharmacokinetics and distribution differ: IgG antibodies have a longer half-life and wider distribution in the body because they can be recycled through neonatal Fc receptors (FcRn). Due to this mechanism, IgG antibodies can maintain high concentrations in the blood and tissues, but this also means that high doses of IgG antibodies may be widely distributed throughout the body, increasing the risk of side effects. IgA antibodies, especially dimeric IgA, primarily exert their immune effects on mucosal surfaces, with distribution more limited to mucosal areas such as the respiratory and digestive tracts. Monomeric IgA has a shorter half-life in the blood, resulting in a shorter time for systemic distribution. Therefore, in certain situations, IgA antibodies can reduce the risk of systemic side effects. Secondly, the mechanisms of immune cell activation differ: IgG antibodies activate various effector cells (such as NK cells, macrophages, and neutrophils) by binding to Fcγ receptors and exert their effects through the complement system. This broad mechanism of action may lead to more severe immune-related adverse events (irAEs) at high doses, such as autoimmune reactions or systemic inflammation. IgA antibodies primarily activate neutrophils and macrophages by binding to FcαRI (CD89). This specific mechanism of action can reduce unwanted immune activation to some extent, thereby lowering the risk of immune-related adverse events. Furthermore, the action of IgA antibodies is more limited to the mucosal surface, which can further limit systemic side effects. Thirdly, the dosage and potency differ: Due to their long half-life and wide distribution, IgG antibodies may require higher doses to achieve sufficient tumor infiltration and therapeutic effect. Such high-dose use may increase the risk of side effects, especially in chronic treatment. In contrast, the specific distribution and mechanism of action of IgA antibodies allow for effective therapeutic concentrations to be achieved at lower doses, particularly in mucosal-associated tumors. This can reduce the risk of dose-related side effects.

[0030] In terms of cost control, IgA antibodies have the potential to become a more cost-effective treatment option. In particular, their application in local treatment can reduce the dosage used systemically, thereby lowering treatment costs.

[0031] Regarding targeted nonspecific effects, IgA antibodies are mainly found on mucosal surfaces (such as the respiratory, digestive, and genitourinary tracts), and exist in two main forms: monomeric IgA in serum and dimeric IgA in mucosa. Dimeric IgA binds to secretory components (SCs) via its J chain, allowing it to reside more effectively on mucosal surfaces, enter systemic circulation less, and reduce systemic distribution; its local action characteristics can significantly reduce nonspecific effects on non-target tissues. Attached Figure Description

[0032] Figure 1 This is a schematic diagram of the structures of the different antibodies (NGF Ab-IgA, BDNF Ab-IgA, NGF Ab-IgG, BDNFAb-IgG, anti-NGF scFv and anti-BDNF scFv) designed in Example 1.

[0033] Figure 2 The results of Western blot analysis for detecting the expression of exogenous IgA antibody fragments using antibodies targeting J chain or IgA are shown in Figure (A). The results of verifying the specific expression of NGF Ab-IgG (~53kDa) and BDNF Ab-IgG antibodies (~53kDa) using anti-human IgG (H+L) antibody are shown in Figure (B). The results of verifying the specific expression of anti-NGF scFv (~25kDa) and anti-BDNF scFv (~25kDa) antibodies using His tag antibody are shown in Figure (C). The results of HEK-293T cells transfected with HEK-293T cells correctly expressing and assembling IgA multimers are shown in Figure (D).

[0034] Figure 3 Results of the high specificity binding of IgA monomers and dimers to the HiTrap ProteinL HP column (A), Western blot and Coomassie Brilliant Blue staining results of enriched and purified IgA antibodies (B and C), Western blot results of overnight incubation of purified IgA antibodies with recombinant human BDNF and NGF proteins (D), and ELISA results of concentration-dependent binding of BDNF Ab-IgA and NGF Ab-IgA antibodies to BDNF and NGF proteins (E).

[0035] Figure 4 The results show that IgA / IgG / scFv antibodies targeting NGF and BDNF inhibit the proliferation of U-87MG cells.

[0036] Figure 5 Image (A) of cell culture scanned and captured using the Evos FLAuto2 automated live-cell imaging system, and statistical results (B) of U-87MG cell debris after antibody treatment.

[0037] Figure 6 Photographs (A) showing the inhibition of U-87MG cell migration and invasion as observed under a phase-contrast microscope, and statistical wound width (B).

[0038] Figure 7 To utilize CellEvent TM A photograph of apoptosis in U-87MG cells obtained from live-cell imaging of Caspase-3 / 7 (A) and the results of statistical analysis of Caspase-3 / 7 activity (B).

[0039] Figure 8 Results of layered scanning of brain tissue after injection of contrast agent into the tail vein of mice (A) and results of tumor volume calculation for each group (B).

[0040] Figure 9 Results of live cell counts in mouse brain tumors analyzed using in vivo imaging software (PerkinElmer Biosciences) (A) and in vivo imaging statistics (B).

[0041] Figure 10 Results of weight and survival analysis of Luci-U-87MG glioma transplanted mice (A) and survival curves of mice in each group (B). Detailed Implementation

[0042] In view of the deficiencies of existing IgG monoclonal antibodies for the treatment of glioma, the present invention provides an IgA antibody that targets neurotrophic factors BDNF and NGF, and provides the application of such antibodies in the treatment of glioma.

[0043] First, in analyzing the expression of BDNF and NGF in the human glioma cell line U-87MG and U-87MG-transplanted nude mouse brain tissue, the inventors found that both U-87MG cells and their glioma tissue highly expressed BDNF and NGF. Human induced pluripotent stem cell (iPSC) differentiated neurons (iPSC-neurons) cultured in vitro can also secrete BDNF and NGF, and their culture supernatant can promote the proliferation of U-87MG cells. This indicates that both neurons and glioma cells can secrete BDNF and NGF, and these neurotrophic factors can promote glioma proliferation through autocrine and paracrine pathways.

[0044] Secondly, this invention constructed IgA antibodies targeting NGF or BDNF (NGF Ab-IgA and BDNF Ab-IgA); simultaneously, it designed IgG antibodies with identical variable region sequences (NGF Ab-IgG and BDNF Ab-IgG) and single-chain variable regions without the Fc fragment (anti-NGF scFv and anti-BDNF scFv). Results showed that after transient transfection of HEK-293T cells with recombinant plasmids, the expression of exogenous antibody fragments was detectable in the supernatant, with IgA assembling to form a dimer structure. The purified IgA antibodies specifically recognized recombinant human BDNF and NGF proteins with strong affinity. These results demonstrate the successful construction and expression of specific, high-affinity IgA antibodies targeting NGF or BDNF.

[0045] This invention validated the antitumor functions of NGF Ab-IgA and BDNF Ab-IgA at the cellular level. Results showed that the addition of recombinant human BDNF or NGF to the culture medium promoted the rapid growth and proliferation of U-87MG cells, while the addition of IgA / IgG / scFv antibodies targeting NGF or BDNF inhibited their proliferation, with IgA antibodies showing the most significant effect. Treatment with NGF Ab-IgA or BDNF Ab-IgA significantly increased U-87MG cell fragmentation and lysis. Recombinant human BDNF and NGF participate in protecting U-87MG cells from apoptosis, while BDNF Ab-IgA and NGF Ab-IgA significantly upregulated the expression of the apoptosis-related protein Caspase-3, inducing stronger apoptosis in U-87MG cells compared to the IgG treatment group. Scratch assay results showed that NGF Ab-IgA and BDNF Ab-IgA significantly inhibited U-87MG cell migration. The above results indicate that BDNF Ab-IgA and NGF Ab-IgA inhibit the growth, proliferation, and survival of glioma cells, induce apoptosis in glioma cells, and reduce cell migration and invasion abilities; among them, BDNF Ab-IgA has a more significant effect than NGF Ab-IgA.

