CD47 blockade has emerged as a promising cancer immunotherapy by promoting phagocytosis of tumor cells. However, this treatment strategy may be limited by the scarcity of macrophages with phagocytic potential at the tumor site. The key myeloid cytokine GM-CSF has been approved as an adjunctive agent in the treatment of cancer by virtue of its ability to stimulate hematopoiesis and particularly induce the differentiation of pro-inflammatory M1 macrophages. Here we report the development of a novel immunocytokine composed of a newly discovered CD47 IgG1 fused with GM-CSF at the C-terminus that exhibited enhanced phagocytosis and M1 macrophage activation, leading to superior antitumor efficacy.
A lead anti-CD47 mAb was identified from panning of a naïve human library and a series of functional screening assays. The human GM-CSF sequence was then fused via various flexible linkers to the C-terminus of 1F8's heavy chain to generate different formats of anti-CD47-GMCSF fusions. The resultant fusion proteins were tested for retention of anti-CD47- and GM-CSF-mediated biological activities. A lead molecule (1F8-GMCSF) was then tested for its ability to reduce tumor growth in immunodeficient NSG mice. In addition, phenotype of tumor associated macrophages (TAM) was investigated in tumor sections by immunohistochemistry (IHC).
In macrophage-tumor cell co-culture, treatment with 1F8-GMCSF fusion molecule led to significantly higher levels of tumor cell phagocytosis than that with anti-CD47 alone. Moreover, higher levels of pro-inflammatory cytokines were detected in co-culture supernatant after treatment with 1F8-GMCSF than that with GM-CSF alone. These data suggest functional cooperation between phagocytosis pathways and GM-CSF-mediated growth and differentiation pathways, leading to synergism for enhanced phagocytosis and increased M1 polarization. Consequently, treatment of 1F8-GMCSF in a tumor xenograft model demonstrated superior efficacy in suppression of tumor growth compared to 1F8 or GM-CSF treatment alone or in combination. IHC analysis of tumor sections confirmed a shift from M2 to M1 phenotype in infiltrating macrophages after 1F8-GMCSF treatment.
We have successfully generated a novel anti-CD47-GM-CSF immunocytokine fusion protein that not only retains key properties of its components but also demonstrates enhanced anti-tumor efficacy and M1 macrophage polarization. This fusion protein represents a second generation anti-CD47 molecule that may be used for treatment of solid tumors and is undergoing preclinical development with an aim to enter clinical studies in 2018.
Citation Format: Zhengyi Wang, Wei Cao, Taylor Guo, Jingwu Zang. A novel immunocytokine fusion protein combining tumor-targeting anti-CD47 antibody with GM-CSF cytokine for enhanced antitumor efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5622.