研究背景

白细胞介素-10（IL-10）是一种多效性细胞因子，具有出独特的免疫调节特性。IL-10不仅能够通过抑制树突状细胞和巨噬细胞的抗原呈递，削弱T细胞活化调控免疫系统的应答反应；还能通过影响多种生长因子和细胞因子的表达水平，降低促炎细胞因子的分泌，促进调节性T细胞的增殖，形成免疫抑制微环境，进而参与调控多种疾病的发生发展过程。因此，IL-10细胞因子相关研究是免疫治疗领域的重要研究方向之一。

虽然基于抗体技术的免疫治疗在肿瘤治疗等领域发展迅速，然而目前尚未有针对人IL-10的理想抗体药物被应用于临床，因此，需要进一步开发与人IL-10特异性结合的抗体应用到相关疾病的诊断和治疗中。

研究方法

以重组人IL-10蛋白作为免疫原免疫小鼠，采用经典的单克隆抗体制备技术，筛选与人IL-10蛋白特异性结合的单克隆抗体，并检测抗体亚型。用ELISA、SDS-PAGE凝胶电泳、Western blot方法对单克隆抗体进行活性鉴定。通过基因克隆方法从杂交瘤细胞中得到轻重链BCR样本，测序获得轻重链可变区序列，将目标基因片段定向克隆至人源IgG1表达载体中，构建重组基因工程抗体表达载体。

研究结果

采用杂交瘤细胞融合技术，成功筛选出对人源IL-10蛋白具有高度亲和性和特异性的单克隆抗体，并利用分子克隆方法完成相应抗体基因的重组表达载体构建，质粒共转染成功诱导表达出抗人IL-10基因工程抗体。抗人IL-10基因工程抗体在体外能与患者标本中IL-10分子特异性结合。

研究结论

本研究成功筛选到一株结合活性和特异性强的抗人IL-10的单克隆抗体。此外，还克隆了抗体的轻重链可变区基因并构建出IgG1型全抗型基因工程抗体—XY-eAbhIL10。XY-eAbhIL10具有与IL-10特异性结合的能力，提示XY-eAbhIL10在肿瘤免疫治疗中具有良好的临床转化前景。

研究背景

随着恶性实体肿瘤及血液系统肿瘤的疾病负担持续攀升，这已经构成全球公共卫生领域的重大挑战。美国癌症研究机构最新发布的《2024年度全球肿瘤流行病学报告》预测全球新发恶性肿瘤患者数量可能突破3500万例。肿瘤早期筛查、诊断技术优化及创新疗法开发始终是学术界长期聚焦的核心议题。因此，选择在靶细胞中高表达并在非恶性组织中有限表达的癌症相关抗原，进而构建单克隆抗体治疗手段，能够保证具有足够的肿瘤特异性和广泛的治疗窗口。前期研究发现神经胶质抗原2（Neuron-Glial Antigen 2，NG2）的分子结构特征及其在各类癌症发生发展中的多重作用，提示NG2是癌症治疗的理想靶标之一。

虽然基于抗体技术的免疫治疗在肿瘤治疗等领域发展迅速，然而目前尚未有针对人NG2的理想抗体药物被应用于临床，因此亟需进一步开发与人NG2特异性结合的抗体来应用到相关疾病的诊断和治疗中。

研究方法

构建高表达人NG2蛋白的细胞株pEF6-NG2-293T，以pEF6-NG2-293T细胞作为免疫原免疫小鼠，采用经典的单克隆抗体制备技术，筛选与人NG2蛋白特异性结合的单克隆抗体，并对抗体的结合活性、结合特异性及亚型进行检测。然后对小鼠腹腔实施液体石蜡预处理，随后采用腹腔注射的方法注入杂交瘤细胞悬浮液。经过约7天的诱导培养后，采集产生的腹水样本，通过蛋白纯化技术对腹水中的抗体进行分离纯化，最终获得高纯度的目标抗体。用流式细胞术、SDS-PAGE凝胶电泳、Western blot等方法对纯品抗体进行活性鉴定，并检测纯品抗体的亲和常数。通过基因克隆方法从杂交瘤细胞中得到轻重链BCR样本，测序获得轻重链可变区序列，将目标基因片段定向克隆至人源IgG1表达载体中，成功构建重组基因工程抗体表达载体。

研究结果

成功构建了高表达NG2抗原的细胞株pEF6-NG2-293T；通过将pEF6-NG2-293T细胞免疫小鼠和杂交瘤抗体制备方法，筛选出一株能够与人NG2蛋白特异性结合的单克隆抗体—XY-hNG2-2H6；随后，又克隆了抗体的轻重链可变区基因并构建出IgG1型全抗型基因工程抗体—XY-eAbhNG2。

