A novel immune-stimulator antibody conjugate (ISAC) against CD47 for cancer immunotherapy

Introduction: Blocking the axis of cluster of differentiation 47 (CD47) and signal regulatory protein-α (SIRPα), known as the "don't eat me" signal, has been a promising approach for cancer treatment. However, monotherapy using monoclonal antibodies against CD47 (aCD47) has shown limited efficacy in various cancer types. Combining aCD47 with tumor-specific monoclonal antibodies has yielded only modest clinical benefits, prompting the need for additional strategies to enhance comprehensive anti-cancer immune responses. Here, we introduce a novel immune-stimulator antibody conjugate (ISAC) comprising aCD47 and a Toll-like receptor 9 (TLR9) agonist, cytidine phosphate guanosine (CpG), aCD47-CpG (Fig. 1A), developed to potentiate robust immune responses against non-Hodgkin lymphoma (NHL).

Materials and

Methods: aCD47-CpG was engineered and characterized via SDS-PAGE and SEC. The binding affinity of the conjugate to CD47 and TLR9 was determined using ELISA. Phagocytosis was confirmed by luminescence- or flow cytometry-based assays using bone marrow-derived macrophage (BMDM) and A20 lymphoma cell line. Quanti-Blue assay was conducted for the evaluation of NF-κB activity. Pro-inflammatory cytokine production was investigated in a macrophage-cancer cell co-culture. In vivo anti-cancer efficacy was assessed using both localized and disseminated mouse lymphoma syngeneic models.

Results, Conclusions, and Discussions: Conjugation pattern of ISAC was confirmed (Fig. 1B–C). Its binding affinity to CD47 and TLR9 was well retained with Kd at a nanomolar scale (Fig. 1D). aCD47-CpG significantly enabled phagocytosis of A20 cells by BMDMs (Fig. 1E–F). Moreover, the conjugate induced enhanced NF-κB activity in monoculture of BMDM and more so in co-culture with A20 cells, indicating the intact biological activity of CpG (Fig. 1G). Importantly, ISAC enhanced the release of pro-inflammatory cytokines, including TNFα, IL-6, and IL-12, in BMDM-A20 cell co-culture to a degree on par with CpG, possibly bridging the innate and adaptive immune responses (Fig. 1H). In both localized and disseminated syngeneic mouse models of lymphoma, aCD47-CpG significantly reduced tumor burden and extended survival (Fig. 1I–L). Surprisingly, mice that had overcome an initial tumor challenge were completely resistant to a rechallenge with A20 cells, suggesting that aCD47-CpG elicits durable systemic immune memory. These data demonstrate that aCD47-CpG can serve as a potent immunomodulatory therapy of NHL and potentially other hematologic tumors.

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