IgA antibody targeting neurotrophic factors BDNF and NGF and application thereof

Abstract

The invention discloses an IgA antibody targeting neurotrophic factors BDNF and NGF and application of the IgA antibody. Wherein the IgA antibodies targeting BDNF and NGF provided by the invention are respectively obtained by expressing and purifying expression plasmids of the IgA antibodies in HEK-293T cells, the two IgA antibodies can respectively and specifically recognize and combine with BDNF and NGF and have high affinity, and the result of the embodiment shows that the IgA antibodies targeting NGF and BDNF provided by the invention can inhibit the growth, proliferation and survival of glioma cells, and can be used for preparing glioma cells. Compared with the IgG antibody, the IgG antibody can induce the apoptosis of glioma cells and reduce the cell migration and invasion ability, and also can permeate the blood brain barrier and reach glioma tissues so as to exert a significantly stronger anti-tumor effect.

Description

Technical Field

[0001] The present 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, a preparation method thereof, and an application thereof in the preparation of a drug for treating glioma. Background Art

[0002] Glioma is the most invasive malignant brain tumor, accounting for about 40% of central nervous system tumors, and the domestic mortality rate is as high as 61.4%. However, there is currently a lack of effective treatment methods. The main obstacle is that the entire brain is diffusely infiltrated by tumor cells, which enables the tumor to escape complete resection by surgery and radiotherapy and chemotherapy. Glioma cells grow in a "root-like" manner and are intertwined with neuronal cells and glial cells. Studies have shown that various 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, I.E. 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 applied to glioma immunotherapy are monoclonal antibodies and all are of the IgG isotype. For example, one of them is Bevacizumab against vascular endothelial growth factor (VEGF), which is a humanized IgG1 monoclonal antibody. It inhibits tumor angiogenesis, reduces tumor blood supply and edema by binding to VEGF to prevent its interaction with receptors. Currently, Bevacizumab has been approved by the US FDA for the treatment of recurrent glioblastoma (GBM). Studies have shown that Bevacizumab can prolong the progression-free survival (PFS), but has limited impact on the overall survival (OS). Nevertheless, for some patients, it can significantly improve the 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 one is Nivolumab against programmed death receptor-1 (PD-1), which is a humanized IgG4 monoclonal antibody. It enhances the immune response of T cells to tumor cells by blocking the interaction between PD-1 and its ligands PD-L1 and PD-L2. Currently, although Nivolumab has not been widely approved for first-line treatment of glioma, in some countries and regions, it has been approved for the treatment of other types of cancer and shows certain potential in clinical trials of recurrent glioblastoma. The results of clinical trials show that the efficacy of using Nivolumab alone or in combination with other therapies is inconsistent, and the overall survival rate of newly diagnosed GBM patients has not been significantly improved. However, in some patients with recurrent GBM, objective responses and disease stability have been observed (Reardon DA et al. Nivolumab in patients with recurrent glioblastoma and cediranib plus nivolumab in patients with recurrent glioblastoma or other solid tumours (CheckMate 143 and CheckMate040): 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 gliomas, they also have some obvious drawbacks and challenges.

[0005] 1. Limited blood-brain barrier (BBB) penetration ability

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

[0007] 2. Tumor heterogeneity and drug resistance

[0008] Glioma cells have a high degree of genetic and phenotypic heterogeneity, which means that not all tumor cells will express the same target, and IgG antibodies targeting a single target may not cover all the variations of tumor cells. In addition, as the treatment progresses, tumors may develop drug resistance to IgG antibody treatment. The existence of this heterogeneity and drug resistance may lead to non-responsiveness or drug resistance in some patients to IgG monoclonal antibody treatment, resulting in poor long-term efficacy.

[0009] 3. Unfavorable therapeutic window

[0010] IgG monoclonal antibodies may require high 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, and high doses may lead to more severe toxic reactions, such as immune-related adverse events (irAEs), limiting the safe use range of the drug.

[0011] 4. Limited disease progression and survival improvement

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

[0013] 5. High cost

[0014] The cost of developing, producing, and using IgG monoclonal antibodies is very high, which not only increases the burden on the healthcare system but also may make the treatment cost unaffordable for some patients. The high price may limit the accessibility of these therapies, especially in regions with limited resources.

[0015] 6. Nonspecific targeting effects

[0016] Some IgG monoclonal antibodies may not only target specific antigens on tumor cells but also bind to similar receptors in normal tissues, causing unnecessary side effects. Nonspecific binding can lead to damage to healthy tissues and increase treatment-related complications. First, IgG antibodies are mainly present in blood and tissue fluids and can activate immune responses through Fc receptors and the complement system. Due to the widespread distribution of IgG antibodies throughout the body, they may bind to similar receptors in normal tissues, triggering nonspecific effects and side effects, especially when high doses are used. Second, IgG antibodies can activate various immune effector mechanisms mediated by Fc receptors, including antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). While this potent immune response enhances the anti-tumor effect, it may also increase damage to normal cells.

[0017] Therefore, there is an urgent need in the art for a new strategy and method for treating tumors, especially gliomas. Summary of the Invention

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

[0019] Another aspect of the present invention provides an IgA antibody targeting neurotrophic factor NGF, named NGF Ab-IgA, which may comprise or consist 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 monomeric or multimeric form.

[0021] In some embodiments, the multimeric form includes dimeric or tetrameric forms.

[0022] Another aspect of the present invention provides an antibody composition comprising the above-mentioned BDNF Ab-IgA and the above-mentioned NGF Ab-IgA.

[0023] Another aspect of the present invention further provides a nucleic acid construct encoding the above-mentioned BDNF Ab-IgA and / or the above-mentioned NGF Ab-IgA.

[0024] The use of BDNF Ab-IgA, or NGF Ab-IgA, or the antibody composition, or the nucleic acid construct provided by the present invention in the preparation of a medicament for treating glioma, in the preparation of a medicament for blocking the trophic delivery signal of nerve-glioma, and in the preparation of a medicament for triggering the ADCC and ADCP effects of microglia also belong to the content of the present invention.

[0025] In some embodiments, the glioma includes glioblastoma.

[0026] In this study, two dimeric IgA antibodies, NGF Ab-IgA and BDNF Ab-IgA, targeting the neurotrophic factors NGF or BDNF were expressed. On the one hand, by antagonizing the neurotrophic factor receptor pathway in the variable region, the trophic delivery signal of nerve-glioma was directly blocked, inhibiting the growth, proliferation, survival and migration of glioma cells and inducing apoptosis. It was demonstrated in a glioma cell model with knockout of the neurotrophic factor receptor that the absence of TRKA, TRKB or NGFR signals directly inhibited glioma growth. On the other hand, the IgA antibody triggered the ADCC and ADCP effects of microglia through cross-linking of FcαRI by the Fc segment, ultimately lysing and phagocytosing glioma cells. At the same time, the IgA antibody may cross the blood-brain barrier or blood-tumor barrier through transcytosis mediated by pIgR, reach the tumor tissue, and bind to pIgR on the surface of tumor cells, thereby promoting tumor cell lysis. The results of this study showed that the IgA antibody was more significant than the IgG antibody in crossing the blood-brain barrier and anti-tumor efficacy. Therefore, the neurotrophic factors BDNF and NGF are expected to become a new target for treating glioma, and using the characteristics of the IgA antibody is also expected to provide a new idea for the immunotherapy of glioma.

