A humanized anti-MSLN×4-1BB bispecific antibody exhibits potent antitumour activity through 4-1BB signaling activation and fc function without systemic toxicity
Abstract 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 immunothera...
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2025-01-01
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| Series: | Journal of Translational Medicine |
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| Online Access: | https://doi.org/10.1186/s12967-025-06107-z |
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| author | Da-yan Zhang Zhi-hua Zhang Wen-ting Liu Wei-ming Zhou Peng-fei Zhou Juan-juan Wei Xue-jing Dai Xiao-li Zeng Yu-qiong Zhou Han-wang Li Heng Zhang Ao-lin Shen Lian-sheng Cheng Guo-dong Shen Yi-fu He |
| author_facet | Da-yan Zhang Zhi-hua Zhang Wen-ting Liu Wei-ming Zhou Peng-fei Zhou Juan-juan Wei Xue-jing Dai Xiao-li Zeng Yu-qiong Zhou Han-wang Li Heng Zhang Ao-lin Shen Lian-sheng Cheng Guo-dong Shen Yi-fu He |
| author_sort | Da-yan Zhang |
| collection | DOAJ |
| description | Abstract 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. |
| format | Article |
| id | doaj-art-1ff571e4bfb94e5788bfba09e0512c6d |
| institution | Kabale University |
| issn | 1479-5876 |
| language | English |
| publishDate | 2025-01-01 |
| publisher | BMC |
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| series | Journal of Translational Medicine |
| spelling | doaj-art-1ff571e4bfb94e5788bfba09e0512c6d2025-01-19T12:37:06ZengBMCJournal of Translational Medicine1479-58762025-01-0123111310.1186/s12967-025-06107-zA humanized anti-MSLN×4-1BB bispecific antibody exhibits potent antitumour activity through 4-1BB signaling activation and fc function without systemic toxicityDa-yan Zhang0Zhi-hua Zhang1Wen-ting Liu2Wei-ming Zhou3Peng-fei Zhou4Juan-juan Wei5Xue-jing Dai6Xiao-li Zeng7Yu-qiong Zhou8Han-wang Li9Heng Zhang10Ao-lin Shen11Lian-sheng Cheng12Guo-dong Shen13Yi-fu He14Department 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 HospitalDepartment 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 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 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 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 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 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 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 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 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 HankeMab Biotechnology Co., Ltd.; Anhui Province Key Laboratory of Gene Engineering Pharmaceutical, Biomedicine Technology Innovation Center of Hefei, Anhui Anke Biotechnology (Group) Co., LtdAnhui Province Key Laboratory of Geriatric Immunotherapy and Nutrition Therapy, Gerontology Institute of Anhui Province Hefei HankeMab Biotechnology Co., Ltd.; Anhui Province Key Laboratory of Gene Engineering Pharmaceutical, Biomedicine Technology Innovation Center of Hefei, Anhui Anke Biotechnology (Group) Co., LtdAnhui Province Key Laboratory of Geriatric Immunotherapy and Nutrition Therapy, Gerontology Institute of Anhui ProvinceDepartment 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 HospitalAbstract 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.https://doi.org/10.1186/s12967-025-06107-z4-1BB/CD137MSLNBispecific antibodyCancer immunotherapyAntitumour immunity |
| spellingShingle | Da-yan Zhang Zhi-hua Zhang Wen-ting Liu Wei-ming Zhou Peng-fei Zhou Juan-juan Wei Xue-jing Dai Xiao-li Zeng Yu-qiong Zhou Han-wang Li Heng Zhang Ao-lin Shen Lian-sheng Cheng Guo-dong Shen Yi-fu He A humanized anti-MSLN×4-1BB bispecific antibody exhibits potent antitumour activity through 4-1BB signaling activation and fc function without systemic toxicity Journal of Translational Medicine 4-1BB/CD137 MSLN Bispecific antibody Cancer immunotherapy Antitumour immunity |
| title | A humanized anti-MSLN×4-1BB bispecific antibody exhibits potent antitumour activity through 4-1BB signaling activation and fc function without systemic toxicity |
| title_full | A humanized anti-MSLN×4-1BB bispecific antibody exhibits potent antitumour activity through 4-1BB signaling activation and fc function without systemic toxicity |
| title_fullStr | A humanized anti-MSLN×4-1BB bispecific antibody exhibits potent antitumour activity through 4-1BB signaling activation and fc function without systemic toxicity |
| title_full_unstemmed | A humanized anti-MSLN×4-1BB bispecific antibody exhibits potent antitumour activity through 4-1BB signaling activation and fc function without systemic toxicity |
| title_short | A humanized anti-MSLN×4-1BB bispecific antibody exhibits potent antitumour activity through 4-1BB signaling activation and fc function without systemic toxicity |
| title_sort | humanized anti msln 4 1bb bispecific antibody exhibits potent antitumour activity through 4 1bb signaling activation and fc function without systemic toxicity |
| topic | 4-1BB/CD137 MSLN Bispecific antibody Cancer immunotherapy Antitumour immunity |
| url | https://doi.org/10.1186/s12967-025-06107-z |
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