Abstract 6076: Non-clinical pharmacology of HX044, a novel FIC CTLA4xCD47 bi-specific antibody

Abstract A bispecific CTLA4-antibody/SIRPα-fusion protein, HX044, was created based on the hypothesis that it would increase antitumor immunity/activities via efficient cis-binding to co-overexpressed CTLA4 and CD47 on regulatory T cells (Treg) within tumor microenvironment while decrease toxicity via lowered affinity to each individual target in peripheral. HX044, as designed as 1+1 asymmetric IgG1 composed of half anti-CTLA4 mAb plus the extracellular CD47 binding domain of SIRPα, binds to human CTLA4 recombinant protein with a EC50 at 1.5 nM or human CD47 recombinant protein with a EC50 at 8.12 nM, which are 30 times weaker than the bi-valent CTLA4 mAb or 160 times weaker than the SIRPα-Fc fusion protein, respectively. In addition, HX044 exhibited reduced blocking activities on the binding of CTLA-4 to CD80 or CD47 to SIRPα, respectively, with an IC50 at 135.8 nM for CTLA4 or 6316 nM for CD47. While HX044 also binds to the respective single-positive cells with reduced binding as compared to the parental single-targeting molecules, HX044 has significantly enhanced binding to the CTLA4hi/CD47hi-double positive cells, suggesting an increased avidity via efficient cis-binding. HX044 showed little binding to the CD47-single-positive RBC and minimized binding to platelets, suggesting its potential minimal hematotoxicity usually associated with other CD47-targeting therapeutics. On the other hand, the minimized blocking function of HX044 showed ina CTLA4-ligand blockade function assay may also potentially contribute to its lower risks of inducing immune-related adverse effect (irAE) usually associated with CTLA4 targeting. HX044 mediates strong ADCC toward double target-expressing cells, which is highly dependent on the CTLA4 expression levels. It has comparable FcRn binding as Ipilimumab (Ipi). In the hCTLA4 x hCD47 x hSIRPα HuGEMM mouse model, HX044 was shown to have strong anti-tumor activity to huCD47-MC38 colorectal tumor (classic “hot” tumor) at significant low dose (<0.2mg/kg) and huCD47-B16F10 melanoma tumor (traditionally “cold” tumor) (6.6mg/kg) accompanied by the reduction of TIL-Treg. Notably, the anti-tumor efficacy of HX044 is significantly superior to Ipilimumab or SIRPα-Fc fusion protein at equivalent molar doses, respectively., In the weekly repeated dosing study in cynomolgus monkeys, HX044 at 1 mg/kg or 6 mg/kg showed no significant drug-related or non-recoverable toxicity findings, while one animal died due to infusion reactions and one animal was euthanized moribund due to electrolyte imbalance and malnutrition caused by prolonged loose stool in the 40mg/kg group. The Highest Non-Severe Toxic Dose (HNSTD) was determined to be 6 mg/kg. A single-dose intravenous infusion of HX044 PK study showed dose-dependent exposure with half-life around 220 hours at low dose, going down with dose increase. Overall, preclinical data of HX044 support its further clinical testing in cancer patients. Citation Format: Hang Ke, Tao Yang, Feiyu Peng, Jialing Li, Cen Chen, Lingxin Xiong, Ruiling Sun, Lei Zhang, Faming Zhang, Henry Qixiang Li. Non-clinical pharmacology of HX044, a novel FIC CTLA4xCD47 bi-specific antibody [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6076.