[0027] In addition, compared with the IgG antibody existing in the prior art, the IgA antibody provided by the present invention also has obvious advantages in terms of tumor heterogeneity and drug resistance, treatment window, cost and non-specific targeting effect.

[0028] Among them, in terms of tumor heterogeneity and drug resistance, the IgA antibody can more effectively activate neutrophils and macrophages, and these immune cells themselves have a broader targeting ability, which may have certain advantages in dealing with tumor heterogeneity; in addition, the IgA antibody can act at the mucosal site, which may have unique advantages for some specific types of cancer; furthermore, the IgA antibody acts through different mechanisms and can avoid the mechanism of IgG antibody drug resistance in some cases. For example, the IgA antibody can more effectively mediate the cytotoxicity of neutrophils, which can provide a new way to overcome certain drug resistances.

[0029] In terms of the therapeutic window, IgA antibodies have some advantages over IgG antibodies in dealing with suboptimal therapeutic windows and the risk of side effects. These advantages are mainly reflected in their restricted distribution, the mechanism of specifically activating immune cells, and the potential to allow lower dosages. First, the pharmacokinetics and distribution are different: IgG antibodies have a long half-life and wide distribution in the body because they can be recycled through the neonatal Fc receptor (FcRn). Due to this mechanism, IgG antibodies can maintain a relatively high concentration 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, mainly play an immune role on mucosal surfaces and are more restricted in distribution to mucosal areas such as the respiratory and digestive tracts. Monomeric IgA has a short half-life in the blood, resulting in a shorter time for systemic distribution. Therefore, in some cases, IgA antibodies can reduce the risk of systemic side effects. Second, the mechanism of activating immune cells is different: IgG antibodies activate various effector cells (such as NK cells, macrophages, and neutrophils) by binding to Fcγ receptors and act through the complement system. This broad mechanism of action may lead to more severe immune-related adverse events (irAEs), such as autoimmune reactions or systemic inflammation, when applied at high doses. IgA antibodies mainly activate neutrophils and macrophages by binding to FcαRI (CD89). This specific mechanism of action can reduce unnecessary immune activation to a certain extent, thereby reducing the risk of immune-related adverse events. In addition, the action of IgA antibodies is more confined to mucosal surfaces, which can further limit systemic side effects. Third, the dosage and potency are different: Due to their long half-life and wide distribution, IgG antibodies may require higher doses to achieve sufficient tumor infiltration and therapeutic effects. This high-dose use may increase the risk of side effects, especially in chronic treatment. The specific distribution and mechanism of action of IgA antibodies can allow effective therapeutic concentrations to be achieved at lower doses, especially in mucosa-associated tumors. This can reduce the risk of dose-related side effects.

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

[0031] In terms of targeting non-specific effects, IgA antibodies are mainly present on mucosal surfaces (such as the respiratory, digestive, and urogenital tracts) and exist in two main forms: monomeric IgA in serum and dimeric IgA in mucosa. Dimeric IgA binds through the J chain and the secretory component (SC), can remain more effectively on the mucosal surface, enter the systemic circulation less, and reduce systemic distribution; the local action characteristics can significantly reduce the non-specific effects on non-target tissues. Description of the Drawings

[0032] Figure 1 Schematic diagrams of different antibodies (NGF Ab-IgA, BDNF Ab-IgA, NGF Ab-IgG, BDNF Ab-IgG, anti-NGF scFv, and anti-BDNF scFv) designed in Example 1.

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

[0034] Figure 3 Results of high-specific binding of monomers and dimers of IgA by the HiTrap ProteinL HP chromatography column (A), Western blot and Coomassie brilliant blue staining results of enriched and purified IgA antibodies (B and C), Western Blot results of IgA purified antibody incubated with recombinant human BDNF and NGF proteins overnight (D), and ELISA experimental results of concentration-dependent binding of BDNF Ab-IgA and NGF Ab-IgA antibodies to BDNF and NGF proteins (E).

[0035] Figure 4 Results of IgA / IgG / scFv antibodies targeting NGF and BDNF inhibiting the proliferation of U-87MG cells.

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

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

[0038] Figure 7 Using CellEvent TM Apoptosis photographs (A) of U-87MG cells imaged by Caspase-3 / 7 live cell imaging and the statistical results (B) of Caspase-3 / 7 activity.

[0039] Figure 8 Results of stratified scanning of brain tissue (A) after intravenous injection of an enhanced contrast agent into the tail vein of mice and the calculated results of tumor volumes in each group (B).

[0040] Figure 9 Results of the number of viable tumor cells in the brains of mice analyzed using in vivo imaging software (PerkinElmer Biosciences) (A) and the statistical results of in vivo imaging (B).

[0041] Figure 10 Results of the analysis of the body weight and survival of mice transplanted with Luci-U-87MG gliomas (A) and the survival curves of mice in each group (B). Detailed implementation manners

[0042] In view of the defects of IgG monoclonal antibodies for treating glioblastoma existing in the prior art, the present invention provides an IgA antibody targeting neurotrophic factors BDNF and NGF, and provides the application of such antibodies in the treatment of glioblastoma.

[0043] First, when the present inventors analyzed the expression of BDNF and NGF in the human glioblastoma cell line U-87MG and the brain tissues of nude mice transplanted with U-87MG gliomas, it was found that both U-87MG cells and their glioma tissues highly expressed BDNF and NGF. Neuronal cells (iPSC-neuron) differentiated from human induced pluripotent stem cells (iPSC) cultured in vitro could also secrete BDNF and NGF, and their culture supernatants could 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, the present invention respectively constructed IgA antibodies targeting NGF or BDNF (NGF Ab-IgA and BDNF Ab-IgA); simultaneously designed IgG antibodies with the same variable region sequences (NGF Ab-IgG and BDNF Ab-IgG) and single-chain variable regions without the Fc segment (anti-NGF scFv and anti-BDNF scFv). The results showed that after transient transfection of HEK-293T cells with the recombinant plasmids, the expression of exogenous antibody fragments could be detected in the supernatant, and IgA could assemble into a dimer structure. The purified IgA antibodies could specifically recognize recombinant human BDNF and NGF proteins with strong affinity. The above results indicated that the construction and expression of specific and high-affinity IgA antibodies targeting NGF or BDNF were successful.

[0045] The present invention verified the anti-tumor functions of NGF Ab-IgA and BDNF Ab-IgA at the cellular level. The results showed that adding recombinant human BDNF or NGF to the medium could promote the rapid growth and proliferation of U-87MG cells, while adding IgA / IgG / scFv antibodies targeting NGF or BDNF could all inhibit their proliferation, but the IgA antibodies had the most significant effect. After treatment with NGF Ab-IgA or BDNF Ab-IgA, the fragmentation and lysis of U-87MG cells were significantly increased. Recombinant human BDNF and NGF were involved in protecting U-87MG cells from apoptosis, while BDNF Ab-IgA and NGF Ab-IgA significantly up-regulated the expression of apoptosis-related protein Caspase-3, inducing more intense apoptosis of U-87MG cells than the IgG treatment group. The results of the scratch assay showed that NGF Ab-IgA and BDNF Ab-IgA significantly inhibited the migration of U-87MG cells. The above results indicated that BDNF Ab-IgA and NGF Ab-IgA inhibited the growth, proliferation and survival of glioma cells, induced apoptosis of glioma cells, and reduced the cell migration and invasion ability; among them, BDNF Ab-IgA had a more obvious effect than NGF Ab-IgA.

