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1214 A Novel anti-MSLN x 4–1BB bispecific antibody with Fc effect function augments the antitumor efficacy
  1. Liansheng Cheng,
  2. Dayan Zhang,
  3. Dayan Zhang,
  4. Lingling Wu,
  5. Weiming Zhou,
  6. Xiaoli Zeng,
  7. Xuejing Dai,
  8. Wenting Liu and
  9. Qun Zhao
  1. Hefei Hankemab Biotechnology CO., LTD, Hefei, Anhui, China


Background Mesothelin (MSLN) is a ~71kDa cell surface glycoprotein that is rarely expressed in normal tissues but overexpressed in a variety of cancers.1 4-1BB is not only expressed on the surface of activated T cells and NK cells but also a marker for Treg.2 Moreover, 4-1BB shows high selectivity for human tumor-derived Tregs and is associated with worse survival outcomes in patients with multiple tumor types, such as bladder cancer, glioblastoma, prostate cancer, or renal clear cell cancer.3 Here, we developed a IgG1-based bi-specific antibody, HK013-1, targeting both MSLN and 4-1BB to achieve better antitumor therapeutic efficacy.

Methods We tested the binding ability of HK013-1 to tumor cells with different expression levels of MSLN, and tested the killing ability of HK013-1-mediated NK cells against these tumor cells in vitro. Moreover, the 4-1BB agonist activity of HK013-1 was detected using CD8+T cells co-cultured with MSLN+ or MSLN- cells. To confirm the safety of HK013-1, non-specific activation of 4-1BB signal mediated by Fc receptor and killing potency to CD8+T cells and Tregs induced by HK013-1 was evaluated. In vivo, we verified the ability to inhibit tumor growth of HK013-1 and examined the effects of HK013-1 on spleen and tumor CD8+T cells and Tregs.

Results HK013-1 could bind to various tumor cells that differentially expressed MSLN and induce NK cells to kill these cells. In co-cultured assay, HK013-1 increased IFN-γ production only in the presence of MSLN+ cells. Compared with anti-4-1BB parent antibody and urelumab, HK013 induced weaker FcγR-mediated 4-1BB activation. Furthermore, HK013-1 engaged NK cells to kill Treg but not CD8+T cells. In 4-1BB humanized transgenic mice, HK013-1 was revealed to reduce the proportion of Treg cells in tumor but had no effect on CD8+T cells, and CD8+T and Treg cells in the spleen. Compared with IgG4-based bi-specific antibody,IgG1-based HK013-1 showed a more significant anti-tumor effect in MC38/MSLN tumor model.

Conclusions IgG1-based HK013-1 prevents tumor development by directly killing tumor cells and depleting Treg to relieve immunosuppression. Preclinical studies have shown that IgG1-based HK013-1 has good antitumor activity and safety, which may further develop its clinical potential.


  1. Weidemann S , Gorbokon N , Hflmayer D, et al. Abstract 2833: Mesothelin expression in human tumor types: a tissue microarray study on more than 13,000 tumor samples. Proceedings: AACR Annual Meeting 2021; April 10-15.

  2. Luu K, Patwardhan MV, Zeng Q, et al. Regulatory T Cells Inhibit T Cell Activity by Downregulating CD137 Ligand via CD137 Trogocytosis. Cells. 2021;10(2):353.

  3. Freeman ZT, Nirschl TR, Hovelson DH, et al. A conserved intratumoral regulatory T cell signature identifies 4-1BB as a pan-cancer target. The Journal of Clinical Investigation. 2020;130(3).

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