[0046] Based on in vitro functional experiments, this invention constructed a mouse striatal orthotopic glioma transplantation model using Luci-U-87MG cells to verify the in vivo antitumor efficacy of NGF Ab-IgA and BDNF Ab-IgA. Results showed that IgA / IgG / scFv antibodies targeting NGF or BDNF could inhibit glioma growth and prolong mouse survival, with the IgA intervention group exhibiting the slowest tumor growth. Magnetic resonance imaging results showed that compared with the PBS and Isotype control groups, the imaging signals in the NGF Ab-IgA and BDNF Ab-IgA intervention groups were significantly reduced, and the tumor mass effect was significantly decreased, with BDNF Ab-IgA showing better efficacy; HE staining indicated a significant reduction in glioma size. In the NGF Ab-IgA and BDNF Ab-IgA antibody intervention groups, IgA infiltration was observed in both normal brain tissue interstitial spaces and tumor tissue; and the average IgA concentration in the mouse brain tissue lysate was significantly higher than that in the IgG group. The above results indicate that IgA antibodies can penetrate the blood-brain barrier and reach glioma tissue, thereby inhibiting glioma growth in mice and demonstrating significant therapeutic effects.

[0047] Finally, this invention explored the possible mechanisms of action of NGF Ab-IgA and BDNF Ab-IgA in vitro and in vivo antitumor activity. Results showed that U-87MG cells highly expressed NGF and BDNF receptors tyrosine kinase receptor A (TrkA), tyrosine kinase receptor B (TrkB), and nerve growth factor receptor (NGFR). Neurotrophic factor receptor knockout U-87MG cell models (TRKA KO, TRKB KO, and NGFR KO) were constructed using CRISPR / Cas9 technology. Experimental results showed that TRKA KO, TRKB KO, and NGFR KO U-87MG cells exhibited decreased proliferation and viability in vitro, and weakened migration ability, with the most significant effect observed in TRKB KO cells. Results of an orthotopic striatal human glioma transplantation model in mice showed that, compared with wild-type U-87MG cells, knockout cells grew more slowly in the mouse cranium, with TRKB KO tumors exhibiting the smallest tumor volume; mouse survival was significantly prolonged. These experimental results indicate that the absence of neurotrophic factor receptor signaling directly inhibits glioma growth. Therefore, NGF Ab-IgA and BDNF Ab-IgA can directly block the neurotrophic factor receptor signaling pathway through the antibody V region, thereby exerting anti-tumor functions.

[0048] Given the differences in the antitumor efficacy of IgA and IgG antibodies, this invention further explored the mechanism of action of the Fc fragment. On one hand, IgA undergoes epithelial transport by binding to pIgR (polymeric immunoglobulin receptor). Flow cytometry confirmed the expression of pIgR on the surface of U-87MG cells. After co-incubating U-87MG cells with NGF Ab-IgA or BDNF Ab-IgA antibodies, IgA was deposited on the surface of pIgR+U-87MG cells. On the other hand, the IgA Fc fragment mediates a series of immune effects by binding to the IgA-specific Fc receptor (FcαRI). Flow cytometry and LDH assays showed that, compared with IgG antibodies, BDNF Ab-IgA antibody mediated stronger ADCC activity in microglia; blocking FcαRI with a specific antibody significantly reduced IgA-mediated microglia cytotoxicity. pHrodo fluorescence flow cytometry and live-cell imaging results showed that BDNF Ab-IgA antibody mediated the chemotaxis and aggregation of microglia towards glioma cells, leading to the gradual internalization and phagocytosis of target cells by microglia. These results indicate that IgA antibody can mediate the ADCC and ADCP effects of microglia through the interaction of its Fc fragment with FcαRI, thereby exerting an anti-tumor effect.

[0049] In summary, this invention demonstrates through in vitro and in vivo experiments that two IgA antibodies, NGF Ab-IgA and BDNF Ab-IgA, targeting neurotrophic factors NGF or BDNF, exhibit good anti-glioma efficacy, with BDNF Ab-IgA showing better results. NGF Ab-IgA and BDNF Ab-IgA can antagonize the neurotrophic factor receptor pathway through their variable regions, directly blocking nutrient transport signals between gliomas and tumors. Furthermore, they can trigger ADCC and ADCP effects in microglia through Fc-linked FcαRI, thus exerting anti-tumor activity. Simultaneously, IgA antibodies may cross the blood-brain barrier or hematoma barrier via pIgR-mediated transcytosis, reaching glioma tissue and binding to pIgR on the surface of tumor cells, promoting tumor cell lysis. The results of this invention suggest that BDNF and NGF neurotrophic factors hold promise as novel targets for glioma treatment, and IgA antibodies are more significant than IgG antibodies in crossing the blood-brain barrier and demonstrating superior anti-tumor efficacy.

[0050] The present invention will now be described in further detail with reference to specific embodiments and accompanying drawings.

[0051] The following disclosure provides many different embodiments or examples for implementing different aspects of the invention. Examples of various specific processes and materials are provided, but those skilled in the art will recognize the application of other processes and / or the use of other materials.

[0052] The embodiments are implemented on the premise of the technical solution of the present invention, and detailed implementation manners and specific operation processes are given. However, the disclosure of the present invention is not limited to the following embodiments.

[0053] Unless otherwise specified, the methods used in the following embodiments are all conventional methods.

[0054] The experimental animals used in the embodiments of the present invention: BALB / c nude mice (Experimental Animal Breeding Center of Nanjing University Model Animal Research Institute): The experimental mice are female, 4 weeks old, with an initial body weight of 15-18 g, and are housed in a specific pathogen-free (SPF) environment [License number: SYXK (Beijing) 2012-0004] at the Experimental Animal Center of the Institute of Basic Medicine, Chinese Academy of Medical Sciences. The animal experiment protocols involved in this research have been reviewed by the Experimental Animal Management and Ethics Committee of the Institute of Basic Medicine, Chinese Academy of Medical Sciences, and all animal experiment operations comply with the standardized requirements of the "Guidelines for the Ethical Review of Laboratory Animal Welfare in China (GB / T 35892-2018)".

[0055] The cell lines used in the embodiments of the present invention:

[0056] U-87MG human glioma cell line: purchased from the Cell Center of the Chinese Academy of Medical Sciences; Luciferase-U-87MG human glioma monoclonal cell line (Luci-U-87MG): purchased from Yikang Beijing Medical Technology Co., Ltd.; adherent culture is carried out in MEM medium (Beijing Xigong Biotechnology Co., Ltd.) containing 10% FBS, 100×NEAA, and 100×NaPyr.

[0057] Human iPSC-neuron: Neurons differentiated from human induced pluripotent stem cells (see the following references: Kondo, T., & Tabar, V. (2009). "Induced pluripotent stem cells: a new era in celltherapy." Nature Reviews Neuroscience; Lujan, E. et al. (2016). "A protocol for the generation of functional neurons from human pluripotent stem cells." Nature Protocols; Maroof, A. et al. (2013). "Directed differentiation of human pluripotent stem cells to functional spinal motor neurons." Cell Stem Cell.), preserved in the applicant's laboratory in B-27 complete culture medium (recombinant human BDNF protein (rhBDNF protein) (Novoprotein, China), 10 μg / mL; recombinant human NGF protein (rhNGF protein) (Novoprotein, China), 10 μg / mL; ascorbic acid... The culture was carried out in Neurobasal medium (Thermo Fisher Scientific, USA) containing 0.2 mM acid, 50 mM cAMP, 10 mM DAPT and 100 × GlutaMAX (0.2 mM acid, 50 mM cAMP, 10 mM DAPT) and 100 × GlutaMAX.

[0058] HMC3 (Human microglia clone 3) human microglia cell line: purchased from the Cell Center of the Chinese Academy of Medical Sciences, and cultured in MEM medium containing 15% FBS.

[0059] HEK-293T cells: a human embryonic kidney epithelial cell line transformed with SV40, preserved in the applicant's laboratory and cultured in DMEM medium (Beijing Xigong Biotechnology Co., Ltd.) containing 10% FBS.

[0060] The strains and plasmids used in the embodiments of this invention are as follows:

[0061] 1) strain

[0062] Escherichia coli DH5α competent cells: used for transformation of general plasmids and recombinant ligation products, genotype F"-p80d lacZAM\5A(lacZYA-argF)U169 end A1 recAl hsdRXl(rk",mk+ )supE44X-thi - gyrA96 relA phoA; purchased from Beijing Quanshijin Biotechnology Co., Ltd.

[0063] 2) Plasmids

[0064] MH-AbVec2.0 expression plasmid (purchased from Addgene): used for eukaryotic expression of IgA antibody in mammalian cells; this plasmid is stored in the applicant's laboratory.

[0065] pCDH-EF1-Luc2-P2A-copGFP (Plasmid#72485): A lentiviral vector for co-expressing luciferase and green fluorescent protein GFP (see CN111961651A).