[0046] Based on in vitro functional experiments, the present invention constructed a glioma transplantation model in the mouse striatum in situ using Luci-U-87MG cells to verify the in vivo anti-tumor efficacy of NGF Ab-IgA and BDNF Ab-IgA. The results showed that IgA / IgG / scFv antibodies targeting NGF or BDNF could inhibit the growth of glioblastoma and prolong the survival of mice, and the tumor growth in the IgA intervention group was the slowest. Magnetic resonance imaging results showed that compared with the PBS and Isotype control groups, the imaging signals of mice in the NGF Ab-IgA and BDNF Ab-IgA intervention groups were significantly reduced, and the tumor mass effect was significantly decreased, with better efficacy in the BDNF Ab-IgA group; HE staining indicated a significant reduction in glioma. In the NGF Ab-IgA and BDNF Ab-IgA antibody intervention groups, IgA infiltration was observed in both the normal brain tissue space and the 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 the glioma tissue, thereby inhibiting the growth of glioblastoma in mice with significant efficacy.

[0047] Finally, the present invention explored the possible mechanisms of action of NGF Ab-IgA and BDNF Ab-IgA in anti-tumor in vitro and in vivo. The results showed that U-87MG cells highly expressed the neurotrophic factor receptor tyrosine kinase receptor A (Tropomyosin-related kinase A, TrkA), tyrosine kinase receptor B (Tropomyosin-related kinase A, TrkB), and nerve growth factor receptor (Nerve growth factor receptor, NGFR). A U-87MG cell model with knockout of neurotrophic factor receptors (TRKA KO, TRKB KO, and NGFR KO) was constructed using CRISPR / Cas9 technology. The experimental results showed that the proliferation and viability of TRKA KO, TRKB KO, and NGFR KO U-87MG cells decreased in vitro, and the migration ability was weakened, with the most obvious effect in TRKB KO cells. The results of the mouse striatum in situ human glioma transplantation model showed that compared with wild-type U-87MG cells, the knockout cells grew more slowly in the mouse intracranial cavity, with the smallest tumor volume in the TRKB KO group; the survival of mice was significantly prolonged. The above experimental results indicate that the lack of neurotrophic factor receptor signals directly inhibits the growth of glioma, so NGF Ab-IgA and BDNF Ab-IgA can directly block the neurotrophic factor receptor signaling pathway through the V region of the antibody to exert anti-tumor functions.

[0048] In view of the differences in the antitumor efficacy of IgA and IgG antibodies, the present invention further conducted an exploratory study on the mechanism of action of the Fc segment. On the one hand, IgA binds to pIgR (polymeric Immunoglobulin Receptor) for epithelial transport. Flow cytometry demonstrated 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 Fc segment of IgA binds to the IgA-specific Fc receptor (FcαRI) to mediate a series of immune effects. The results of flow cytometry and LDH experiments showed that compared with IgG antibodies, the BDNF Ab-IgA antibody mediated stronger ADCC activity of microglia; after blocking FcαRI with a specific antibody, the cytotoxic activity of IgA-mediated microglia was significantly reduced. The results of pHrodo fluorescence flow cytometry and live cell imaging showed that the BDNF Ab-IgA antibody mediated the chemotaxis and aggregation of microglia towards glioma cells, and caused the target cells to be gradually internalized and phagocytosed by microglia. The above results indicate that IgA antibodies can interact with FcαRI through the Fc segment to mediate the ADCC and ADCP effects of microglia and play an antitumor role.

[0049] In summary, the present invention demonstrated through in vitro and in vivo experiments that the two IgA antibodies NGF Ab-IgA and BDNF Ab-IgA targeting neurotrophic factors NGF or BDNF have good anti-glioma efficacy, with BDNF Ab-IgA having a better effect. On the one hand, NGF Ab-IgA and BDNF Ab-IgA can antagonize the neurotrophic factor receptor pathway through the variable region to directly block the nutrient delivery signal of neurons-gliomas; on the other hand, they crosslink FcαRI through the Fc segment to trigger the ADCC and ADCP effects of microglia and exert antitumor activity. At the same time, IgA antibodies may cross the blood-brain barrier or blood-tumor barrier through transcytosis mediated by pIgR, reach the glioma tissue, bind to pIgR on the surface of tumor cells, and promote tumor cell lysis. The results of the present invention suggest that the neurotrophic factors BDNF and NGF are expected to become new targets for the treatment of gliomas, and IgA antibodies are more significant than IgG antibodies in crossing the blood-brain barrier and antitumor efficacy.

[0050] The present invention will be further described in detail below with reference to specific examples and drawings.

[0051] The following disclosure provides many different embodiments or examples for implementing different aspects of the present invention. The present invention provides examples of various specific processes and materials, but those of ordinary skill in the art can be aware of 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] The methods used in the following embodiments are all conventional methods unless otherwise specified.

[0054] The experimental animals used in the embodiments of the present invention: BALB / c nude mice (Experimental Animal Breeding Center, Institute of Model Animals, Nanjing University): The experimental mice are female, 4 weeks old, with an initial body weight of 15-18 g, and are raised in a specific pathogen free (SPF) environment [Permit No.: SYXK (Jing) 2012-0004] in 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 with 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 (which can be obtained with reference to the following literature: Kondo, T., & Tabar, V. (2009). "Induced pluripotent stem cells: a new era in cell therapy." 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, and cultured adherently in Neurobasal medium (Thermo Fisher Scientific, USA) containing 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, 0.2 mM; cAMP, 50 mM; DAPT, 10 mM) and 100× GlutaMAX.

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

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

[0060] Strains and plasmids used in the examples of the present invention:

[0061] 1) Strains

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

[0063] 2) Plasmid

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

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

[0066] eSpCas9-LentiCRISPR v2 (Plasmid#52961): enhanced specificity SpCas9 [Enhancedspecificity SpCas9 (eSpCas9)], also known as SpCas9 (K848A / K1003A / R1060A). This plasmid was constructed by Feng Zhang's laboratory and replaced the original lentiCRISPR v1 (Plasmid#49535). eSpCas9 can improve the specificity of the Cas9 protein for target genes, reduce off-target effects on target genes by more than 10 times, and at the same time maintain a strong editing efficiency for target genes.

[0067] psPAX2 and pMD2.G (see CN102174471A): lentiviral packaging plasmids, which use the three-plasmid Lenti-X packaging system to increase vector stability and safely produce high-titer and 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] The 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 each contained the complete antigen-recognition variable domains (VH and VL). The IgA sequence was derived from NCBI-Immunoglobulin heavy constant alpha 1 (Homo sapiens); its constant domains (CH1-CH2-CH3) and the tailpiece structural partition were analyzed by the 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] The secretion of the two IgA antibodies into the supernatant was driven by the Signal Peptide signal peptide; they sequentially contained the antibody VH and VL. The middle of the antibody variable regions was connected by a flexible fragment. The Linker was 15 amino acids long and consisted of glycine (Gly) and serine (Ser) [(G4S)×3], which had a certain elasticity and protease resistance, enabling the functional regions of VH and VL to pair after folding and form an antigen-binding site. Its constant region was also connected to IgA1 CH2-CH3-Tailpiece and J chain, thereby ensuring its stable assembly into a dimer dIgA (IgA dimer) in vitro. An Fc fragment (CH2-CH3) was designed at the end to bind to the FcαRI on the surface of effector cells, thereby initiating a series of downstream activation signals. At the same time, 4 groups of control antibodies were designed and 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 segment). The specific structures of the antibodies are as Figure 1As shown, Panel A is a schematic diagram of the structures of NGF Ab-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), Panel B is a schematic diagram of the structures of monomeric IgA and dimeric IgA, Panel C is 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 Panel D is 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 vector is 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 histidines (6×His) were introduced at the C-terminus of the scFv fragment for antibody capture and purification. Isotype Controls, namely IgA Isotype and IgG Isotype, were set for IgA and IgG respectively.

