CD73是一种细胞外酶，参与将由应激下癌细胞分泌的促炎细胞外ATP（eATP）转化为抗炎的腺苷（ADO）。已经显示多种实体癌类型会利用CD73过度表达作为一种免疫抑制性检查点。因此，CD73拮抗抗体，尤其是oleclumab，目前正在多个多中心试验中评估其临床适用性。然而，传统单克隆CD73抑制抗体的疗效可能受限于对正常细胞上CD73的靶向/非肿瘤结合。因此，应该采取一种新方法，更有选择性地将CD73免疫检查点抑制导向癌细胞。为了解决这个问题，我们构建了一种新型四价双特异性抗体（bsAb），命名为bsAb CD73xEGFR。随后，使用体外和体内肿瘤模型评估了bsAb CD73xEGFR的抗癌活性。体外处理多种癌细胞类型以bsAb CD73xEGFR以EGFR导向方式强烈抑制CD73酶活性（大约为71%）。在这个过程中，bsAb CD73xEGFR诱导了抗原/抗体复合物的快速内化，导致同时将CD73和EGFR从癌细胞表面驱逐。此外，bsAb CD73xEGFR使癌细胞对多种化疗药物的细胞毒性活性敏感，并强烈抑制了癌细胞的增殖/迁移能力（约为40%）。出人意料的是，我们发现用oleclumab处理癌细胞似乎增强了一些促癌特性，包括EGFR的上调和磷酸化、肿瘤细胞增殖（约为20%）以及对细胞毒性药物和电离辐射的耐药性（约为39%）。重要的是，在使用合成的CD73pos/EGFRpos CT26癌细胞接种的免疫能力BALB/c小鼠肿瘤模型中，与oleclumab相比，bsAb CD73xEGFR的治疗效果更好（肿瘤体积减小65%对31%）。与oleclumab相比，bsAb CD73xEGFR的治疗增强了CD8pos T细胞和M1巨噬细胞在肿瘤内的存在。bsAb CD73xEGFR优于oleclumab，因为它以EGFR导向的方式抑制了CD73/ADO免疫检查点，并同时对抗EGFR和CD73的多种促癌活性。因此，bsAb CD73xEGFR在多种难治性实体癌类型的临床应用潜力显著。© 作者（或其雇主）2023年。在CC BY-NC下允许重复使用。不允许商业再利用。BMJ出版。

CD73 is an ecto-enzyme that is involved in the conversion of pro-inflammatory extracellular ATP (eATP) excreted by cancer cells under stress to anti-inflammatory adenosine (ADO). A broad variety of solid cancer types was shown to exploit CD73 overexpression as a suppressive immune checkpoint. Consequently, CD73-antagonistic antibodies, most notably oleclumab, are currently evaluated in several multicenter trials for clinical applicability. However, the efficacy of conventional monospecific CD73-inhibiting antibodies may be limited due to on-target/off-tumor binding to CD73 on normal cells. Therefore, a novel approach that more selectively directs CD73 immune checkpoint inhibition towards cancer cells is warranted.To address this issue, we constructed a novel tetravalent bispecific antibody (bsAb), designated bsAb CD73xEGFR. Subsequently, the anticancer activities of bsAb CD73xEGFR were evaluated using in vitro and in vivo tumor models.In vitro treatment of various carcinoma cell types with bsAb CD73xEGFR potently inhibited the enzyme activity of CD73 (~71%) in an EGFR-directed manner. In this process, bsAb CD73xEGFR induced rapid internalization of antigen/antibody complexes, which resulted in a prolonged concurrent displacement of both CD73 and EGFR from the cancer cell surface. In addition, bsAb CD73xEGFR sensitized cancer to the cytotoxic activity of various chemotherapeutic agents and potently inhibited the proliferative/migratory capacity (~40%) of cancer cells. Unexpectedly, we uncovered that treatment of carcinoma cells with oleclumab appeared to enhance several pro-oncogenic features, including upregulation and phosphorylation of EGFR, tumor cell proliferation (~20%), and resistance towards cytotoxic agents and ionizing radiation (~39%). Importantly, in a tumor model using immunocompetent BALB/c mice inoculated with syngeneic CD73pos/EGFRpos CT26 cancer cells, treatment with bsAb CD73xEGFR outperformed oleclumab (65% vs 31% tumor volume reduction). Compared with oleclumab, treatment with bsAb CD73xEGFR enhanced the intratumoral presence of CD8pos T cells and M1 macrophages.BsAb CD73xEGFR outperforms oleclumab as it inhibits the CD73/ADO immune checkpoint in an EGFR-directed manner and concurrently counteracts several oncogenic activities of EGFR and CD73. Therefore, bsAb CD73xEGFR may be of significant clinical potential for various forms of difficult-to-treat solid cancer types.© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.