Background/Objectives: Canine Diffuse Large B-cell Lymphoma (cDLBCL) is characterized by a high prevalence of MHC II DR (DLA-DR) antigen overexpression. Murine anti-pan-DLA-DR monoclonal antibodies (mAbs) B5 and E11 have been previously observed to promote death of cDLBCL cells in vitro and in vivo. Consequently, DLA-DR antigens are considered a prospective target for passive immunotherapy aside from CD20. While infusion of anti-pan MHC II mAbs has demonstrated tumor suppression in cDLBCL xenografted immunodeficient mice, the relative contributions of direct cellular versus immune-mediated mechanisms to this therapeutic effect remain undefined. This study aimed to dissect these potential mechanisms of mAb E11. Methods: Canine lymphoma and leukemia cell lines CLBL1 and CLB70 were incubated with full E11 antibody or its F(ab′)2 and Fab fragments and cell viability was assessed with sub-G1 assay then, NOD-SCID mice were xenotransplanted with 1.5 × 107canine CLBL1 cells expressing nanoluciferase and were infused either with mAb E11 or its fragments, each at 1 mg/kg body mass, twice weekly for three consecutive weeks. Tumor burden was monitored by assessing body weight, nanoluciferase activity in blood, and by flow cytometric analyses of bone marrow tumor cell content. Time to tumor progression (TTP) was calculated based on weight loss and luminescence measurements. Results: We observed cytotoxic activity of monovalent E11-Fab fragments in vitro and in vivo. The mean TTP for mice treated with irrelevant mouse IgG antibodies was 9.8 ± 4.65 days. In contrast, treatment with E11 Fab fragments resulted in a TTP of 19.1 ± 2.67 days, which was similar to that achieved with the full E11 mAb (19.5 ± 1.73 days) and E11 F(ab′)2 fragments (18.1 ± 2.9 days). Conclusions: Our findings demonstrate a potent antibody cytotoxicity mechanism that operates in vivo and is independent of cell surface MHC II crosslinking or Fc engagement. These data support the promising potential of E11-Fab fragments for further clinical development as a therapeutic agent in canine lymphoma.
背景/目的：犬弥漫性大B细胞淋巴瘤（cDLBCL）以高频率表达MHC II DR（DLA-DR）抗原为特征。先前研究发现，鼠源抗泛DLA-DR单克隆抗体B5和E11在体外及体内均能促进cDLBCL细胞死亡。因此，除CD20外，DLA-DR抗原被视为被动免疫治疗的潜在靶点。尽管在cDLBCL异种移植免疫缺陷小鼠模型中，输注抗泛MHC II单抗已显示肿瘤抑制作用，但其治疗效应中直接细胞毒性机制与免疫介导机制的相对贡献尚未明确。本研究旨在解析单抗E11的潜在作用机制。方法：将犬淋巴瘤/白血病细胞系CLBL1和CLB70分别与完整E11抗体及其F(ab′)2、Fab片段共培养，通过亚G1期检测评估细胞活力；随后向NOD-SCID小鼠异种移植1.5×10⁷个表达纳米荧光素酶的犬CLBL1细胞，并分别以1 mg/kg体重的剂量输注E11单抗或其片段，每周两次连续三周。通过体重监测、血液纳米荧光素酶活性检测及骨髓肿瘤细胞流式分析评估肿瘤负荷，依据体重下降和发光强度测定计算肿瘤进展时间。结果：我们观察到单价E11-Fab片段在体外和体内均具有细胞毒性活性。对照组（无关小鼠IgG抗体处理）小鼠的平均肿瘤进展时间为9.8±4.65天，而E11 Fab片段治疗组达到19.1±2.67天，与完整E11单抗（19.5±1.73天）及E11 F(ab′)2片段（18.1±2.9天）的治疗效果相当。结论：本研究揭示了一种在体内发挥作用的强效抗体细胞毒性机制，该机制不依赖于细胞表面MHC II交联或Fc段介导作用。这些数据为E11-Fab片段作为犬淋巴瘤治疗药物的临床转化潜力提供了有力支持。
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