The Generation of Dual-Targeting Fusion Protein PD-L1/CD47 for the Inhibition of Triple-Negative Breast Cancer

Abstract

Triple-negative breast cancer (TNBC) is a highly aggressive subset of breast cancer with limited therapeutic options. However, its immune evasion mechanisms, characterized by the over-expression of the immune checkpoint molecules PD-L1 and CD47, can be targeted in order to facilitate cancer elimination by cells of innate and adaptive immunity. In this paper, we describe the design, preparation, and evaluation of three novel dual-targeting fusion proteins that were based on the structure frame of prototype IAB (innate and adaptive dependent bispecific fusion protein) and the “Orcutt-type IgG-scFv” molecular model. Three molecules with different spatial conformations were designed to improve antigen–antibody affinity by the addition of Ag–Ab binding sites from the variable region sequences of the anti-PD-L1 monoclonal antibody (mAb) atezolizumab and CV1, a high-affinity receptor of CD47. The results showed that the best-performing among the three proteins designed in this study was protein Pro3; its CV1 N-terminus and Fc domain C-terminus were not sterically hindered. Pro3 was better at boosting T cell proliferation and the engulfment of macrophages than the IAB prototype and, at the same time, retained a level of ADCC activity similar to that of IAB. Through improved design, the novel constructed dual-targeting immunomodulatory protein Pro3 was superior at activating the anti-tumor immune response and has thus shown potential for use in clinical applications.

Engineering high‐affinity dual targeting cellular nanovesicles for optimised cancer immunotherapy

“…We firstly performed flow cytometry to detect the expression levels of PD‐L1 and CD47 on triple‐negative breast cancer (TNBC) cells MDA‐MB‐231 and 100 ng/mL IFN‐γ pre‐treated lung adenocarcinoma cells A549. We verified that PD‐1 and CD47 were highly expressed on these cells as previously reported (Bian et al., 2022; Lee et al., 2019) (Figures 3a and S2). Next, we employed coimmunoprecipitation (Co‐IP) assay to examine the interaction of tumour cells with the engineered fusion WT NVs.…”

“…Therefore, we used HEK293 cells (human embryonic kidney cells) to produce NVs in our study, which were easy to expand in vitro, perform engineering and could function on different types of tumour cells with high biological safety (Zhang et al., 2018). Additionally, there are significant differences in the expression levels of PD‐L1 and CD47 on tumour cells with different malignancy and NVs internalised into cells after anchoring to ligands on the surface of target high malignance cells by high‐affinity PD‐1 and SIRPα proteins, suggesting that these fusion HAC NVs were potential for targeted drug delivery application (Bian et al., 2022; Li et al., 2023; Zhang et al., 2018).…”

Engineered high-affinity dual targeting cellular nanovesicles for optimized cancer immunotherapy

“…We rstly performed ow cytometry to detect the expression levels of PD-L1 and CD47 on triple-negative breast cancer (TNBC) cells MDA-MB-231 and 100 ng/mL IFN-γ pre-treated lung adenocarcinoma cells A549. We veri ed that PD-1 and CD47 were highly expressed on these cells as previously reported [22,23] (Fig. 3A and Figure S2, Supporting Information).…”

Opportunities and challenges of CD47-targeted therapy in cancer immunotherapy

“…To overcome these challenges, several strategies are being investigated. One approach is to combine CD47-targeted therapies with other immune checkpoint inhibitors, such as anti-PD-1/PD-L1 antibodies, to overcome the immune evasion mechanisms employed by tumor cells [12,105]. Another approach is to develop bispecific antibodies that can target both CD47 and another immune checkpoint molecule simultaneously [12,91,106].…”