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Background: The survived cancer cells after cytotoxic chemotherapy are known to promote immune evasion phenotype by increasing the expression of PD-L1 and CD47. Targeting of CD47 and PD-L1 is predicted to overcome immune evasion by coordinately restoring each innate and adaptive immunity. IOH-001, a CD47/PD-L1 dual-targeting bispecific antibody, has been shown to activate immune cells in tumor microenvironment by blocking PD-1/PD-L1 signals that inhibit cytotoxic T-cells, while also blocking interaction with CD47/SIRPa between cancer cells and macrophages. We have investigated whether IOH-001 inhibits tumor growth via co-targeting tumor cells or not. Methods: To assess the function of IOH-001, a series of in vitro functional assays including cell surface binding, antibody-dependent cellular cytotoxicity (ADCC), phagocytosis assays and mixed lymphocyte reaction (MLR) assay were performed compared with the parent antibody. In vivo efficacy of IOH-001 was tested in colon cancer mouse models with human genes KI. Results: IOH-001 has been shown to be bound strongly to various types of PD-L1/CD47-expressing cancer cells including the ones in solid and hematological cancers. In most cancer cells, IOH-001 was demonstrated to have a lower EC50 than the parental antibodies and to be bound in a dose-dependent manner as well. Interestingly, IOH-001 also has shown the selectivity to bind only to cancer cells even under the conditions of co-culturing RBC and cancer cells. IOH-001 has induced phagocytosis of cancer cells by human blood CD14+ monocyte-derived macrophages. Since IOH-001 is an IgG1 type antibody, ADCC and IFN-r expression have been increased compared with the parental antibodies. Consistent with the in vitro data, IOH-001 has more strongly suppressed tumor growth than the combination treatment of the parental antibodies in a dose-dependent manner in syngeneic animal models. Moreover, the tumor of the CR mouse has been identified not to be re-generated in the re-challenge model. Conclusion: IOH-001, dual-blockage of anti-CD47 and anti-PD-L1, has shown the benefits in treating some solid tumors. Bispecific antibody IOH-001 is more likely to work better in targeting tumor cells than the combination of anti-CD47 and anti-PD-L1. Preclinical efficacy results of IOH-001 provide a strong rationale for assessing therapeutic potential in clinical studies. Citation Format: Jeong-kook Kim, A-Ra Jeon, Jihyun Park, Ji Yea Choi, Ji Eun Park, Sun Kwang Song, Ji Hye Choi, Heewook Shin, Ji Hye Lee, Ji Hye Yun, Yoen Hee Ahn, Heung Tae Kim. IOH-001, a novel CD47/PD-L1 bispecific antibody, enhances anti-tumor activity in solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 596.

RASSF1A is a tumor suppressor implicated in many tumorigenic processes; however, the basis for its tumor suppressor functions are not fully understood. Here we show that RASSF1A is a novel antagonist of protumorigenic RhoA activity. Direct interaction between the C-terminal amino acids (256–277) of RASSF1A and active GTP-RhoA was critical for this antagonism. In addition, interaction between the N-terminal amino acids (69-82) of RASSF1A and the ubiquitin E3 ligase Smad ubiquitination regulatory factor 1 (Smurf1) disrupted GTPase activity by facilitating Smurf1-mediated ubiquitination of GTP-RhoA. We noted that the RhoA-binding domain of RASSF1A displayed high sequence homology with Rho-binding motifs in other RhoA effectors, such as Rhotekin. As predicted on this basis, RASSF1A competed with Rhotekin to bind RhoA and to block its activation. RASSF1A mutants unable to bind RhoA or Smurf1 failed to suppress RhoA-induced tumor cell proliferation, drug resistance, epithelial–mesenchymal transition, migration, invasion, and metastasis. Clinically, expression levels of RASSF1A and RhoA were inversely correlated in many types of primary and metastatic tumors and tumor cell lines. Collectively, our findings showed how RASSF1A may suppress tumorigenesis by intrinsically inhibiting the tumor-promoting activity of RhoA, thereby illuminating the potential mechanistic consequences of RASSF1A inactivation in many cancers. Cancer Res; 76(7); 1847–59. ©2016 AACR.