Introduction CD43s, a unique sialylated form of CD43 expressed by myeloid malignancies, is a novel target and AT1413, an antibody targeting CD43s was shown to have therapeutic potential against acute myeloid leukemia (AML) and myelodysplastic syndrome. CD43s is recognized by the human antibody AT1413, that was isolated from a high-risk AML patient who successfully cleared the leukemia after allogeneic stem cell transplantation. Because CD43 is also expressed in non-hematopoietic cells we studied whether CD43s is also present and can also be targeted on non-hematopoietic tumors.
Materials and methods AT1413 binding on a panel of tumor cell lines was analyzed by flow cytometry. AT1413 was constructed into a bispecific T-cell engaging format (AT1413 bTCE) by linking the full-length AT1413 IgG to two single chain variable fragments against CD3ϵ with a combination of site-specific enzymatic and chemical coupling. A monovalent T-cell engager was produced as heterotrimer consisting of one AT1413 monovalent chain and one anti-CD3 scFV fused to AT1413 monovalent chain. Point mutations in the IgG heavy chain were introduced to prevent interactions between AT1413 T-cell engagers and Fc-receptors. The cytotoxicity-inducing activities were established using PBMCs as effector and tumor cells as target cells using standard cytotoxic assays in vitro and in vivo in a mouse model carrying human immune cells.
Results We observed that AT1413 binds to non-hematopoietic tumor cells, such as melanoma and breast cancer. AT1413 immune precipitated CD43s from melanoma cells confirming that it recognizes the same target on melanoma as on AML. AT1413 induced antibody dependent cellular cytotoxicity against melanoma cell lines and primary melanoma samples. However, AT1413 was unable to affect growth of melanoma cells in vivo. To increase the efficacy of AT1413 it was formatted as a bispecific T-cell engaging antibody (TCE): one binding bivalently (bTCE) to CD43s and monovalently to CD3ϵ and the other monovalently (KiH) to both CD43s and CD3ϵ. In vitro, these TCEs redirected T-cell cytotoxicity against melanoma cells with different potencies. To investigate their effects in vivo, we grafted mice that harbor a human immune system with the melanoma cell line A375. Treatment with both AT1413 bTCE and AT1413 KiH significantly reduced tumor outgrowth.
Conclusion AT1413 recognizes a sialylated epitope on CD43 shared by melanoma, AML and MDS cells. Two different bispecific TCE forms of AT1413 induce strong anti-tumor cytotoxic activities in vitro and in vivo. These data indicate a broad therapeutic potential of AT1413.
Citation Format: Greta de Jong, Lina Bartels, Martijn Kedde, Els Verdegaal, Marijn A. Gillissen, Sophie E. Levie, Madalina G. Cercel, Susan E. van Hal-van Veen, Christien Fatmawati, Dorien van de Berg, Etsuko Yasuda, Yvonne Claassen, Arjen Q. Bakker, Remko Schotte, Julien Villaudy, Koen Wagner, Hergen Spits, Mette D. Hazenberg, Pauline M. van Helden. T-cell engager bispecific formats of an AML patient-derived antibody targeting a unique sialylated CD43 epitope induce kill of melanoma cells in vitro and in vivo [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 542.