[0072] The following aspects were mainly considered in the design of the antibodies of the present invention:

[0073] 1. Selection of IgA as the antibody type

[0074] In view of the fact that most current antibody therapies use IgG antibodies and have many deficiencies, the present invention recognizes that IgA antibodies have unique biological properties, such as the ability to form a dimeric structure, which is linked by the J chain, enhancing stability, and can initiate activation signals by binding to the effector cell surface receptor through FcαRI. Also considering the role of IgA antibodies in mucosal immunity and their possible better ability to penetrate the blood-brain barrier, IgA antibodies were designed.

[0075] 2. Optimized variable region source

[0076] The present invention utilizes the antigen recognition variable domains (VH and VL) that have been verified effective in the patent documents WO 2006 / 110883A2 and US2016 / 0207991A1 to ensure a high degree of specificity of the antibodies for the target antigens (NGF and BDNF). This selection based on existing successful cases reduces the development risk and increases the probability of success.

[0077] 3. Well-designed Linker

[0078] The present invention introduces a flexible segment composed of glycine and serine as a connector ((G4S)×3) during antibody design, which 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 an antigen binding site. This specific length and composition of the linker is designed to maximize the functionality and stability of the antibody while providing sufficient flexibility to adapt to different application environments.

[0079] 4. Construction of complete dimeric dIgA

[0080] The present invention ensures that the IgA antibody can be stably assembled into a dimer in vitro by including the CH2-CH3-Tailpiece and J chain sequences, which is essential 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 higher stability and a wider range of potential applications.

[0081] 5. Application of Fc fragment

[0082] The present invention designs 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 antibodies. The addition of the Fc fragment provides the antibody with additional functions, enabling it to not only block the effects of neurotrophic factors, but also further affect target cells through immune system-mediated mechanisms.

[0083] 6. Design of control plasmid

[0084] In addition to the main IgA antibody design, the present invention also designs a variety of control plasmids, including IgG antibodies, single-chain variable fragments (scFv), and isotype controls (Isotype Control), which are helpful for comprehensively evaluating the performance of new IgA antibodies.

[0085] (II) Extraction of target plasmid in small quantities

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

[0087] 2. Inoculate 7 strains containing the following expression plasmids: 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 respectively in LB medium containing ampicillin resistance, and culture them in a shaker at 37 °C and 200 r / min for 12 - 16 hours. Extract the bacteria in the growth plateau phase.

[0088] 3. Take 5 mL of each target bacterial liquid (the dosage of each reagent can be doubled accordingly), centrifuge at 4000 rpm for 1 minute at room temperature, and carefully aspirate and 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, immediately gently invert and mix several times to avoid breaking the plasmid DNA. At this time, the solution should be uniformly transparent blue, otherwise there is an excessive amount of bacteria.

[0090] 5. Add 350 μL of Buffer P3, immediately gently invert and mix several times; at this time, the solution should return to colorless, otherwise the mixing is not thorough. Let it stand at room temperature for 2 minutes to completely remove RNA.

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

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

[0093] 8. Sterilely aspirate 3 mL of Elution Buffer, place the adsorption columns into 7 clean centrifuge tubes respectively, and vertically add 50 - 100 μL of Elution Buffer preheated to 60 °C to the center of the adsorption membrane, and let it bind fully at room temperature for about 10 minutes.

[0094] 9. Finally, centrifuge and elute at 9000×g for 1 minute to obtain the target plasmid extracted in small amounts by the kit. Use a Nanodrop 2000 ultraviolet spectrophotometer to detect its concentration and label it. 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 IgA, IgG, and scFv antibody expression plasmids obtained above in a -20°C refrigerator for subsequent experiments such as double digestion.

[0095] (III) Double digestion reaction

[0096] Perform double digestion reactions on the eukaryotic expression vector MH-AbVec2.0 and 6 groups of PUC57 plasmids.

[0097] 1. Use EcoR I and Hind III to double digest the empty MH-AbVec2.0 plasmid according to the digestion system shown in Table 1 below.

[0098] Table 1:

[0099]

[0100] 2. Use EcoR I and Hind III to double digest the target PUC57 plasmid according to the digestion system shown in Table 2 below.

[0101] Table 2:

[0102]

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

[0104] 1. Use 1% agarose gel electrophoresis to identify the size of the digestion reaction bands. Prepare the agarose gel solution and pour it into a beaker. Heat it at a high temperature until the agarose melts and shake it evenly. After cooling slightly, add 0.5 μg / mL ethidium bromide until the solution turns light red. Pour an appropriate volume of the solution into a horizontal electrophoresis tank and insert the sample comb as soon as possible to avoid generating bubbles.

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

[0106] 3. After mixing the above double digestion products with the Purple loading dye DNA staining solution in proportion, form a purple and uniform DNA mixture, and carefully inject it into the sample wells; prevent cross-contamination of samples in different wells.

[0107] 4. Electrophorese at a constant voltage of 110V for about 45 minutes. Stop electrophoresis when it reaches about 2 / 3 according to the color of the indicator, and try to distinguish the target DNA fragment from other fragments as much as possible.

[0108] 5. Use PE gloves to gently remove the gel from the electrophoresis tank. A blunt knife can be used to scrape the edges to prevent breakage.

[0109] The double-digested products were analyzed by 1% agarose gel electrophoresis. The results showed that the DNA fragment sizes of the above-mentioned antibody plasmids were all consistent with the expected results. Predicted by SignalP 4.1, the cleavage sites of the anti-BDNF antibody were between the 30th and 31st amino acids (ASA-AV), and the cleavage sites of the anti-NGF antibody were between the 26th and 27th amino acids (ASA-QV), indicating that the N-terminal signal peptide design of the antibody was correct and could direct the newly synthesized protein to be secreted into the supernatant.

[0110] (V) Column DNA Gel Extraction

[0111] 1. Use the column gel extraction method to purify the eukaryotic expression vector of MH-AbVec2.0 and the double-digested products of 6 groups of PUC57 plasmids. First, preheat the water bath to 50 °C. Place the digested products under an ultraviolet lamp and quickly cut out the target gel block to reduce the exposure time. Put the gel block in a 15 mL centrifuge tube, weigh the centrifuge tube before and after and record the net weight of the gel block.

[0112] 2. Add 6 times the volume of Buffer B2 of the net weight of the gel block. Place the centrifuge tube in a 50 °C water bath until the gel is completely dissolved, and continuously shake it during the process to make the gel block dissolve evenly.