[0066] eSpCas9-LentiCRISPR v2 (Plasmid#52961): Enhanced specificity SpCas9 (eSpCas9), also known as SpCas9 (K848A / K1003A / R1060A). This plasmid was constructed by Zhang Feng's lab and replaced the original lentiCRISPR v1 (Plasmid#49535). eSpCas9 can improve the specificity of the Cas9 protein to the target gene, reduce the off-target effect of the target gene by more than 10 times, while maintaining strong editing efficiency of the target gene.

[0067] psPAX2 and pMD2.G (see CN102174471A): Lentiviral packaging plasmids that utilize the three-plasmid Lenti-X packaging system to increase vector stability and safely produce high-titer, highly infectious lentiviral particles.

[0068] Example 1: Construction of expression plasmids for IgA antibodies (NGF Ab-IgA and BDNF Ab-IgA) targeting neurotrophic factors BDNF and NGF.

[0069] (I) Design and Construction of IgA Antibodies

[0070] IgA antibodies targeting NGF and BDNF were designed using the Snapgene program and named NGF Ab-IgA and BDNF Ab-IgA, respectively. The variable region sequences of the antibodies were derived from patent documents WO 2006 / 110883A2 and US2016 / 0207991A1, respectively, and contain complete antigen recognition variable domains (VH and VL). The IgA sequence was derived from NCBI-Immunoglobulin heavy constant alpha 1 (Homo sapiens); its constant domain (CH1-CH2-CH3) and tailpiece structure partitions were analyzed using IMGT Repertoire, RecName, and HGNC libraries. The J CHAIN ​​sequence was derived from NCBI-Dna-joining chain of multimeric IgA and IgM (Homo sapiens). The antibody sequences were synthesized by Shanghai Sangon Biotech Co., Ltd.

[0071] Two IgA antibodies were secreted from their supernatant by a signal peptide, VH and VL, respectively. The variable regions of the antibodies were linked by a flexible linker, a 15-amino acid linker composed of glycine (Gly) and serine (Ser) (G4S) × 3, possessing elasticity and protease resistance, allowing the functional regions of VH and VL to still pair after folding, forming antigen-binding sites. The constant region simultaneously connects the IgA1 CH2-CH3-Tailpiece and the J chain, ensuring its stable assembly into the dimer dIgA (IgA dimer) in vitro. An Fc fragment (CH2-CH3) was designed at the terminal to bind to FcαRI on the effector cell surface, thereby initiating a series of downstream activation signals. Four control antibodies were designed, named NGF Ab-IgG and BDNF Ab-IgG (with the same variable region sequences as NGF Ab-IgA and BDNF Ab-IgA, respectively), anti-NGF scFv, and anti-BDNF scFv (both single-chain variable regions without the Fc region). The specific structures of the antibodies are shown below. Figure 1As shown, A is a schematic diagram of the structure of NGFAb-IgA (the amino acid sequence of which is shown in SEQ ID NO:1) and BDNF Ab-IgA (the amino acid sequence of which is shown in SEQ ID NO:2), B is a schematic diagram of the structure of monomeric IgA and dimer IgA, C is a schematic diagram of the structure of NGF Ab-IgG (the amino acid sequence of which is shown in SEQ ID NO:3) and BDNF Ab-IgG (the amino acid sequence of which is shown in SEQ ID NO:4), and D is a schematic diagram of the structure of anti-NGF scFv (the amino acid sequence of which is shown in SEQ ID NO:5) and anti-BDNF scFv (the amino acid sequence of which is shown in SEQ ID NO:6); the vectors are all MH-AbVec2.0-IGHG1 (Tiller, T. et al., Efficient generation of monoclonal antibodies from single human B cells by single cell RT-PCR and expression vector cloning. J Immunol Methods, 2008, 329(1-2): p.112-24). Six histidine residues (6×His) were introduced at the C-terminus of the scFv fragment for antibody capture and purification. Isotype controls were set up for IgA and IgG, namely IgA Isotype and IgG Isotype.

[0072] The following aspects were mainly considered when designing the antibody in this invention:

[0073] 1. IgA was selected as the antibody type.

[0074] Given that most current antibody therapies use IgG antibodies, which have many drawbacks, this invention recognizes that IgA antibodies have unique biological characteristics, such as the ability to form a dimer structure, enhanced stability through J-chain linkage, and the ability to initiate activation signals by binding to effector cell surface receptors via FcαRI. Considering the role of IgA antibodies in mucosal immunity and their potential for better blood-brain barrier penetration, IgA antibodies were designed.

[0075] 2. Optimized variable region source

[0076] This invention utilizes the proven and effective antigen-recognizing variable domains (VH and VL) described in patent documents WO 2006 / 110883A2 and US2016 / 0207991A1 to ensure high specificity of the antibody for the target antigens (NGF and BDNF). This selection based on existing successful cases reduces development risks and increases the probability of success.

[0077] 3. A meticulously designed Linker

[0078] This invention introduces a flexible linker ((G4S)×3) composed of glycine and serine as a connector during antibody design. This not only ensures the appropriate distance between VH and VL but also increases protease resistance, allowing the two functional regions to fold correctly and pair to form antigen-binding sites. This specific length and composition of the linker design is intended to maximize the preservation of antibody functionality and stability while providing sufficient flexibility to adapt to different application environments.

[0079] 4. Constructing the complete dimer dIgA

[0080] This invention, by incorporating the CH2-CH3-Tailpiece and J chain sequences, ensures that IgA antibodies can be stably assembled into a dimer form in vitro, which is crucial for maintaining the unique biological activity of IgA. The construction of the dimer structure increases the molecular weight and complexity of the antibody, but also brings greater stability and a wider range of potential applications.

[0081] 5. Application of Fc fragments

[0082] This invention incorporates an Fc fragment (CH2-CH3) at the end of the constant region to bind to FcαRI on the surface of effector cells, thereby activating downstream signaling pathways. This design enhances the functional diversity of the antibody. The addition of the Fc fragment provides the antibody with additional functionality, enabling it not only to block the action of neurotrophic factors but also to further influence target cells through immune system-mediated mechanisms.

[0083] 6. Design of control plasmids

[0084] In addition to the main IgA antibody design, this invention also designs a variety of control plasmids, including IgG antibodies, single-chain variable fragments (scFv), and isotype controls, which helps to comprehensively evaluate the performance of novel IgA antibodies.

[0085] (II) Small-scale extraction of target plasmid

[0086] 1. First, preheat the water bath to 65℃.

[0087] 2. Seven bacterial strains containing the following expression plasmids were inoculated into LB medium containing ampicillin resistance: PUC57-NGF Ab-IgA, PUC57-BDNF Ab-IgA, PUC57-NGF Ab-IgG, PUC57-BDNF Ab-IgG, PUC57-anti-NGF scFv, PUC57-anti-BDNF scFv, and MH-AbVec2.0 expression plasmid. The strains were cultured at 37°C and 200 rpm for 12–16 hours, and bacterial cells in the plateau phase were extracted.

[0088] 3. Take 5 mL of each target bacterial culture (the amount of each reagent can be increased accordingly), centrifuge at 4000 rpm for 1 minute at room temperature, and carefully discard the supernatant. Add 250 μL of Buffer P1 and 1 μL of VisualLyse to the bacterial pellet and shake to obtain a bacterial suspension.

[0089] 4. Add 250 μL of Buffer P2 to the above 7 suspensions, and immediately and gently invert several times to mix, avoiding plasmid DNA breakage. The solution should be a uniform, clear blue color at this point; otherwise, there is an excess of bacterial cells.

[0090] 5. Add 350 μL of Buffer P3 and immediately gently invert several times to mix; the solution should return to colorless at this point, otherwise the mixing is incomplete. Let stand at room temperature for 2 minutes to completely remove RNA.

[0091] 6. Prepare 7 labeled adsorption columns. Carefully transfer the supernatant obtained after centrifugation at 18000×g for 10 minutes at room temperature into each column, with a maximum of 750 μL each time. Centrifuge at 9000×g for 30 seconds and carefully discard the supernatant. Add 500 μL of Wash Solution to each column and repeat the washing step once.

[0092] 7. Centrifuge the empty adsorption column and the matching collection tube at 9000×g for 1 minute, and let stand for 10 minutes until all the residual ethanol evaporates.

