(1) Zhang DY (2) Zhang ZH (3) Liu WT (4) Zhou WM (5) Zhou PF (6) Wei JJ (7) Dai XJ (8) Zeng XL (9) Zhou YQ (10) Li HW (11) Zhang H (12) Shen AL (13) Cheng LS (14) Shen GD (15) He YF

Journal of translational medicine

BACKGROUND: Agonistic monoclonal antibodies targeting 4-1BB/CD137 have shown preclinical promise, but their clinical development has been limited by severe liver toxicity or limited efficacy. Therefore, a safe and efficient immunostimulatory molecule is urgently needed for cancer immunotherapy. METHODS: A novel anti-MSLN_4-1BB bispecific antibody (bsAb) was generated via antibody engineering, and its affinity and activity were detected via enzyme-linked immunosorbent assay (ELISA), flow cytometry, and T-cell activation and luciferase reporter assays. In vivo antitumour activity was assessed by establishing humanized mice bearing human MSLN-expressing MC38 (MC38/hMSLN) or CT26 (CT26/hMSLN) cells, and safety was further evaluated in cynomolgus monkeys. RESULTS: We generated two humanized anti-MSLN_4-1BB bsAbs (HK013-G1/G4) by fusing an anti-4-1BB scFv to the C-terminus of an anti-MSLN VHH with an intact Fc fragment from human IgG1 or IgG4. The two bsAbs were able to block the binding of CA125 to MSLN and stimulate 4-1BB signaling pathway, which was strictly dependent on MSLN expression. In particular, HK013-G1 retained Fc function and induced ADCC effect in tumour cells, whereas HK013-G4 did not. Strikingly, HK013-G1 showed superior antitumour activity to HK013-G4 both in vitro and in vivo and remained effective even in the presence of soluble MSLN. HK013-G1 enhanced antitumour immunity and induced durable antigen-specific immune memory to prevent rechallenged tumour growth, even at a dose as low as 1 mg/kg. Furthermore, HK013-G1 did not induce nonspecific production of proinflammatory cytokines and showed good tolerability up to the highest tested dose (30 mg/kg weekly) for 5 weeks, with no HK013-G1-related adverse effects observed in cynomolgus monkeys. In addition, the mean half-life of HK013-G1 was approximately 61 and 97 h at single doses of 3 and 30 mg/kg, respectively. CONCLUSION: The optimal anti-MSLN_4-1BB bsAb HK013-G1 exhibited synergistic antitumour effects by inducing an ADCC effect (innate immunity) and stimulating the 4-1BB signaling pathway (adaptive immunity) upon cross-bridging with MSLN with no systemic toxicity, which may offer the promise of an improved therapeutic window relative to that of 4-1BB agonists.

Author Info: (1) Department of Medical Oncology, Division of Life Sciences and Medicine, The First Affiliated Hospital of USTC, University of Science and Technology of China, Anhui Provincial C