[0113] 3. Transfer all the dissolved gel solution into the labeled adsorption column. The sample loading volume each time is 750 μL. Centrifuge at 8000×g at room temperature for 30 seconds to transfer 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 adsorption column of the target DNA solution, centrifuge at 9000×g at room temperature for 30 seconds, and discard the liquid in the collection tube.

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

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

[0117] 7. Take the Elution Buffer and preheat it to 60 °C. Vertically add 35 μL of Elution Buffer to the center of the adsorption membrane respectively, and let it bind fully at room temperature for about 10 minutes.

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

[0119] (VI) Ligation Reaction

[0120] 1. According to the ligation system shown in Table 3 below, ligate the double-digested products obtained by gel recovery at 25°C for 1 hour or at 16°C overnight under the action of T4 DNA ligase, and transform the ligation products into Escherichia coli DH5α competent cells.

[0121] Table 3:

[0122]

[0123] (VII) Screening for Positive Recombinant Plasmids

[0124] 1. Pick a single colony in the LB plate, number it, and directly pipette the picking tip into 7 mL of LB liquid medium containing ampicillin, place it on a shaker at 37°C, and shake the bacteria at 200 r / min until it becomes turbid, about 12 - 16 hours, to avoid damaging the DNA due to too long time.

[0125] 2. Extract the above bacterial liquid according to the requirements of the plasmid miniprep kit. Perform double digestion identification and first-generation sequencing on the plasmid. Analyze the sequencing results with Snapgene software to obtain positive recombinant plasmids with correct sequencing, and name them 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 HEK-293T cells in a 6-well plate, and use them for transfection when the cell state is good and the density reaches 80% - 90%. Transiently transfect 6 groups of recombinant plasmids and their isotype controls into HEK-293T cells, and prepare whole cell lysates.

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

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

[0132] 4. Uniformly pipette 200 μL of the DNA transfection mixture into a 6-well plate and gently shake to mix well.

[0133] 5. Incubate in an incubator for 24 - 48 hours. Collect the cell culture supernatants of different transfection groups and the control group, aliquot and store them at -80 °C in a refrigerator to avoid repeated freezing and thawing.

[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 samples (IgA, IgG, scFv, and negative control) and loading buffer to a 1.5 mL centrifuge tube, pipette thoroughly to mix well. The loading buffer contains bromophenol blue, etc., which is used to indicate the electrophoresis position.

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

[0138] 4. Pour 1.5 L of electrophoresis buffer into the electrophoresis tank with the sample loading rack installed. After loading the samples in sequence, perform electrophoresis at a constant voltage of 75 V. When the pre-stained protein Marker bands are significantly separated, adjust to a constant voltage of 120 V for electrophoresis. When the bromophenol blue indicates that the electrophoresis position is close to the end, turn off the voltage.

[0139] 5. Disassemble the glass plates, gently take out the electrophoresis gel and cut off the excess parts. Cut the nitrocellulose membrane into 6.6 cm × 8 cm, and soak it thoroughly in the electrotransfer buffer. Stack them in the order of positive electrode - filter paper - NC membrane - electrophoresis gel - filter paper - negative electrode, wet them in the electrotransfer buffer and filter out the bubbles.

[0140] 6. Install the electrotransfer rack, pour 1.5 L of electrotransfer buffer into the tank, perform electrotransfer at a constant current of 200 mA for 2 hours in an ice box to reduce heat dissipation.

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

[0142] 8. Incubate with primary antibody: Prepare the corresponding target antibody (mouse anti-human Immunoglobulin J polypeptide), and incubate the NC membrane with the primary antibody solution with shaking 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 membrane washing 5 times by shaking, 5 minutes each time.

[0144] 10. Secondary antibody incubation: Horseradish peroxidase-labeled goat anti-mouse IgG antibody. Additionally, Horseradish peroxidase-labeled Anti-Human IgA alpha-Chain antibody and Horseradish peroxidase-labeled goat anti-human IgG (H+L) antibody can also be used for verification. Dilute the antibody proportionally with the blocking solution; Incubate with shaking at room temperature for 1 hour.

[0145] Wash the membrane 5 times by shaking with 1×TBST washing buffer, 5 minutes each time.

[0146] 12. Prepare the Luminal developing solution, react with each group of NC membranes respectively, place them in the ECL autoradiography instrument for exposure imaging and analysis in sequence.

[0147] The protein expression of the antibody was identified by SDS-PAGE immunoblotting. The recombinant plasmids of IgA, IgG, and scFv against BDNF and NGF were transiently transfected into HEK-293T cells respectively, and an equal amount of cell culture supernatant was collected. Antibodies targeting Jchain or IgA were used to specifically detect the expression of exogenous IgA antibody fragments, such as Figure 2 As shown in Western blot results in Panel A, the cell culture supernatant of the transfected group of HEK-293T cells could specifically express NGF Ab-IgA and BDNF Ab-IgA antibodies, and target bands were detected at around ~73 kDa (monomer) and ~140 kDa (dimer), which were consistent with the expected sizes.

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

[0149] ·Hepes Native-PAGE

[0150] To identify whether the IgA antibody can complete the assembly of in vitro multimers, the supernatant non-denatured protein samples were detected by Native-PAGE.

[0151] 1. Use BeyoGel TMThe Plus PAGE precast gel (Hepes, 4 - 20%, 10 wells) was used to verify the multimer assembly of IgA antibodies. Polyacrylamide gel electrophoresis (PAGE) technology is widely used for protein separation, purification, detection, identification, molecular weight analysis, etc.

[0152] 2. First, take out the precast gel from the packaging bag; fix it in the electrophoresis tank and gently and slowly pull out the comb.

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

[0154] 4. Use a 1 mL pipette to suck out the residual storage buffer in the precast gel wells, and gently blow and rinse the loading wells with the electrophoresis buffer. Vertically insert the tip of a 10 μL pipette tip into the loading well for loading. Cover the lid of the electrophoresis tank and insert the power cord plug into the power socket of the electrophoresis instrument (red to red, black to black).

[0155] 5. Control the voltage at 150 V and perform electrophoresis for 40 - 50 minutes; zero the voltage when the bromophenol blue band runs to near the bottom of the gel.

[0156] 6. Take out the glass gel plate, cut the adhesive between the two glass plates, and gently pry open the glass gel plate with a scraper and take it out. Stack them in the order of positive electrode - filter paper - NC membrane - electrophoresis gel - filter paper - negative electrode.

[0157] 7. Install the electro - transfer rack and transfer the membrane at a constant current of 300 mA for 60 minutes.

[0158] 8. For subsequent steps, refer to the above - mentioned Western blot.

[0159] The results are as Figure 2 shown in Panel D below. It can be seen that the transfected HEK - 293T cells can correctly express and assemble IgA multimers, and target bands are detected at around ~140 kDa (dimer) and ~280 kDa (tetramer), which are consistent with the expected sizes.

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

[0161] · Purify IgA antibodies with Peptide M / Agarose

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

[0163] 2. Prepare the following buffers:

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

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

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

[0167] 4) Regeneration / Storage Buffer: 20% (V / V) ethanol in phosphate-buffered saline.

