A novel fully human anti-CD47 antibody as a potential therapy for human neoplasms with good safety.

Strategies for targeting CD47 are becoming a hot spot of cancer immunotherapy. However the ubiquitous expression of CD47, especially on the RBC, makes the targeted therapy facing safety risk issues. So, how to balance the safety and efficacy during CD47 inhibition is currently a major question. We had reported an anti-CD47 antibody ZF1 with potent anti-tumor effect. In this study, we further developed and assessed a novel fully human anti-CD47 antibody, AMMS4-G4, derived from ZF1 using affinity maturation. AMMS4-G4 exhibited equivalent anticancer effects with Hu5F9-G4, a humanized anti-CD47 antibody in clinical trial, on the potential of inducing significant phagocytosis of tumor cells in vitro and prolonging the survival of leukemia xenografted mice. Additionally, AMMS4-G4 significantly inhibited the growth of grafted solid tumors by enhancing macrophage infiltration and modestly enhanced the anti-tumor activity of opsonizing antibody and antiangiogenic therapy. In cynomolgus monkeys, AMMS4-G4 was safely administered, was well tolerated at doses of 30 and 60 mg/kg, and did not produce serious adverse events, except for the reversible anemia, which was observed after 3 days and started to recover from 9 days later. Remarkably, it was proved by in vitro assay that Hu5F9-G4 induced RBC hemagglutination which wasn't observed in AMMS4-G4. On the whole, AMMS4-G4 was demonstrated to be a promising candidate with great potential and safe profile for cancer immunotherapy.

Establishment of a novel cell-based assay for screening small molecule antagonists of human interleukin-6 receptor.

AIM: Blockade of interleukin-6 (IL-6) or its receptor (IL-6R) is effective in preventing the progression of autoimmune diseases, such as systemic lupus erythematosus and rheumatoid arthritis. In the present study, we established a novel cell-based assay for identifying small molecule IL-6R antagonists. METHODS: HEK293A cells were transfected with recombinant plasmids pTaglite-SNAP-IL6R and pABhFc-IL6 to obtain membrane-bound IL-6R and recombinant human IL-6 coupled with human Fc fragment (rhIL-6), respectively. A novel screening assay based on the interaction between IL-6R and rhIL-6 was established, optimized and validated. The stability of the assay was also assessed by calculating the Z'-factor. RESULTS: RhIL-6 dose-dependently bound to IL-6R expressed at HEK293A cell surface. The IC50 value of the known antagonist ab47215 was 0.38±0.08 µg/mL, which was consistent with that obtained using the traditional method (0.36±0.14 µg/mL). The value of Z'-factor was 0.68, suggesting that the novel assay was stable for high throughput screening. A total of 474 compounds were screened using the novel screening assay, and 3 compounds exhibited antagonistic activities (IC50=8.73±0.28, 32.32±9.08, 57.83±4.24 µg/mL). Furthermore, the active compounds dose-dependently inhibited IL-6-induced proliferation of 7TD1 cells, and reduced IL-6-induced STAT3 phosphorylation in U937 cells. CONCLUSION: A novel cell-based screening assay for identifying small molecule IL-6R antagonists was established, which simplifies the procedures in traditional cellular ELISA screening and profiling and reduces the costs.