[0093] 8. Aseptically aspirate 3 mL of Elution Buffer and place the adsorption column into 7 clean centrifuge tubes. Vertically add 50–100 μL of preheated Elution Buffer (60°C) to the center of each adsorption membrane and allow to bind fully at room temperature for about 10 minutes.

[0094] 9. Finally, centrifuge at 9000×g for 1 minute to obtain the target plasmid obtained from the kit's small-scale extraction. Detect and label its concentration using a Nanodrop2000 UV spectrophotometer. The OD260 / OD280 ratio should be between 1.8 and 1.9, and the OD260 / OD230 ratio should be greater than 2.0. Store the obtained IgA, IgG, and scFv antibody expression plasmids at -20°C for subsequent experiments such as double enzyme digestion.

[0095] (III) Double Enzyme Digestion Reaction

[0096] Double enzyme digestion was performed on the MH-AbVec2.0 eukaryotic expression vector and 6 groups of PUC57 plasmids.

[0097] 1. Digest the empty vector plasmid MH-AbVec2.0 with EcoRI and HindIII according to the enzyme digestion system shown in Table 1 below.

[0098] Table 1:

[0099]

[0100] 2. The PUC57 target plasmid was digested with EcoRI and HindIII using the enzyme digestion system shown in Table 2 below.

[0101] Table 2:

[0102]

[0103] (iv) Identification and purification of double enzyme digestion products

[0104] 1. Identify the size of enzyme digestion bands using 1% agarose gel electrophoresis. Prepare the agarose gel solution by pouring it into a beaker and heating it at high temperature until the agarose melts and the mixture is thoroughly shaken. After slightly cooling, add 0.5 μg / mL ethidium bromide until the solution turns light red. Pour an appropriate volume of solution into a horizontal electrophoresis tank and insert the sample comb as soon as possible, avoiding the formation of air bubbles.

[0105] 2. Let stand at room temperature for about 30 minutes, allowing the agarose gel to cool naturally until it solidifies, then remove the sample comb. Keep the gel moist before loading to prevent it from drying out and cracking.

[0106] 3. Mix the above double enzyme digestion products with Purple laoding dye DNA staining solution in the specified ratio to form a uniform purple DNA mixture. Carefully inject the mixture into the sample wells to prevent cross-contamination between samples from different wells.

[0107] 4.110V constant voltage electrophoresis for about 45 minutes. Stop electrophoresis when it reaches 2 / 3 of the way through, based on the indicator color, and try to distinguish the target DNA fragment from other fragments.

[0108] 5. Using PE gloves, gently remove the gel from the electrophoresis tank. You can use a blunt knife to scrape off the edges to prevent breakage.

[0109] Analysis of the double enzyme digestion products by 1% agarose gel electrophoresis showed that the DNA fragment sizes of the antibody plasmids were consistent with the expected results. SignalP 4.1 prediction indicated that the cleavage site of the BDNF-targeting antibody was between amino acids 30 and 31 (ASA-AV), and the cleavage site of the NGF-targeting antibody was between amino acids 26 and 27 (ASA-QV), suggesting that the N-terminal signal peptide of the antibodies was correctly designed and could guide the newly synthesized protein to be secreted into the supernatant.

[0110] (V) Column-based DNA gel recovery

[0111] 1. The double-enzyme digestion products of the MH-AbVec2.0 eukaryotic expression vector and six groups of PUC57 plasmids were purified using a column gel extraction method. First, the water bath was preheated to 50℃. The digestion products were placed under a UV lamp, and the target gel block was cut as soon as possible to minimize exposure time. The gel block was placed in a 15mL centrifuge tube, and the weight of the centrifuge tube before and after centrifugation was measured and recorded as the pure weight of the gel block.

[0112] 2. Add Buffer B2 at 6 times the pure weight of the gel block, and place the centrifuge tube in a 50°C water bath until the gel is completely dissolved, shaking constantly to ensure the gel block dissolves evenly.

[0113] 3. Transfer the entire sol solution into the labeled adsorption column, with a sample volume of 750 μL each time. Centrifuge at 8000 × g for 30 seconds at room temperature to separate the target DNA solution into the adsorption membrane and discard the liquid in the collection tube.

[0114] 4. Add 300 μL of Buffer B2 to the target DNA solution adsorption column, centrifuge at 9000×g for 30 seconds at room temperature, and discard the liquid in the collection tube.

[0115] 5. Add 500 μL of Wash Solution to the adsorption column, centrifuge at 9000 × g for 30 seconds at room temperature, discard the liquid in the collection tube; repeat the washing step once.

[0116] 6. Centrifuge the empty adsorption column and the matching collection tube at 9000×g for 1 minute, and let stand for 10 minutes until all residual ethanol evaporates.

[0117] 7. Preheat the Elution Buffer to 60°C, and vertically add 35 μL of Elution Buffer to the center of the adsorption membrane. Allow it to fully bind at room temperature for about 10 minutes.

[0118] 8. Finally, centrifuge at 9000×g for 1 minute to elute the target plasmid and vector obtained by column gel recovery; store in a -20℃ freezer for subsequent experiments such as ligation reactions.

[0119] (vi) Connection reaction

[0120] 1. The double enzyme digestion products obtained from gel recovery were ligated according to the ligation system shown in Table 3 below at 25°C for 1 hour or at 16°C overnight using T4 DNA ligase, and the ligation products were then transformed into E. coli DH5α competent cells.

[0121] Table 3:

[0122]

[0123] (vii) Screening for positive recombinant plasmids

[0124] 1. Pick a single colony from an LB plate and number it. Then, directly pipette the colony pick into 7 mL of LB liquid medium containing ampicillin. Place the plate on a shaker at 37°C and 200 rpm until the culture becomes turbid, which takes about 12 to 16 hours. Avoid prolonged shaking to prevent DNA damage.

[0125] 2. Extract the bacterial culture according to the instructions of the plasmid mini-extraction kit. Perform double enzyme digestion identification and first-generation sequencing on the plasmids. Analyze the sequencing results using Snapgene software to obtain correctly sequenced positive recombinant plasmids, which are named NGF Ab-IgA plasmid, BDNF Ab-IgA plasmid, NGF Ab-IgG plasmid, BDNF Ab-IgG plasmid, anti-NGF scFv plasmid, and anti-BDNF scFv plasmid, respectively.

[0126] Example 2: Expression and identification of NGF Ab-IgA and BDNF Ab-IgA

[0127] (I) Detection of IgA antibody expression and assembly by immunoblotting

[0128] Protein sample preparation

[0129] 1. Pre-inoculate 1.5 × 10⁻⁶ cells into 6-well plates. 6 HEK-293T cells were selected and transfected when they were in good condition and reached a density of 80%–90%. Six groups of recombinant plasmids and their isotype controls were transiently transfected into HEK-293T cells, and whole-cell lysates were prepared.

[0130] 2. Prepare a centrifuge tube, add 200 μL jetPRIME buffer to dilute 2 μg of DNA, and gently mix.

[0131] 3. Add 4 μL of jetPRIME transfection reagent to the total DNA dilution buffer and incubate at room temperature for 10 minutes to form a DNA transfection mixture.

[0132] 4. Add 200 μL of DNA transfection mixture evenly to each well of a 6-well plate and gently shake to mix.

[0133] 5. Incubate in an incubator for 24–48 hours, collect the cell culture supernatant from different transfection groups and the control group, and store in aliquots at -80°C to avoid repeated freeze-thaw cycles.

[0134] Western blot

[0135] 1. Prepare SDS-PAGE separating gel and stacking gel, see the reagent preparation section for details.

[0136] 2. Add 20 μL of the above cell culture supernatant sample (IgA, IgG, scFv and negative control) and loading buffer to a 1.5 mL centrifuge tube, mix thoroughly by pipetting. The loading buffer contains bromophenol blue and other substances to indicate the electrophoresis position.

[0137] 3. Prepare boiling water in advance, boil the sample for 10 minutes to fully denature the proteins and expose the antigen binding sites, and then cool it on ice.

[0138] 4. Pour 1.5L of electrophoresis buffer into the electrophoresis tank with the sample holder installed. After loading the samples sequentially, proceed with electrophoresis at a constant voltage of 75V. When the pre-stained protein marker bands are clearly separated, adjust the voltage to 120V. When the bromophenol blue indicator is nearing the end of the electrophoresis, zero the voltage.