[0168] 3. Immunoglobulin IgA purification procedure

[0169] 1) Pack 1 mL of immobilized 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 filtering and dialyzing the IgA sample using a 0.2 μm filter, load it onto the chromatographic column. Wash the column with 10 mL of equilibration / wash buffer;

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

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

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

[0174] Use HiTrap ProteinL and ProteinA pre-packed ready-to-use columns to prepare the purified above-mentioned IgA and IgG target antibodies. After transient transfection of HEK-293T cells, continue to culture at 37°C, 5% CO 2Cultivate for 24 - 48 h, and then purify the cell culture supernatant according to the instructions of AktaAdvant 25 (GE - Instructions). Among them, the binding buffer is 20 mM sodium phosphate, and the elution buffer is 0.1 M citric acid. All samples are filtered through a 0.45 μm filter to prevent column blockage.

[0175] The antibody purification procedure is as follows:

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

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

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

[0179] 4) Load the IgA antibody or IgG antibody using the injection pump;

[0180] 5) Wash the pipeline with 5 - 10 column volumes of the binding buffer;

[0181] 6) Elute with 2 - 5 column volumes of the 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] After that, after loading and binding, rinsing non - specific proteins, and eluting with 0.1 M citric acid, four pure proteins, namely NGF Ab - IgA, BDNF Ab - IgA, NGF Ab - IgG, and BDNF Ab - IgG, are obtained respectively, and a part of the supernatant is retained as a control for subsequent experiments.

[0184] · Affinity detection of IgA antibody

[0185] 1. Coating the ELISA plate with IgA antibody: Coat NGF Ab - IgA and BDNF Ab - IgA onto the ELISA plate respectively overnight at 4°C; the next day, discard the solution in the wells and wash the plate 3 times with 10 mM PBS buffer (pH 7.4) for 3 minutes each time;

[0186] 2. Adding samples: Add serially diluted rhNGF protein or rhBDNF protein to each well of the ELISA plate; the protein is diluted with 0.05 M carbonate coating buffer (pH 9.0);

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

[0188] 4. At the same time, set up blank wells, negative control wells, positive control wells with commercially available NGF antibody (Abcam) and commercially available BDNF antibody (Thermo);

[0189] 5. Add 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 washing solution;

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

[0191] 7. Stop the reaction and measure the OD450 value in an enzyme-linked immunosorbent assay reader.

[0192] The HiTrap ProteinL HP column can specifically bind monomers and dimers of IgA. In this study, affinity chromatography was used to purify IgA antibodies from the culture supernatant of HEK-293T cells. The results are shown in Figure 3 Panel A. The results of Western blot and Coomassie brilliant blue staining showed that the supernatant IgA antibodies were effectively enriched and purified, as shown in Figure 3 Panels B and C. The purified IgA antibodies were used to incubate recombinant human BDNF and NGF proteins overnight. The Western Blot results showed that specific bands appeared at 14 kDa, as shown in Figure 3 Panel D, indicating that the purified BDNF Ab-IgA and NGF Ab-IgA antibodies can specifically recognize recombinant human BDNF and NGF proteins.

[0193] Furthermore, the affinity of the purified IgA antibodies was detected. BDNF Ab-IgA and NGF Ab-IgA were coated on the enzyme-linked immunosorbent assay plate and bound to gradient-diluted recombinant human BDNF and NGF proteins respectively. The ELISA experimental results showed that BDNF Ab-IgA and NGF Ab-IgA antibodies had concentration-dependent binding to BDNF and NGF proteins, with strong affinity, lower than that of commercially available anti-BDNF and anti-NGF antibodies, as shown in Figure 3 Panel E.

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

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

[0196] (1) Effects of IgA antibodies on the proliferation of glioma cells

[0197] 1. Use the Cell Counting Kit 8 (CCK-8) method to detect the effects of antibodies targeting NGF or BDNF on the proliferation of U-87MG cells.

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

[0199] 3. After aspirating the MEM medium, add 100 μL of different groups of antibodies 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, IgG Isotype. Incubate the culture plate in the incubator for 24 hours.

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

[0201] 5. Measure the absorbance at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

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

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

[0204] After treatment with IgA / IgG / scFv antibodies targeting NGF and BDNF, the number of viable U-87MG cells decreased, suggesting that the antibodies can inhibit the proliferation of U-87MG cells; among them, NGF Ab-IgA and BDNF Ab-IgA antibodies were the most significant in inhibiting the proliferation of U-87MG cells, and the results were as Figure 4 shown.

[0205] (2) Effects of IgA antibodies on the growth and survival of glioma cells

[0206] 1. Detect the effect of antibodies targeting NGF or BDNF on the proliferation of U-87MG cells using TO-PRO-3 and Hoechst 33342 dyes. TO-PRO-3 is a cyanine monomer dye with very strong binding affinity for dsDNA. It can be used as a nuclear counterstain and dead cell indicator, and is one of the most sensitive nucleic acid detection probes; the 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 Hoechst 33342 binds to double-stranded DNA, the maximum excitation / emission wavelength is 350 / 461 nm.

[0207] 2. Seed U-87MG cells in a 24-well plate in advance and place it in an incubator at 37 °C and 5% CO 2 to allow them to adhere. Discard half of the MEM complete medium, and supplement the culture plate with 200 μL of different groups of antibodies targeting NGF or BDNF (the same groups as set above), and incubate the culture plate in the incubator for 24 hours.

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

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

[0210] 5. Dilute the TO-PRO-3 stock solution and Hoechst 33342 stock solution (1:1000) in 1×PBS to prepare the 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 3 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 the proliferation of glioma cells, this study further explored the relationship between the antibodies and cell growth and survival. U-87MG cells were treated with different proteins for 72 hours, and the cell culture plate was observed using Evos. The results showed that adding recombinant human BDNF or NGF to the culture medium was helpful for the growth and survival of U-87MG cells. The cells were plump with clear outlines and high growth density, as Figure 5As shown in Panel A. After treatment with IgA / IgG / scFv antibody, the state of U-87MG cells deteriorated; among them, the density of U-87MG cells treated with NGF Ab-IgA and BDNF Ab-IgA was the lowest, their growth stagnated, the cell edges were unclear, the refraction was poor, and a large number of cell debris appeared, such as Figure 5 shown in Panels A and B.

[0214] TO-PRO-3 was used to perform real-time labeling of dead cells. The lysed cells released DNA-binding fluorescent carriers that emitted bright red fluorescence. The Evos observation results showed that compared with the untreated group, the number of TO-PRO-3+ U-87MG cells treated with IgA / IgG / scFv antibody increased, indicating that U-87MG cells lysed after antibody treatment; the lysis signals in the NGF Ab-IgA and BDNF Ab-IgA treatment groups were the most significant, suggesting that they most significantly inhibited the survival of U-87MG cells. In addition, death signals were also found in the IgA Isotype and IgG Isotype treatment groups.

[0215] The above results indicate that after treatment with NGF Ab-IgA or BDNF Ab-IgA, the fragmentation and lysis of U-87MG cells increased significantly, which was not conducive to cell growth and survival.

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

[0217] The scratch assay (Wound Healing) was used to detect the effects of different antibodies targeting NGF and BDNF on the migration and invasion abilities of glioma cells.

[0218] 1. The scratch assay was used to detect the effect of antibodies targeting NGF or BDNF on the migration of U-87MG cells. A 10 μL pipette tip was used to scratch the confluent monolayer of U-87MG cells. The cells were untreated (0 hours) or treated with rhBDNF / rhNGF protein or antibody for 24 hours.

[0219] 2. Representative results were observed under a phase contrast microscope and photographed at the same position.

