527 Preclinical dose-pharmacokinetic-efficacy modeling of botensilimab using a mouse surrogate of the Fc-enhanced anti-CTLA-4 antibody

<h3>Background</h3> CTLA-4 antagonist antibodies were the first immune checkpoint inhibitors demonstrating durable clinical responses.¹ However, their expansion to new indications has been limited. Preclinical murine studies have demonstrated that anti-CTLA-4 activity depends on co-engaging Fcγ receptor (FcγR)-dependent mechanisms to promote T-cell priming, Treg depletion, and myeloid activation.² These findings extended to patients treated with botensilimab, an Fc-enhanced multifunctional anti-CTLA-4 antibody, with clinical activity across multiple poorly immunogenic and treatment-refractory cancers, including microsatellite stabile (MSS) colorectal cancer (CRC).³ To support the clinical development of botensilimab, we modelled efficacious doses and exposure parameters using mouse tumor models treated with a pharmacologically matched mouse surrogate antibody (botensilimab^ms). <h3>Methods</h3> The potency and efficacy of botensilimab^msversus a conventional CTLA-4 surrogate antibody were evaluated in the MSS CT26 CRC model. The CTLA-4 antibody, clone 9D9, engineered on a murine IgG2b Fc backbone was selected for its functional comparability to conventional human IgG1 whereas an Fc-engineered murine IgG2b, with S239D/A330L/I332E mutations was used as a surrogate for botensilimab. Dose-response relationships were assessed using single administrations. Binding affinities for CTLA-4 and FcγRs were determined by flow cytometry and surface plasmon resonance. A multi-dose pharmacokinetic (PK) study correlated drug plasma concentrations with preclinical response. PK parameters were derived using a non-compartmental analysis. Pharmacodynamic effects were assessed by flow cytometry. <h3>Results</h3> Botensilimab^ms demonstrated pharmacological equivalence to botensilimab, with comparable CTLA-4 binding affinities and comparable ratios of binding to activating and inhibiting Fc gamma receptors. In tumor models, botensilimab^ms exhibited superior efficacy and potency compared to the conventional anti-CTLA-4^ms. The therapeutic dose range for botensilimab^ms (5 – 50 µg) was lower than that of conventional anti-CTLA-4^ms (10 – 100 µg). At equivalent doses, botensilimab^ms elicited stronger intratumoral pharmacodynamic effects, including more pronounced Treg depletion and expansion of tumor-reactive T cell clones. Half-lives were comparable for both antibodies (50.6 hours for botensilimab^ms versus 55.5 hours for conventional CTLA-4^ms). Exposure-response analysis revealed that the therapeutic range corresponded to average plasma concentrations (C_avg) of 1.1 – 11.1 µg/mL for botensilimab^ms, consistent with botensilimab’s recommended Phase 2 dose (RP2D) of 75 mg Q6W, which yielded a C_avg of 4.49 ± 1.39 µg/mL. In comparison, the C_avg range for conventional CTLA-4^ms was 1.7 – 17.9 µg/mL. <h3>Conclusions</h3> This study demonstrated the enhanced potency and efficacy of an Fc-enhanced anti-CTLA-4 antibody (botensilimab^ms) versus conventional anti-CTLA-4 in CT26 CRC. The observed exposure-response relationship predicted the clinically efficacious doses of botensilimab, validating this modeling approach for guiding clinical development of Fc-engineered antibodies. <h3>References</h3> Maker AV, Phan GQ, Attia P, <i>et al</i>. Tumor regression and autoimmunity in patients treated with cytotoxic T lymphocyte-associated antigen 4 blockade and interleukin 2: a phase I/II study. <i>Ann Surg Oncol</i> 2005;<b>12</b>:1005–16. Waight JD, Chand D, Dietrich S,<i> et al</i>. Selective FcγR Co-engagement on APCs Modulates the Activity of Therapeutic Antibodies Targeting T Cell Antigens.<i> Cancer Cell</i> 2018;<b>33</b>:1033–1047. Bullock AJ, Schlechter BL, Fakih MG <i>et al</i>. Botensilimab plus balstilimab in relapsed/refractory microsatellite stable metastatic colorectal cancer: a phase 1 trial. <i>Nat Med</i> 2024 (Online ahead of print). <h3>Ethics Approval</h3> Human materials were obtained following informed consent under Western Institutional Review Board® Protocol #20233227. Animal data was approved by an institutional review board Agenus-Neosome IACUC Protocols #NLS18-016 and NLS21-015.