[0139] 5. Remove the glass plate, gently remove the electrophoresis gel and cut off any excess. Cut the nitrocellulose membrane into 6.6cm × 8cm pieces and soak them thoroughly in the electrophoresis solution. Stack the membranes in the following order: positive electrode - filter paper - NC membrane - electrophoresis gel - filter paper - negative electrode. Wet the stacks in the electrophoresis solution and filter out any air bubbles.

[0140] 6. Install the electro-rotator rack, pour 1.5L of electro-rotator fluid into the tank, and start the 200mA constant current electro-rotator for 2 hours. Electro-rotator inside the ice box to reduce heat dissipation.

[0141] 7. Prepare blocking buffer: Add 5% skim milk powder for blocking to 1×TBST buffer. Place the NC membrane in the blocking buffer and incubate at room temperature with shaking for 2 hours.

[0142] 8. Primary antibody incubation: Prepare the corresponding target antibody (mouse anti-human Immunoglobulin J polypeptide), and incubate the NC membrane with the primary antibody solution at room temperature for 2 hours or overnight at 4°C.

[0143] Soak the NC membrane in 9.1×TBST washing buffer, and repeat the washing process by shaking the membrane 5 times, each time for 5 minutes.

[0144] 10. Secondary antibody incubation: Horseradish enzyme-labeled goat anti-mouse IgG antibody. Alternatively, horseradish enzyme-labeled Anti-Human IgA alpha-Chain antibody and horseradish enzyme-labeled goat anti-human IgG (H+L) antibody can be used for verification. Antibodies are diluted proportionally with blocking buffer and incubated at room temperature with shaking for 1 hour.

[0145] Wash the membrane repeatedly with 11.1×TBST washing buffer, shaking for 5 minutes each time.

[0146] 12. Prepare Luminal developer, react it with each group of NC films, place them in an ECL autodeveloper for sequential exposure imaging and analysis.

[0147] The protein expression of antibodies was identified using SDS-PAGE immunoblotting. HEK-293T cells were transiently transfected with recombinant plasmids containing anti-BDNF and NGF IgA, IgG, and scFv, and equal volumes of cell culture supernatant were collected. The expression of exogenous IgA antibody fragments was specifically detected using antibodies targeting Jchain or IgA. Figure 2 The Western blot results shown in Figure A indicate that the supernatant of the transfected HEK-293T cells specifically expressed NGF Ab-IgA and BDNF Ab-IgA antibodies. The target bands were detected at approximately 73 kDa (monomer) and 140 kDa (dimer), consistent with the expected size.

[0148] The specific expression of NGF Ab-IgG (~53kDa) and BDNF Ab-IgG antibodies (~53kDa) was verified using anti-human IgG (H+L) antibodies, and the results are as follows: Figure 2 As shown in Figure B. The specific expression of anti-NGF scFv (~25kDa) and anti-BDNF scFv (~25kDa) antibodies was verified using His tag antibodies, and the results are shown below. Figure 2 As shown in section C.

[0149] ·Hepes Native-PAGE

[0150] To determine whether IgA antibodies can complete the assembly of in vitro multimers, native-PAGE was used to detect non-denatured protein samples in the supernatant.

[0151] 1. Using BeyoGel TMPlus, pre-cast PAGE gels (Hepes, 4-20%, 10 wells) are used to verify the multimeric assembly of IgA antibodies. Polyacrylamide gel electrophoresis (PAGE) is widely used for protein separation, purification, detection, identification, and molecular weight analysis.

[0152] 2. First, remove the pre-cast adhesive from the packaging bag; fix it in the electrophoresis tank, and slowly and steadily pull out the comb.

[0153] 3. Prepare the electrophoresis buffer. For non-denatured proteins, use BeyoGel. TM Plus Native-PAGE Hepes electrophoresis solution (20×). Fill the inner tank with electrophoresis solution, and add electrophoresis solution to the outer tank until it covers the anode.

[0154] 4. Use a 1mL pipette to remove any remaining storage buffer from the pre-cast gel wells, and gently rinse the wells with electrophoresis buffer. Insert the tip of a 10μL pipette vertically into each well to load the sample. Close the electrophoresis tank lid and plug the power cord into the electrophoresis apparatus's power socket (red to red, black to black).

[0155] 5. Control the voltage to 150V and electrophoresis for 40-50 minutes; when the bromophenol blue stripe reaches near the bottom of the gel, adjust the voltage to zero.

[0156] 6. Remove the glass adhesive strip, cut the adhesive between the two glass plates, gently pry the glass adhesive strip apart with a scraper and remove it. Stack the plates in the following order: positive electrode - filter paper - NC membrane - electrophoresis gel - filter paper - negative electrode.

[0157] 7. Install the electric transfer frame and transfer the film for 60 minutes at a constant current of 300mA.

[0158] 8. Follow up with the Western blot procedure as described above.

[0159] The results are as follows Figure 2 As shown in Figure D, the transfected HEK-293T cells can correctly express and assemble IgA multimers. The target bands were detected at approximately 140 kDa (dimer) and 280 kDa (tetramer), consistent with the expected size.

[0160] (II) Purification and identification of NGF Ab-IgA and BDNF Ab-IgA

[0161] • Peptide M / Agarose for purifying IgA antibodies

[0162] 1. Peptide M is a synthetic peptide derived from the 50-amino acid Streptococcus M protein, containing an additional C-terminal cysteine ​​residue. Peptide M binds to both monomeric and dimeric human IgA subclasses (IgA1 and IgA2) with high specificity and affinity, exhibiting a binding capacity of 4–6 mg of human IgA per milliliter of gel. Peptide M is used for one-step affinity purification of IgA and for the specific detection of antigen-binding IgA1.

[0163] 2. Prepare the following buffer solution:

[0164] 1) Equilibration / washing buffer: 10mM sodium phosphate, 150mM sodium chloride, pH 7.2

[0165] 2) Elution buffer: 0.1M glycine, pH 2.0

[0166] 3) Neutralization buffer: 0.75M sodium phosphate or 1M Tris, pH 9.0

[0167] 4) Regeneration / storage buffer: 20% (v / v) ethanol dissolved in phosphate buffered saline.

[0168] 3. Immunoglobulin IgA purification procedure

[0169] 1) Pack 1 mL of fixed Peptide M / Agarose into a suitable column. Perform all chromatographic steps at a flow rate of 0.5–1 mL / min or under gravity flow. Equilibrate the column with 5 mL of equilibration / wash buffer;

[0170] 2) After dialysis, the IgA sample was filtered through a 0.2 μm filter and then loaded onto a chromatographic column. The column was washed with 10 mL of equilibration / wash buffer.

[0171] 3) Elute the column with 10 mL of elution buffer; immediately adjust the pH of the eluent to 7.5 by adding neutralization buffer.

[0172] 4) Finally, wash the column with 10 mL of equilibration / wash buffer. Store Peptide M / Agarose in regeneration / storage buffer at 4°C.

[0173] · Purification of IgA and IgG antibodies using liquid chromatography system

[0174] The aforementioned IgA and IgG target antibodies were prepared and purified using pre-packaged ready-to-use columns containing HiTrap Protein L and Protein A. After transient transfection of HEK-293T cells, the cells were cultured at 37°C and 5% CO2 for 24–48 h. The cell culture supernatant was then purified according to the AktaAdvant 25 (GE-Instructions) instructions. The binding buffer was 20 mM sodium phosphate, and the elution buffer was 0.1 M citrate. All samples were filtered through a 0.45 μm filter to prevent column clogging.

[0175] The antibody purification procedure is as follows:

[0176] 1) First, add 150 μL of 1M Tris HCl (pH 9.0) to the collection tube;

[0177] 2) After cleaning the tubing, fill all tubing and the syringe pump with binding buffer. Connect the Protein A or Protein L purification column to the syringe pump and test the flow path to prevent air introduction;

[0178] 3) Equilibrate the tubing with 10 column volumes of binding buffer at a flow rate of 2 mL / min;

[0179] 4) Use a syringe pump to load IgA or IgG antibodies onto the sample;

[0180] 5) Wash the tubing with 5 to 10 column volumes of binding buffer;

[0181] 6) Elute with 2 to 5 column volumes of elution buffer at a flow rate of 1 mL / min;

[0182] 7) Wash the affinity chromatography column with 5 column volumes of 20% ethanol to prevent microbial growth. Store it in 20% ethanol at 4°C.