[0220] 3. The following formula was used to calculate the relative migration distance: Relative migration distance = (A - B) / A, where A represents the average width of the cell scratch before treatment and B represents the average width of the cell scratch after treatment. The results were expressed as mean ± SE.

[0221] The statistical results showed that adding exogenous neurotrophic factors helped improve wound closure activity, suggesting that BDNF or NGF could 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 the migration and invasion of U-87MG cells, and the inhibitory effect of BDNF Ab-IgA was stronger, as Figure 6 shown in Figures A and B below.

[0222] (IV) Effects of IgA antibodies on glioma cell apoptosis

[0223] 1. Use CellEvent TM Caspase-3 / 7 to detect the effects of antibodies targeting NGF or BDNF on the apoptosis of U-87MG cells. This dye is a novel fluorescent substrate for activating Caspase-3 / 7, composed of a tetra-amino acid peptide (DEVD) conjugated to a nucleic acid-binding dye; after activating Caspase-3 or Caspase-7 in apoptotic cells, the DEVD peptide is cleaved, enabling the dye to bind to DNA and produce a bright fluorescence reaction, with an absorption / emission maximum of approximately 502 / 530 nm.

[0224] 2. Seed U-87MG cells in 24-well plates in advance and place them in an incubator at 37°C and 5% CO 2 to allow them to adhere.

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

[0226] 4. Dilute CellEvent TM Caspase-3 / 7 Green Detection Reagent in complete medium to a final concentration of 5 μM and directly add it to the adherent cells. Return the cells to the incubator and incubate at 37°C for at least 30 minutes.

[0227] 5. Use the channels suitable for FITC and Alexa Fluor 488 dyes to scan and photograph the cell culture plates through an Evos FL Auto2 automated live cell imaging system; simultaneously, count the apoptotic Caspase3 / 7 activity (%).

[0228] The correlation between NGF Ab-IgA and BDNF Ab-IgA antibodies and glioma cell apoptosis was detected. 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 process of cell apoptosis. The results showed that compared with the untreated group, the 17 kDa cleaved (active) form of Caspase-3 appeared in U-87MG cells after treatment with IgA / IgG / scFv antibodies, indicating that apoptosis occurred in U-87MG cells. Recombinant human BDNF and NGF were involved in protecting U-87MG cells from apoptosis.

[0229] Statistical results showed that compared with IgG antibody, 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 highly expressed 17 kDa Caspase-3, further verifying the pro-apoptotic function of the variable region of IgA antibodies. The Fc fragment of the antibody (IgA Isotype and IgG Isotype) could induce a small amount of cell apoptosis, showing a statistical difference compared with the untreated group.

[0230] Using CellEvent TM Caspase-3 / 7 live cell imaging was used to further detect the apoptosis of U-87MG cells. The observation results under the Evos microscope showed that compared with the untreated group, after treatment with IgA / IgG / scFv for 24 hours, the apoptotic U-87MG cells activated Caspase-3 / 7 to produce a bright fluorescence reaction, as shown in Figure 7 Panel A. Statistical analysis of Caspase-3 / 7 activity showed that NGF Ab-IgA and BDNF Ab-IgA induced the most significant apoptosis in U-87MG cells, as shown in Figure 7 Panel B.

[0231] The above results indicate that exogenous supplementation of recombinant human BDNF or NGF contributes to the growth, proliferation and survival of glioma cells, is involved in protecting cells from apoptosis, and improves the in vitro migration ability of glioma cells.

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

[0233] Example 4: In Vivo Antitumor Experiments of NGF Ab-IgA and BDNF Ab-IgA

[0234] (I) Construction of a Mouse Model of U-87MG Malignant Glioma

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

[0236] 1. Digest Luci-U-87MG cells stably expressing exogenous luciferase with 0.25% trypsin and wash the cells 2 times with 1×PBS. Count and adjust the suspension concentration to 1.0×10 5 cells / μL, and place it on ice for later use.

[0237] 2. Install a stereotaxic apparatus (the red port is connected to an injection pump, and the other three holes, red, yellow, and green, are respectively connected to the positioning apparatus corresponding to the X, Y, and Z axes).

[0238] 3. Anesthetize BALB / c nude mice by intraperitoneal injection of tribromoethanol. Pad cotton on the ventral side of the mice to keep them warm, fix them on the mouse adapter platform using ear rods, and wipe the head hair with an alcohol cotton ball.

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

[0240] 5. Locate the bregma of the mouse as the origin (X = 0, Y = 0) and make a mark. Use a cranial drill to drill a hole 1 mm above the bregma and 2 mm to the left (X = -2, Y = 1). Gently drill until there is a feeling of falling through and then stop.

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

[0242] 7. Wait for 5 minutes after all the injection is completed, then slowly withdraw the Hamilton micro-injection needle; immediately soak it in saline for cleaning.

[0243] 8. Drop freshly prepared bone wax on the cranial hole to prevent the outflow of the cell suspension; then aspirate 2 μL of 3M medical glue to adhere and sew the mouse scalp.

[0244] 9. After the mice regain consciousness, continue to raise them in the SPF-class animal room, and observe and record the status of the mice at any time.

[0245] (2) NGF Ab-IgA and BDNF Ab-IgA antibody immunotherapy

[0246] Randomly divide 90 female BALB / c nude mice into 9 groups (3 mice in each group, repeated 3 times), 4 weeks old, with a body weight of about 15-18 g. Construct a malignant glioma mouse model according to the above steps, and perform different antibody immunotherapies about 14 days later. One day apart, inject NGF Ab-IgA and BDNF Ab-IgA, NGF Ab-IgG and BDNF Ab-IgG, anti-NGF scFv and anti-BDNF scFv, IgA Isotype and IgA Isotype through the tail vein respectively, and use PBS as a negative control at the same time, and continue the treatment 10 times. Set up multiple groups of experiments to record the following data: perform magnetic resonance imaging (MRI) and small animal in vivo imaging on the tumor-bearing mice of the same batch. The mice need to be injected with an enhanced contrast agent (Injection dimeglumine gadopentetate) in advance, and the MRI / enhanced T1 nuclear magnetic parameters are set as TE / 17.23 ms; Read Fov 20.0, Offset 0.1 mm; Phase 20.0, Offset -0.1 mm; Slices 15; Thk 1.00 mm. Obtain the brain tissues of the mice in different antibody treatment groups and fix them with 4% paraformaldehyde for HE staining or other pathological experiments such as immunofluorescence and immunohistochemistry. Separate glioma mouse in vivo models are set up to record the body weight and survival data (10 mice in each group). The specific grouping and intervention plan are shown in Table 4.

[0247] Table 4. Experimental grouping and treatment plan

[0248]

[0249] In order to further clarify the in vivo tumor suppression effect of IgA antibodies, multi-directional three-dimensional imaging of glioma mice was performed using magnetic resonance imaging (MRI). After injecting the enhanced contrast agent into the tail vein of the mice, the brain tissues were scanned in layers, and the results were as Figure 8As shown in Panel A, the mice in the PBS group, IgA Isotype group, and IgG Isotype group showed bright high signals with uniform enhancement or nodular enhancement, presenting signs of round tumor occupancy; suggesting that the tumor tissues had vigorous proliferation and absorbed and metabolized the contrast agent relatively quickly. In contrast, the mice in the NGF Ab-IgA and BDNF Ab-IgA intervention groups showed low and weak signals in the striatum of the brain, and the tumor occupancy effect was significantly reduced; this result indicates that IgA-targeted antibody drugs significantly inhibited the growth of gliomas in mice.

