Actin binding-related genes (Myh4, Actn3, Acta1, Mybpc2, Tnnt3, and Tnni2) were upregulated after IBI188 treatment (Supplementary Fig.?1). the anti-tumor efficacy of IBI188. IBI188 treatment upregulated cell movement- and inflammation-related genes in macrophages. Synergism was observed when combined with an anti-CD20 therapeutic antibody, whose function depends on antibody-dependent cellular cytotoxicity/phagocytosis (ADCC/ADCP). CD47 expression was evaluated following azacytidine (AZA) treatment, a standard-of-care for patients with multiple myeloma; enhanced anti-tumor efficacy was observed in the combination group in AML xenograft models. Notably, IBI188 treatment increased vascular endothelial growth factor-A (VEGF-A) levels in a solid tumor model, and combined treatment with an anti-VEGF-A antibody and IBI188 resulted in an enhanced anti-tumor effect. These data indicate that IBI188 is a therapeutic anti-CD47 antibody with anti-tumor potency, which can be enhanced when used in combination with standard-of-care drugs for cancer treatment. Supplementary Information The online version contains supplementary material available at 10.1007/s00262-021-02989-2. Keywords: IBI188, CD47, Tumor inhibition, Anti-CD20, Azacytidine, VEGF-A Introduction Immunotherapy is a powerful tool for the treatment of cancer. Directly targeting the immune system triggers a strong memory immune response than conventional chemotherapy, which leads to substantial survival benefits [1]. The overall response rate observed with programmed cell death protein (PD)-1-targeted therapy varies between cancer types and generally remains low [2]; therefore, new combination therapies are needed to maximize the anti-tumor efficacy of these therapies. CD47, also known as integrin-associated protein (IAP), is a widely expressed transmembrane protein. Tumor cells expressing CD47 directly inhibit macrophage or dendritic cell phagocytosis of tumor cells via interaction with signal regulatory protein (SIRP)- expressed on phagocytes. High expression of CD47 has been reported in numerous hematologic and solid cancers [3C7], suggesting that CD47 participates in tumor immune escape. The clinical prognostic outcome is strongly negatively correlated with CD47 expression [4]. Blocking the CD47/SIRP- connection may enhance the phagocytotic function of antigen-presenting cells, and has shown strong anti-tumor potency in multiple preclinical models, either Methylprednisolone through macrophages or dendritic cells [5, 8, 9]. Based on this, restorative antibodies and fusion proteins focusing on the CD47-SIRP- pathway have been recognized and tested PALLD clinically. Vascular endothelial growth element (VEGF)-A regulates blood vessel development and homeostasis. VEGF-A is definitely secreted by tumor cells and the surrounding stromal cells, advertising endothelial cell proliferation or survival and subsequent angiogenesis [10C12]. These blood vessels then provide tumor cells with nutrients. In addition, VEGF-A was recently shown to possess immune-suppressive function. VEGF-A can directly inhibit Methylprednisolone the Methylprednisolone maturation of dendritic cells and the cytotoxic function of T cells [13C15]. Moreover, CD47 deficiency in T cells or tumor stromal cells raises VEGF-A manifestation in T cells and at tumor sites, which contributes to the Methylprednisolone state of immune suppression. It is unclear whether obstructing the CD47 pathway in tumor cells would elevate VEGF-A manifestation inside the tumor. In this Methylprednisolone study, we screened a highly potent anti-CD47 obstructing antibody named IBI188, which can promote the phagocytosis of tumor cells by macrophages in vitro. The anti-tumor effectiveness of IBI188 has been shown in NHL and AML/MDS xenograft mouse models, when given as monotherapy and in combination with an anti-CD20 antibody or azacytidine (AZA). During AZA treatment, bad feedback was observed with upregulation of CD47, which inhibited the phagocytotic ability of macrophages. Moreover, in a solid tumor model, VEGF-A manifestation was elevated following anti-CD47 antibody treatment, which suggests that angiogenesis limits the efficacy of this antibody in solid tumors. Materials and methods Cell collection, cell line building, and transfection Raji, MDA-MB-231, MV-4-11, CCRF-CEM, and HL-60 cells were purchased from ATCC (Manassas, VA). CHO-S manifestation cell lines were generated according to the manufacturers instructions using the Freedom? CHO-S? Kit (Invitrogen). Full-length human being CD47 coding sequences (CDS) were inserted into the pCHO 1.0 vector to generate CHO-S cells overexpressing CD47. Antibody manifestation and purification Hu5F9 is definitely a human being immunoglobulin (Ig)G4 CD47 antibody that utilizes weighty and light chain sequences from a publicly available source (World Health Corporation Proposed INN List 120). IBI301 is definitely a bio-similar of Rituximab (World Health Corporation Proposed INN List 77). IBI305 is definitely a bio-similar of bevacizumab (World Health Corporation Proposed INN List 83). All practical.