研究结论

本研究成功筛选到一株特异性抗人NG2的单克隆抗体—XY-hNG2-2H6，成功构建出IgG1型全抗型基因工程抗体—XY-eAbhNG2，提示XY-eAbhNG2在肿瘤免疫治疗中展现出较好的临床转化潜力和应用前景。

Background 

Interleukin-10 (IL-10) is a pleiotropic cytokine with distinct immunoregulatory properties. It modulates immune responses by suppressing antigen presentation by dendritic cells and macrophages, thereby reducing T cell activation. IL-10 also alters the expression of various growth factors and cytokines, decreases pro-inflammatory cytokine secretion, and promotes the proliferation of regulatory T cells, ultimately shaping an immunosuppressive microenvironment. These functions contribute to the pathogenesis and progression of numerous diseases. As such, IL-10 has emerged as a key focus in immunotherapy research.

Although antibody-based immunotherapies have rapidly advanced, particularly in oncology, there is currently no clinically available therapeutic antibody targeting human IL-10. Therefore, there is an urgent need to develop IL-10-specific antibodies for potential use in the diagnosis and treatment of related diseases..

Methods

Recombinant human IL-10 protein was used as the immunogen to immunize mice. Classical monoclonal antibody production techniques were applied to screen for hybridomas producing IL-10-specific antibodies, and antibody isotyping was performed. The monoclonal antibodies were validated using ELISA, SDS-PAGE, and Western blot. The variable regions of the antibody light and heavy chains were amplified from hybridoma cells using molecular cloning techniques, sequenced, and subsequently inserted into human IgG1 expression vectors to construct recombinant gene-engineered antibody expression plasmids.

Results

Through hybridoma cell fusion, monoclonal antibodies with high affinity and specificity for human IL-10 were successfully screened. The corresponding antibody genes were cloned, and recombinant expression vectors were constructed. Following co-transfection, a gene-engineered anti-IL-10 antibody was successfully expressed. This engineered antibody demonstrated the ability to specifically bind IL-10 molecules present in patient samples in vitro.

Conclusion

This study successfully identified a monoclonal antibody with strong binding activity and high specificity against human IL-10. Furthermore, the variable regions of the antibody’s light and heavy chains were cloned, and a full-length IgG1 gene-engineered antibody—XY-eAbhIL10—was constructed. XY-eAbhIL10 exhibited specific binding to IL-10, indicating its promising potential for clinical translation in tumor immunotherapy.

Background 

With the rising global burden of malignant solid tumors and hematologic malignancies, cancer has become a major public health challenge worldwide. According to the latest 2024 Global Cancer Epidemiology Report released by the American Cancer Research Institute, the number of new cancer cases is projected to exceed 35 million globally. Early tumor screening, improved diagnostic technologies, and the development of novel therapeutic strategies remain central themes in cancer research. Selecting tumor-associated antigens that are highly expressed in cancer cells but minimally expressed in normal tissues is key to ensuring tumor specificity and expanding the therapeutic window of monoclonal antibody therapies. Previous studies have revealed that Neuron-Glial Antigen 2 (NG2), due to its structural features and multiple roles in cancer progression, represents a promising target for cancer therapy.

Although antibody-based immunotherapies have made significant progress in cancer treatment, no ideal therapeutic antibody specifically targeting human NG2 has yet been approved for clinical use. Therefore, it is urgently necessary to develop NG2-specific monoclonal antibodies for use in the diagnosis and treatment of NG2-related cancers.

Methods

A human NG2-overexpressing cell line, pEF6-NG2-293T, was constructed and used as an immunogen to immunize mice. Classical hybridoma technology was employed to screen for monoclonal antibodies specifically binding to human NG2. The binding activity, specificity, and isotype of the antibodies were characterized. Mice were pre-treated intraperitoneally with liquid paraffin, followed by intraperitoneal injection of hybridoma cells. After approximately seven days of in vivo cultivation, ascitic fluid was collected. The target antibodies were purified from the ascites using protein purification techniques. The purified antibodies were evaluated for binding activity and affinity using flow cytometry, SDS-PAGE, and Western blot. Subsequently, the variable regions of the heavy and light chain genes were amplified from hybridoma cells, sequenced, and cloned into human IgG1 expression vectors to construct recombinant gene-engineered antibody plasmids.

Results

A stable NG2-overexpressing cell line (pEF6-NG2-293T) was successfully established. Using this cell line for immunization and hybridoma screening, a monoclonal antibody with high specificity for human NG2, designated XY-hNG2-2H6, was identified. The corresponding variable regions of the light and heavy chains were cloned, and a full-length IgG1-type gene-engineered antibody, XY-eAbhNG2, was successfully constructed.

Conclusion

This study successfully identified a monoclonal antibody—XY-hNG2-2H6—with high specificity against human NG2 and developed a full-length IgG1-type recombinant antibody—XY-eAbhNG2. The findings suggest that XY-eAbhNG2 has strong potential for clinical translation and application in tumor immunotherapy..