TJC4, a Differentiated Anti-CD47 Antibody with Novel Epitope and RBC Sparing Properties

Introduction Tumor cells overexpress CD47 which engages signal-regulatory protein (SIRPa) on macrophages to deliver a "do not eat" signal to avoid being phagocytosed. Blocking CD47 using SIRPa-Fc or anti-CD47 antibodies (Ab) has emerged as a promising strategy to neutralize CD47 and promote tumor eradication. However, targeting CD47 led to significant anemia and thrombocytopenia in both pre-clinical studies and phase I trials as CD47 is also expressed on normal red blood cells (RBCs) and platelets. I-Mab has developed a novel CD47 antibody, TJC4 also known as TJ011133, which was endowed with an RBC sparing property and unique binding epitope, may have better safety profile based on the pre-clinical data. Methods A naïve human single chain variable fragment (ScFv) library was subjected for the binders to human CD47-extracellular domain (ECD). All the binders with unique sequences were converted to full antibodies and screened against human RBCs and tumor cells, leading to the discovery of TJC4. A series of head to head experiments have been performed with other CD47 antibodies to compare the in vitro RBC binding and hemagglutination, ability to block the CD47-SIRPa interaction and enhance the macrophage mediated phagocytosis of tumor cells. Different in vivo tumor models were employed to evaluate the anti-tumor efficacy of TJC4 either by mono or combination treatment. In addition, a comprehensive analysis of the hematological parameters was assessed in cynomolgus monkeys receiving a single intravenous infusion or weekly repeated injections. To explore the underlined mechanism of the RBC sparing properties of TJC4, the binding pose and epitope were identified by X-ray crystallography and the influence of CD47 glycosylation in RBCs were further examined. Results TJC4 is a fully human anti-CD47 IgG4 antibody that shares a similar binding affinity to human and cynomolgus monkey CD47. Like other anti-CD47 antibodies, TJC4 blocks the interaction of CD47 and SIRPa, leading to the enhanced macrophage phagocytosis of various CD47+ tumor cell lines and primary AML cells. Mono-treatment of TJC4 completely eradicated tumor cells in a Raji cell xenograft model and significantly extended the overall survival of treated mice in an AML model. When combined with Rituximab, TJC4 showed a superior efficacy in a DLBCL model over the mono-treatment group. TJC4 has unique RBC sparing properties as evident by the negligible binding to healthy human RBCs and platelets respectively. Single dose or repeat dose treatment of TJC4 minimally and transiently impacts RBCs in cynomolgus monkeys and no other safety findings were observed up to the highest dose (100 mg/kg). No impact was observed in platelets. The unique functional properties of TJC4 can be explained in part by its structure when in complex with CD47, which reveals an almost straight head-to-head binding and a novel conformational epitope that is distinct from other CD47 antibodies. Upon the structural analysis of the binding epitope, we identified a potential N-linked glycosylation site located nearby the two critical epitopes on the CD47 protein. Due to the nature of the high glycosylation degree of membrane proteins by RBCs, the N-linked glycan is hypothesized to function as a "shield" to block the exposure of the epitopes and prevent the TJC4 binding to human RBCs. This hypothesis is validated by the restoration of TJC4 binding to the deglycosylated RBCs after the PNGase treatment. Conclusion In summary, TJC4 is a next generation therapeutic anti-CD47 antibody that is devoid of the hematological liabilities while maintaining anti-tumor efficacy. These attributes of TJC4 differentiate it from other CD47 targeting agents currently in clinical evaluation. Disclosures Meng: I-Mab Biopharma: Employment, Equity Ownership. Wang:I-Mab Biopharma: Employment, Equity Ownership. Guo:I-Mab Biopharma: Employment, Equity Ownership. Cao:I-Mab Biopharma: Employment, Equity Ownership. Shen:I-Mab Biopharma: Employment, Equity Ownership.