[0183] Subsequently, after sample loading, non-specific protein washing, and elution with 0.1M citric acid, four purified proteins, namely NGF Ab-IgA, BDNF Ab-IgA, NGF Ab-IgG, and BDNF Ab-IgG, were obtained, and a portion of the supernatant was retained as a control for subsequent experiments.

[0184] • Affinity detection of IgA antibodies

[0185] 1. IgA antibody coating of ELISA plates: NGF Ab-IgA and BDNF Ab-IgA were coated onto ELISA plates and incubated overnight at 4°C; the next day, the solution in the wells was discarded, and the plates were washed three times with 10mM PBS buffer (pH 7.4) for 3 minutes each time.

[0186] 2. Sample loading: Add serially diluted rhNGF or rhBDNF protein to each well of the microplate; the protein is diluted with 0.05M carbonate coating buffer at pH 9.0;

[0187] Incubate at 37°C for 3 hours; wash the plate three times with washing buffer PBS, 3 minutes each time;

[0188] 4. Simultaneously prepare blank wells, negative control wells, and positive control wells containing commercially available NGF antibody (Abcam) and commercially available BDNF antibody (Thermo);

[0189] 5. Add the HRP-conjugated enzyme-labeled antibody Peroxidase AffiniPure Goat Anti-Human IgA to each reaction well and incubate for 45 minutes. After the reaction, wash 5 times with PBST wash buffer;

[0190] 6. TMB color development: Add ELISA substrate and incubate at 37°C for 15 minutes for color development;

[0191] 7. Terminate the reaction and measure the OD450 value using an enzyme-linked immunosorbent assay (ELISA) reader.

[0192] The HiTrap ProteinL HP column can specifically bind to both monomers and dimers of IgA. This study used affinity chromatography to purify IgA antibodies from HEK-293T cell culture supernatant, and the results are as follows: Figure 3 As shown in Figure A, Western blot and Coomassie Brilliant Blue staining results indicate that the IgA antibody in the supernatant was effectively enriched and purified, as shown in Figure A. Figure 3 Figures B and C are shown in the diagram. Recombinant human BDNF and NGF proteins were incubated overnight using the IgA-purified antibody described above. Western blot results showed specific bands at 14 kDa, as shown in Figures B and C. Figure 3 As shown in Figure D, the purified BDNF Ab-IgA and NGF Ab-IgA antibodies can specifically recognize recombinant human BDNF and NGF proteins.

[0193] The affinity of the purified IgA antibodies was further tested. BDNF Ab-IgA and NGF Ab-IgA were coated onto ELISA plates and then bound to serially diluted recombinant human BDNF and NGF proteins, respectively. ELISA results showed that the BDNF Ab-IgA and NGF Ab-IgA antibodies bound to BDNF and NGF proteins in a concentration-dependent manner, exhibiting strong affinity, although lower than that of commercially available anti-BDNF and anti-NGF antibodies. Figure 3 As shown in Figure E.

[0194] The above results indicate that the construction and expression of specific, high-affinity IgA antibodies targeting NGF and BDNF were successful.

[0195] Example 3: In vitro cell experiments of NGF Ab-IgA and BDNF Ab-IgA

[0196] (I) Effects of IgA Antibody on Glioma Cell Proliferation

[0197] 1. The effects of antibodies targeting NGF or BDNF on the proliferation of U-87MG cells were detected using the Cell Counting Kit 8 (CCK-8) assay.

[0198] 2. Prepare 100 μL of U-87MG cell suspension in a 96-well plate. Pre-culture the plate at 37°C in a 5% CO2 incubator for 24 hours to allow it to adhere.

[0199] 3. After discarding the MEM medium, add 100 μL of different antibody groups targeting NGF or BDNF to the culture plate, namely NGF Ab-IgA, BDNF Ab-IgA, NGF Ab-IgG, BDNF Ab-IgG, anti-NGF scFv, anti-BDNF scFv, IgA Isotype, and IgG Isotype. Incubate the culture plate in an incubator for 24 hours.

[0200] 4. Add 10 μL of CCK-8 solution to each well; incubate the culture plate in the dark for 4 hours.

[0201] 5. Measure the absorbance at 450 nm using an ELISA reader.

[0202] 6. Cell proliferation calculation formula: Cell viability = [(As-Ab] / (Ac-Ab)] × 100%; Cell inhibition rate = [(Ac-As] / (Ac-Ab)] × 100%. Where As is the experimental well containing glioma cells, CCK8 and target antibody; Ac is the control well without target antibody; and Ab is the blank well without cells.

[0203] The results showed that, compared with the untreated group, the number of U-87MG viable cells in the culture medium supplemented with NGF or BDNF was significantly increased, with the BDNF group showing a greater number, and the difference was statistically significant; suggesting that neurotrophic factors NGF and BDNF can promote the proliferation of U-87MG cells.

[0204] Treatment with IgA / IgG / scFv antibodies targeting NGF and BDNF reduced the number of viable U-87MG cells, indicating that both antibodies inhibited U-87MG cell proliferation. Among them, the NGF Ab-IgA and BDNF Ab-IgA antibodies showed the most significant inhibition of U-87MG cell proliferation, as shown in the results below. Figure 4 As shown.

[0205] (II) Effects of IgA antibodies on the growth and survival of glioma cells

[0206] 1. The effects of antibodies targeting NGF or BDNF on the proliferation of U-87MG cells were detected using TO-PRO-3 and Hoechst 33342 dyes. TO-PRO-3 is a carbocyanine monomeric dye with a very strong binding affinity for dsDNA. It can be used as a nuclear counterstain and a dead cell indicator, and is one of the most sensitive nucleic acid detection probes; its maximum excitation / emission wavelength is 642 / 661 nm. Hoechst 33342 is a blue fluorescent dye that can penetrate cell membranes and can be directly used for staining the nuclei of live cells. After binding to double-stranded DNA, the maximum excitation / emission wavelength of Hoechst 33342 is 350 / 461 nm.

[0207] 2. Beforehand, seed U-87MG cells onto 24-well plates and place them in a 37°C, 5% CO2 incubator to allow them to adhere. Discard half of the MEM complete culture medium and add 200 μL of different groups of antibodies targeting NGF or BDNF to each culture plate (set the groups as above). Incubate the culture plates in the incubator for 24 hours.

[0208] 3. First, heat both dyes to room temperature. Before each use, briefly centrifuge the DMSO solution to the bottom of the vial.

[0209] 4. Wash the adherent cells 1–3 times in 1×PBS.

[0210] 5. Dilute TO-PRO-3 stock solution and Hoechst 33342 stock solution in 1×PBS (1:1000) to prepare staining working solution.

[0211] 6. Add enough staining solution to cover the cells; incubate in the dark for 30 minutes.

[0212] 7. Discard the staining solution, wash the cells three times with 1×PBS, and scan and photograph the cell culture plate using the Evos FLAuto2 automated live cell imaging system.

[0213] After observing the direct effects of NGF Ab-IgA and BDNF Ab-IgA antibodies on glioma cell proliferation, this study further explored the relationship between antibodies and cell growth and survival. U-87MG cells were treated with different proteins for 72 hours, and cell culture plates were observed using Evos. The results showed that the addition of recombinant human BDNF or NGF to the culture medium promoted the growth and survival of U-87MG cells, resulting in plump cells with clear outlines and high growth density. Figure 5As shown in Figure A, U-87MG cell condition deteriorated after treatment with IgA / IgG / scFv antibodies; among them, U-87MG cells treated with NGF Ab-IgA and BDNF Ab-IgA showed the lowest cell density, growth arrest, indistinct cell edges, poor refractive properties, and a large amount of cell debris, such as... Figure 5 As shown in images A and B.

[0214] TO-PRO-3 was used to label dead cells in real time; lysed cells released DNA that bound to a fluorescent carrier, emitting bright red fluorescence. Evos observations revealed an increase in TO-PRO-3+U-87MG cells treated with IgA / IgG / scFv antibodies compared to the untreated group, indicating cell lysis after antibody treatment. The lysis signals were most significant in the NGF Ab-IgA and BDNF Ab-IgA treatment groups, suggesting that these treatments most effectively inhibited U-87MG cell survival. Furthermore, death signals were also observed in the IgA Isotype and IgG Isotype treatment groups.