Author Info: (1) Department of Medical Oncology, Division of Life Sciences and Medicine, The First Affiliated Hospital of USTC, University of Science and Technology of China, Anhui Provincial Cancer Hospital, Hefei, 230031, Anhui, China. Hefei HankeMab Biotechnology Co., Ltd.; Anhui Province Key Laboratory of Gene Engineering Pharmaceutical, Biomedicine Technology Innovation Center of Hefei, Anhui Anke Biotechnology (Group) Co., Ltd, Hefei, 230031, Anhui, China. Anhui Province Key Laboratory of Geriatric Immunotherapy and Nutrition Therapy, Gerontology Institute of Anhui Province, Hefei, 230001, Anhui, China. (2) Department of Medical Oncology, Division of Life Sciences and Medicine, The First Affiliated Hospital of USTC, University of Science and Technology of China, Anhui Provincial Cancer Hospital, Hefei, 230031, Anhui, China. Anhui Province Key Laboratory of Geriatric Immunotherapy and Nutrition Therapy, Gerontology Institute of Anhui Province, Hefei, 230001, Anhui, China. (3) Hefei HankeMab Biotechnology Co., Ltd.; Anhui Province Key Laboratory of Gene Engineering Pharmaceutical, Biomedicine Technology Innovation Center of Hefei, Anhui Anke Biotechnology (Group) Co., Ltd, Hefei, 230031, Anhui, China. Anhui Province Key Laboratory of Geriatric Immunotherapy and Nutrition Therapy, Gerontology Institute of Anhui Province, Hefei, 230001, Anhui, China. (4) Hefei HankeMab Biotechnology Co., Ltd.; Anhui Province Key Laboratory of Gene Engineering Pharmaceutical, Biomedicine Technology Innovation Center of Hefei, Anhui Anke Biotechnology (Group) Co., Ltd, Hefei, 230031, Anhui, China. Anhui Province Key Laboratory of Geriatric Immunotherapy and Nutrition Therapy, Gerontology Institute of Anhui Province, Hefei, 230001, Anhui, China. (5) Hefei HankeMab Biotechnology Co., Ltd.; Anhui Province Key Laboratory of Gene Engineering Pharmaceutical, Biomedicine Technology Innovation Center of Hefei, Anhui Anke Biotechnology (Group) Co., Ltd, Hefei, 230031, Anhui, China. Anhui Province Key Laboratory of Geriatric Immunotherapy and Nutrition Therapy, Gerontology Institute of Anhui Province, Hefei, 230001, Anhui, China. (6) Hefei HankeMab Biotechnology Co., Ltd.; Anhui Province Key Laboratory of Gene Engineering Pharmaceutical, Biomedicine Technology Innovation Center of Hefei, Anhui Anke Biotechnology (Group) Co., Ltd, Hefei, 230031, Anhui, China. Anhui Province Key Laboratory of Geriatric Immunotherapy and Nutrition Therapy, Gerontology Institute of Anhui Province, Hefei, 230001, Anhui, China. (7) Hefei HankeMab Biotechnology Co., Ltd.; Anhui Province Key Laboratory of Gene Engineering Pharmaceutical, Biomedicine Technology Innovation Center of Hefei, Anhui Anke Biotechnology (Group) Co., Ltd, Hefei, 230031, Anhui, China. Anhui Province Key Laboratory of Geriatric Immunotherapy and Nutrition Therapy, Gerontology Institute of Anhui Province, Hefei, 230001, Anhui, China. (8) Hefei HankeMab Biotechnology Co., Ltd.; Anhui Province Key Laboratory of Gene Engineering Pharmaceutical, Biomedicine Technology Innovation Center of Hefei, Anhui Anke Biotechnology (Group) Co., Ltd, Hefei, 230031, Anhui, China. Anhui Province Key Laboratory of Geriatric Immunotherapy and Nutrition Therapy, Gerontology Institute of Anhui Province, Hefei, 230001, Anhui, China. (9) Hefei HankeMab Biotechnology Co., Ltd.; Anhui Province Key Laboratory of Gene Engineering Pharmaceutical, Biomedicine Technology Innovation Center of Hefei, Anhui Anke Biotechnology (Group) Co., Ltd, Hefei, 230031, Anhui, China. Anhui Province Key Laboratory of Geriatric Immunotherapy and Nutrition Therapy, Gerontology Institute of Anhui Province, Hefei, 230001, Anhui, China. (10) Hefei HankeMab Biotechnology Co., Ltd.; Anhui Province Key Laboratory of Gene Engineering Pharmaceutical, Biomedicine Technology Innovation Center of Hefei, Anhui Anke Biotechnology (Group) Co., Ltd, Hefei, 230031, Anhui, China. Anhui Province Key Laboratory of Geriatric Immunotherapy and Nutrition Therapy, Gerontology Institute of Anhui Province, Hefei, 230001, Anhui, China. (11) Hefei HankeMab Biotechnology Co., Ltd.; Anhui Province Key Laboratory of Gene Engineering Pharmaceutical, Biomedicine Technology Innovation Center of Hefei, Anhui Anke Biotechnology (Group) Co., Ltd, Hefei, 230031, Anhui, China. Anhui Province Key Laboratory of Geriatric Immunotherapy and Nutrition Therapy, Gerontology Institute of Anhui Province, Hefei, 230001, Anhui, China. (12) Anhui Province Key Laboratory of Geriatric Immunotherapy and Nutrition Therapy, Gerontology Institute of Anhui Province, Hefei, 230001, Anhui, China. shenaolin@ustc.edu.cn. Department of General Surgery, Division of Life Sciences and Medicine, The First Affiliated Hospital of USTC, University of Science and Technology of China, Hefei, 230001, Anhui, China. shenaolin@ustc.edu.cn. (13) Hefei HankeMab Biotechnology Co., Ltd.; Anhui Province Key Laboratory of Gene Engineering Pharmaceutical, Biomedicine Technology Innovation Center of Hefei, Anhui Anke Biotechnology (Group) Co., Ltd, Hefei, 230031, Anhui, China. charlson@ustc.edu.cn. Anhui Province Key Laboratory of Geriatric Immunotherapy and Nutrition Therapy, Gerontology Institute of Anhui Province, Hefei, 230001, Anhui, China. charlson@ustc.edu.cn. (14) Anhui Province Key Laboratory of Geriatric Immunotherapy and Nutrition Therapy, Gerontology Institute of Anhui Province, Hefei, 230001, Anhui, China. gdshen@ustc.edu.cn. Department of Geriatrics, Division of Life Sciences and Medicine, The First Affiliated Hospital of USTC, University of Science and Technology of China, Hefei, 230001, Anhui, China. gdshen@ustc.edu.cn. (15) Department of Medical Oncology, Division of Life Sciences and Medicine, The First Affiliated Hospital of USTC, University of Science and Technology of China, Anhui Provincial Cancer Hospital, Hefei, 230031, Anhui, China. yifuhe@ffyy.ustc.edu.cn. Anhui Province Key Laboratory of Geriatric Immunotherapy and Nutrition Therapy, Gerontology Institute of Anhui Province, Hefei, 230001, Anhui, China. yifuhe@ffyy.ustc.edu.cn.

Citation: J Transl Med 2025 Jan 13 23:53 Epub01/13/2025

Link to PUBMED: http://www.ncbi.nlm.nih.gov/pubmed/39806351

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