[0250] The three-dimensional fitting technique was used to calculate the volume of brain tumors. The results showed that after 10 cumulative interventions, the volume of brain tumors in the PBS group of mice increased to approximately 378.8 ± 31.0 mm 3 ; there was no significant difference between the IgA Isotype intervention group (359.1 ± 11.8 mm 3 ) and the IgG Isotype intervention group (341.7 ± 44.2 mm 3 ) compared with the PBS group.

[0251] The tumor volumes in the NGF Ab-IgA intervention group (51.9 ± 33.9 mm 3 ) and the BDNF Ab-IgA intervention group (36.8 ± 11.6 mm 3 ) were significantly reduced. Among them, the effect of BDNF Ab-IgA was better, and the brain tumor shrank to about 1 / 10 of that in the PBS group. The average tumor volumes in the intervention groups of the same target IgG and scFv antibodies decreased to 160.8 ± 28.6 mm 3 and 129.3 ± 46 mm 3 , as shown in Panel B. Figure 8 As shown in Panel B.

[0252] Continuous paraffin sections were prepared from the intact brain tissues of tumor-bearing mice. After hematoxylin-eosin (HE) staining, it was found that the brain tumors in the PBS group, IgA Isotype group, and IgG Isotype group of mice occupied more layers and had a larger area, and dense tumor cell nucleus regions were visible; the regions with erythrocyte aggregation inside the brain tissues were hemorrhagic necrosis foci. The areas of brain tumors in other antibody intervention groups were reduced to varying degrees, with the most obvious reduction in the NGF Ab-IgA and BDNF Ab-IgA groups, and small-volume tumors or no tumors were visible in the brain tissue sections. This result further indicates that IgA antibodies targeting NGF or BDNF significantly inhibit the growth of gliomas.

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

[0254] 1. First, anesthetize Luci-U-87MG glioma mice by perfusing isoflurane in the gas anesthesia chamber. Initialize the small animal in vivo imager. Data acquisition can be performed when the adapter temperature measurement strip shows a green bar (platform temperature -80 °C). Select the 25-cm platform for animal imaging to collect images.

[0255] 2. Inject the luciferase detection substrate D-luciferin into the peritoneal cavity of the mice (see the reagent preparation section for details). Each mouse should be injected with at least 150 mg / Kg body weight.

[0256] 3. Collect images 15 minutes after injecting the substrate, and keep the exposure time consistent among groups.

[0257] Use in vivo imaging software (PerkinElmer Biosciences) for analysis. Its signal intensity reflects the number of viable tumor cells in the mouse brain, as shown in Figure 9 Panel A.

[0258] The statistical results of in vivo imaging show that compared with the PBS group, after 10 interventions with IgA / IgG / scFv antibodies targeting NGF or BDNF, the Luciferase fluorescence intensity in the mouse brain tissue decreased, indicating that the above measures can all inhibit the growth of glioma in mice. Among them, the Luciferase fluorescence intensity in the NGF Ab-IgA and BDNF Ab-IgA intervention groups was the lowest, indicating that their tumor growth was the slowest and the treatment effect was the best; BDNF Ab-IgA was slightly better than NGF Ab-IgA, as shown in Figure 9 Panel B.

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

[0260] Analyze the body weight and survival of Luci-U-87MG glioma transplanted mice (10 mice in each group). After 56 days of observation and recording, it was found that the mice in the PBS group showed varying degrees of hunchback, rigidity, swelling of the head tumor, motor retardation, and imbalance at the 3rd week; and the body weight of the mice decreased rapidly, as shown in Figure 10 Panel A. The NGF Ab-IgA and BDNF Ab-IgA intervention groups had less impact on the body weight of the mice.

[0261] The survival curve results show that all the mice in the PBS group died within 35 days. After intervention with IgA / IgG / scFv antibodies targeting NGF or BDNF, the survival time of glioma mice can be prolonged; among them, the survival time of the tumor-bearing mice in the NGF Ab-IgA and BDNF Ab-IgA antibody intervention groups was the longest, with a significant difference. The longest time for all the mice in the BDNF Ab-IgA group to die was the longest, as shown in Figure 10As shown in Panel B.

[0262] In summary, in vitro and in vivo experiments have demonstrated that two IgA antibodies, NGF Ab-IgA and BDNF Ab-IgA, targeting neurotrophic factors NGF or BDNF, have good anti-glioma efficacy, with BDNF Ab-IgA showing better results. Specifically, BDNF Ab-IgA and NGF Ab-IgA significantly inhibit the growth, proliferation, and survival of U-87MG cells, induce strong apoptosis in target cells, and significantly reduce cell migration and invasion ability. In a mouse orthotopic glioma transplantation model, NGF Ab-IgA and BDNF Ab-IgA significantly inhibit the progression of glioma and prolong the survival of mice, with BDNF Ab-IgA showing better efficacy; IgA antibodies are superior to IgG and scFv antibodies. Dimeric NGF Ab-IgA and BDNF Ab-IgA can penetrate the blood-brain barrier or blood-tumor barrier, reach the glioma tissue, bind to pIgR on the surface of tumor cells, and promote tumor cell lysis. In addition, IgA antibodies can interact with FcαRI through the Fc segment, recruit microglia to mediate ADCC and ADCP effects, and exert a stronger anti-tumor effect than IgG antibodies. The results of this invention suggest that BDNF and NGF neurotrophic factors have the potential to become new targets for the treatment of glioma, and IgA antibodies are more significant than IgG antibodies in crossing the blood-brain barrier and anti-tumor efficacy.

[0263] Finally, it should be noted that the above are only preferred embodiments of the present invention and are not used 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 perform equivalent replacements for some of the technical features. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

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

2.

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

1.

3. The IgA antibody according to claim 1 or 2, characterized in that The IgA antibody is in the form of monomer or polymer.

4. The IgA antibody according to claim 3, characterized in that The multimeric form includes a dimer or a tetramer form. 5 . An antibody composition comprising the BDNF Ab-IgA according to claim 1 and the NGF Ab-IgA according to claim 2 . 6 . A nucleic acid construct encoding the BDNF Ab-IgA according to claim 1 and / or the NGFAb-IgA according to claim 2 .

7. Use of the BDNF Ab-IgA according to claim 1, or the NGF Ab-IgA according to claim 2, or the IgA antibody according to claim 3 or 4, or the antibody composition according to claim 5, or the nucleic acid construct according to claim 6 in the preparation of a drug for treating glioma.

8. Use of the BDNF Ab-IgA according to claim 1, or the NGF Ab-IgA according to claim 2, or the IgA antibody according to claim 3 or 4, or the antibody composition according to claim 5, or the nucleic acid construct according to claim 6 in the preparation of a drug for blocking nutrient delivery signals of neuro-glioma.

9. Use of the BDNF Ab-IgA according to claim 1, or the NGF Ab-IgA according to claim 2, or the IgA antibody according to claim 3 or 4, or the antibody composition according to claim 5, or the nucleic acid construct according to claim 6 in the preparation of a medicament for triggering microglial ADCC and ADCP effects.

10. The use according to claim 7 or 8, wherein the glioma comprises a brain glioma.

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