[0215] The above results indicate that treatment with NGF Ab-IgA or BDNF Ab-IgA significantly increased the fragmentation and lysis of U-87MG cells, which was detrimental to cell growth and survival.

[0216] (III) Scratch assay (IgA antibody targeting NGF and BDNF inhibits glioma cell migration)

[0217] The effects of different antibodies targeting NGF and BDNF on the migration and invasion abilities of glioma cells were detected using a scratch healing assay.

[0218] 1. The effect of antibodies targeting NGF or BDNF on the migration of U-87MG cells was detected using a scratch assay. Confluent monolayers of U-87MG cells were scraped with the tip of a 10 μL pipette. Cells were either untreated (0 h) or treated with rhBDNF / rhNGF protein or antibody for 24 h.

[0219] 2. Observe representative results under a phase contrast microscope and take pictures at the same location.

[0220] 3. Calculate the relative migration distance using the following formula: Relative migration distance = (AB) / A, where A represents the average width of the cell scratches before treatment, and B represents the average width of the cell scratches after treatment. The result is expressed as mean ± SE.

[0221] Statistical results showed that the addition of exogenous neurotrophic factors helped improve wound closure activity, suggesting that BDNF or NGF can enhance the in vitro migration ability of U-87MG cells. The migration distance of U-87MG cells in the IgA antibody treatment group was significantly shortened, indicating that NGF Ab-IgA and BDNF Ab-IgA antibodies significantly inhibited U-87MG cell migration and invasion, with BDNF Ab-IgA showing a stronger inhibitory effect. Figure 6 As shown in images A and B.

[0222] (IV) Effects of IgA Antibody on Glioma Cell Apoptosis

[0223] 1.Use CellEvent TM The effect of Caspase-3 / 7 assay on apoptosis in U-87MG cells using antibodies targeting NGF or BDNF. This dye is a novel fluorescent substrate for activating Caspase-3 / 7, consisting of a tetraamino acid peptide (DEVD) conjugated to a nucleic acid-binding dye. Upon activation of Caspase-3 or Caspase-7 in apoptotic cells, the DEVD peptide is cleaved, allowing the dye to bind to DNA and produce a bright fluorescent reaction with a maximum absorption / emission of approximately 502 / 530 nm.

[0224] 2. Beforehand, plate U-87MG cells in 24-well plates and place them in a 37°C, 5% CO2 incubator to allow them to adhere to the plates.

[0225] 3. Discard half of the MEM complete culture medium, add 200 μL of different groups of antibodies targeting NGF or BDNF (set the groups as above) to treat the target cells, and incubate the culture plate in an incubator for 24 hours.

[0226] 4. Set CellEvent TM Caspase-3 / 7 Green Detection Reagent was diluted in complete culture medium to a final concentration of 5 μM and added directly to adherent cells. The cells were then incubated at 37°C for at least 30 minutes.

[0227] 5. Using channels compatible with FITC and Alexa Fluor 488 dyes, scan and image cell culture plates using the Evos FLAuto2 automated live-cell imaging system; simultaneously, determine the Caspase 3 / 7 activity (%).

[0228] The correlation between NGF Ab-IgA and BDNF Ab-IgA antibodies and glioma cell apoptosis was investigated. U-87MG cells were treated with different proteins for 24 hours, and Western blot was used to specifically detect Caspase-3, a key cleavage enzyme in the terminal apoptosis process. The results showed that, compared with the untreated group, U-87MG cells treated with IgA / IgG / scFv antibodies all showed the presence of a 17kDa cleaved (active) form of Caspase-3, indicating that U-87MG cells underwent apoptosis. Recombinant human BDNF and NGF are involved in protecting U-87MG cells from apoptosis.

[0229] Statistical results showed that, compared with IgG antibodies, BDNF Ab-IgA and NGF Ab-IgA antibodies induced stronger target cell apoptosis; comparing the two IgA antibodies, BDNF Ab-IgA was superior to NGF Ab-IgA. U-87MG cells treated with Anti-BDNF scFv and Anti-NGF scFv also showed high expression of 17kDa Caspase-3, further validating the pro-apoptotic function of the IgA antibody variable region. The antibody Fc fragment (IgA Isotype and IgG Isotype) could induce a small amount of apoptosis, with a statistically significant difference compared to the untreated group.

[0230] Using CellEvent TM Caspase-3 / 7 live-cell imaging was further used to detect apoptosis in U-87MG cells. Evos microscopy revealed that, compared to the untreated group, after 24 hours of IgA / IgG / scFv treatment, apoptotic U-87MG cells activated Caspase-3 / 7, producing a bright fluorescent reaction, such as... Figure 7 As shown in Figure A. Statistical analysis of Caspase-3 / 7 activity revealed that NGF Ab-IgA and BDNF Ab-IgA most significantly induced apoptosis in U-87MG cells. Figure 7 As shown in image B.

[0231] The above results indicate that exogenous supplementation with recombinant human BDNF or NGF helps glioma cells grow, proliferate, and survive, participates in protecting cells from apoptosis, and enhances the in vitro migration ability of glioma cells.

[0232] BDNF Ab-IgA and NGF Ab-IgA significantly inhibited glioma cell proliferation and survival, induced strong apoptosis, and reduced cell migration and invasion abilities; among them, BDNF Ab-IgA was stronger than NGF Ab-IgA in inducing apoptosis and inhibiting glioma cell survival and migration.

[0233] Example 4: In vivo antitumor experiment of NGF Ab-IgA and BDNF Ab-IgA

[0234] (I) Constructing a mouse model of U-87MG malignant glioma

[0235] Based on in vitro functional experiments, this study seeded Luci-U-87MG cells stably expressing exogenous luciferase into the striatum of BALB / c nude mice to construct a human glioma mouse transplantation model (3 mice per group, repeated 3 times) to verify the in vivo antitumor efficacy of NGF Ab-IgA and BDNF Ab-IgA antibodies.

[0236] 1. Luci-U-87MG cells stably expressing exogenous luciferase were digested with 0.25% trypsin and washed twice with 1×PBS. The cells were counted and the suspension concentration was adjusted to 1.0×10⁻⁶. 5 Cells / μL, keep on ice until ready to use.

[0237] 2. Install the stereotaxic device (connect the injection pump to the red port, and connect the other three ports (red, yellow, and green) to the stereotaxic device for the X, Y, and Z axes, respectively).

[0238] 3. Anesthetize BALB / c nude mice by intraperitoneal injection of tribromoethanol. Place cotton pads on the ventral side of the mice for warmth, secure them to the mouse adapter platform using ear rods, and wipe their head fur with alcohol-soaked cotton balls.

[0239] 4. Make a sagittal incision with a scalpel and curved forceps, and wipe the skull with 3% hydrogen peroxide to remove the fascia.

[0240] 5. Locate the anterior fontanelle of the mouse as the origin (X=0, Y=0) and mark it. Use a skull drill to drill a hole 1 mm above the anterior fontanelle and 2 mm to the left (X=-2, Y=1). Drill lightly until you feel a sense of emptiness and stop.

[0241] 6. Mix the Luci-U-87MG cell suspension again, fix the Hamilton microinjection needle and remove air bubbles. Set the microinjection pump to slowly and evenly insert the needle to a depth of 3.5 mm, and gently withdraw the needle to 3 mm; then inject the cell suspension at a constant rate of 1 μL / min, setting the target volume to 5 μL.

[0242] 7. After the needle has been fully inserted, wait 5 minutes, then slowly withdraw the Hamilton microinjection needle; immediately rinse with saline solution.

[0243] 8. Apply freshly prepared bone wax to the skull opening to prevent the cell suspension from leaking out; then apply 2 μL of 3M medical adhesive to the mouse scalp sutures.

[0244] 9. After the mice have recovered, continue to house them in an SPF-grade animal room, and observe and record their condition at any time.

[0245] (II) Immunotherapy with NGF Ab-IgA and BDNF Ab-IgA antibodies

[0246] Ninety female BALB / c nude mice, aged 4 weeks and weighing approximately 15–18 g, were randomly divided into 9 groups (n=3 per group, replicated 3 times). A malignant glioma mouse model was constructed following the steps described above. Approximately 14 days later, mice were treated with different antibody immunotherapies. One day apart, NGF Ab-IgA and BDNF Ab-IgA, NGF Ab-IgG and BDNF Ab-IgG, anti-NGF scFv and anti-BDNF scFv, and IgA Isotype and IgA Isotype were injected via the tail vein. PBS was used as a negative control. Treatment was continued for 10 cycles. Multiple experiments were conducted to record the following data: magnetic resonance imaging (MRI) and small animal in vivo imaging of the same batch of tumor-bearing mice. Mice were injected with a contrast agent (Injection dimeglumine gadopentetate) beforehand. The MRI / contrast T1 parameters were set as follows: TE / 17.23ms; Read FOV 20.0, Offset 0.1mm; Phase 20.0, Offset -0.1mm; Slices 15; Thk 1.00mm. Brain tissue was obtained from mice in different antibody treatment groups and fixed with 4% paraformaldehyde for HE staining or other immunofluorescence and immunohistochemical pathological experiments. A separate mouse model of glioma was established for recording body weight and survival data (n=10 per group). Specific grouping and intervention protocols are shown in Table 4.

[0247] Table 4. Experimental Groups and Treatment Regimens

[0248]

[0249] To further clarify the in vivo tumor-suppressing effect of IgA antibodies, multi-planar stereoscopic imaging of mice with gliomas was performed using magnetic resonance imaging (MRI). After intravenous injection of contrast agent into the tail vein of mice, layered scanning of the brain tissue was conducted, and the results are as follows: Figure 8As shown in Figure A, mice in the PBS, IgA, and IgG Isotype groups exhibited bright high-signal intensity with uniform or nodular enhancement, indicating round or oval tumor lesions; suggesting vigorous tumor proliferation and rapid absorption and metabolism of contrast agents. In contrast, mice in the NGF Ab-IgA and BDNF Ab-IgA intervention groups showed weak low-signal intensity in the striatum, with significantly reduced tumor lesion effects; these results suggest that IgA-targeting antibody drugs significantly inhibit the growth of gliomas in mice.

[0250] The brain tumor volume was calculated using three-dimensional fitting technology. The results showed that after 10 cumulative interventions, the brain tumor volume in the PBS group mice increased to approximately 378.8 ± 31.0 mm. 3 IgA Isotype intervention group (359.1±11.8mm) 3 ) and the IgG Isotype intervention group (341.7±44.2mm) 3 There was no statistically significant difference compared to the PBS group.

[0251] NGF Ab-IgA intervention group (51.9±33.9mm) 3 ) and BDNF Ab-IgA intervention group (36.8±11.6mm) 3 Tumor volume was significantly reduced, with BDNF Ab-IgA showing better results, shrinking brain tumors to approximately 1 / 10 of that in the PBS group. In the same target IgG and scFv antibody intervention groups, the average tumor volume decreased to 160.8 ± 28.6 mm. 3 and 129.3±46mm 3 ,like Figure 8 As shown in image B.

[0252] Serial paraffin sections were prepared from intact brain tissue of tumor-bearing mice. Hematoxylin-eosin (HE) staining revealed that the brain tumors in the PBS, IgA, and IgG Isotype groups occupied multiple layers and covered a large area, with dense tumor cell nuclei visible; areas of erythrocyte aggregation within the brain tissue were hemorrhagic necrotic foci. The brain tumor area decreased to varying degrees in other antibody intervention groups, with the most significant reduction observed in the NGF Ab-IgA and BDNF Ab-IgA groups, where small-volume tumors or no tumors were visible in the brain tissue sections. These results further demonstrate that IgA antibodies targeting NGF or BDNF significantly inhibit glioma growth.

[0253] (III) IVIS Spectrum small animal in vivo imaging (NGF Ab-IgA and BDNF Ab-IgA significantly inhibited the progression of malignant glioma)

[0254] 1. First, anesthetize Luci-U-87MG glioma mice by perfusion of isoflurane in the gas anesthesia chamber. Initialize the small animal in vivo imaging system; data acquisition can begin when the adapter temperature bar shows green (platform temperature -80℃). Select a 25 cm platform for animal imaging for image acquisition.

[0255] 2. The D-luciferin substrate for luciferase detection was injected intraperitoneally into mice (see the reagent preparation section for details). Each mouse should be injected with at least 150 mg / kg body weight.

[0256] 3. Images were acquired 15 minutes after substrate injection and fixation, with consistent exposure times between groups.

[0257] Analysis using in vivo imaging software (PerkinElmer Biosciences) showed that the signal intensity reflected the number of live tumor cells in the mouse brain, such as... Figure 9 As shown in image A.

[0258] In vivo imaging results showed that, compared with the PBS group, after 10 treatments with IgA / IgG / scFv antibodies targeting NGF or BDNF, the fluorescence intensity of Luciferase in mouse brain tissue was reduced, indicating that these measures could inhibit the growth of mouse gliomas. Among them, the NGF Ab-IgA and BDNF Ab-IgA intervention groups showed the lowest Luciferase fluorescence intensity, indicating the slowest tumor growth and the best treatment effect; BDNF Ab-IgA was slightly better than NGF Ab-IgA. Figure 9 As shown in image B.

[0259] (iv) NGF Ab-IgA and BDNF Ab-IgA prolong the survival of glioma mice

[0260] The body weight and survival of Luci-U-87MG glioma transplanted mice (n=10 per group) were analyzed. After 56 days of observation and recording, mice in the PBS group showed varying degrees of arched back, rigidity, enlarged head tumors, bradykinesia, and ataxia at week 3; and their body weight decreased rapidly, such as... Figure 10 As shown in Figure A, the NGF Ab-IgA and BDNFAb-IgA intervention groups had a milder effect on mouse body weight.

[0261] Survival curve results showed that all mice in the PBS group died within 35 days. Intervention with IgA / IgG / scFv antibodies targeting NGF or BDNF prolonged the survival of glioma-bearing mice; among them, the NGF Ab-IgA and BDNF Ab-IgA antibody intervention groups showed the longest survival, with significant differences. The BDNF Ab-IgA mice had the longest overall mortality time. Figure 10As shown in image B.

[0262] In summary, this invention demonstrates through in vitro and in vivo experiments that two IgA antibodies targeting neurotrophic factors NGF or BDNF, NGF Ab-IgA and BDNF Ab-IgA, have good anti-glioma efficacy, with BDNF Ab-IgA showing better results. Specifically, BDNF Ab-IgA and NGF Ab-IgA significantly inhibited the growth, proliferation, and survival of U-87MG cells, induced strong apoptosis in target cells, and significantly reduced cell migration and invasion abilities. In a mouse orthotopic glioma transplantation model, NGF Ab-IgA and BDNF Ab-IgA significantly inhibited glioma progression and prolonged mouse survival, with BDNF Ab-IgA showing better efficacy; the IgA antibodies were superior to IgG and scFv antibodies. Dimerized NGF Ab-IgA and BDNF Ab-IgA can penetrate the blood-brain barrier or hematoma barrier to reach glioma tissue, bind to pIgR on the surface of tumor cells, and promote tumor cell lysis. Furthermore, IgA antibodies can interact with FcαRI via the Fc fragment, recruiting microglia to mediate ADCC and ADCP effects, exhibiting a stronger anti-tumor effect than IgG antibodies. The results of this invention suggest that BDNF and NGF neurotrophic factors may become new targets for the treatment of gliomas, and that IgA antibodies are more significant than IgG antibodies in crossing the blood-brain barrier and in terms of anti-tumor efficacy.

[0263] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An IgA antibody targeting the neurotrophic factor BDNF, named BDNF Ab-IgA antibody, characterized in that, The amino acid sequence of the BDNF Ab-IgA antibody is shown in SEQ ID NO:

2.

2. An IgA antibody targeting the neurotrophic factor NGF, named NGF Ab-IgA antibody, characterized in that, The amino acid sequence of the NGF Ab-IgA antibody is shown in SEQ ID NO:

1.

3. The IgA antibody according to claim 1 or 2, characterized in that, The IgA antibody is in monomeric form.

4. An antibody composition comprising the BDNF Ab-IgA antibody of claim 1 and the NGFAb-IgA antibody of claim 2.

5. A nucleic acid construct encoding the BDNF Ab-IgA antibody of claim 1 and / or the NGF Ab-IgA antibody of claim 2.

6. The use of the BDNF Ab-IgA antibody of claim 1, or the NGF Ab-IgA antibody of claim 2, or the IgA antibody of claim 3, or the antibody composition of claim 4, or the nucleic acid construct of claim 5 in the preparation of a medicament for treating glioma.

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