1200 A clinically validated pH-sensitive nanomedicine platform for encapsulating therapeutic bispecific T cell engagers for tumor specific delivery and activation

Background Bispecific antibodies (BsAbs) are an important class of therapeutics for immune-oncology applications. T cell engagers (TCEs) target tumor-associated antigens and cytotoxic T cells to eradicate antigen-expressing tumor cells. Blinatumomab (CD19 X CD3 bispecific) is approved for CD19-positive B cell acute lymphoblastic leukemia,1 but its toxicity may be limiting, with one-third of patients in the pivotal Phase 3 study requiring treatment interruption for adverse events.2 TCEs for solid tumors have likewise demonstrated encouraging clinical efficacy but shown dose-limiting toxicities due to on-target/off-tumor effects3,4 For instance, patients receiving solitomab (EpCAM X CD3 bispecific) experienced severe gastrointestinal toxicity which precluded its further development.5 To minimize the off-tumor effects, we have developed ON-BOARD, an ultra-pH sensitive nanoparticle platform, has shown utility in cytokine and monoclonal antibody encapsulation and targeted delivery to the acidic tumor microenvironment.6,7 The clinical safety and feasibility of ON-BOARD has been demonstrated by effective delivery of fluorophores to solid tumors for imaging of multiple tumor types in Phase I and II clinical trials with pegsitacianine.8 Herein we expand the utility of ON-BOARD platform for the encapsulation and pH-specific activation of bispecific antibodies with potential for anticancer therapy. Methods A panel of BsAbs (including biosimilar equivalents of blinatumomab, solitomab, and others) was used to demonstrate encapsulation by the ON-BOARD platform and pH-dependent activation. Formulations of ON-BOARD with BsAbs were purified by size exclusion chromatography and the encapsulation efficiencies were quantified by HPLC. Particle size and uniformity were studied by dynamic light scattering. ON-BOARD/BsAb formulations were assessed in vitro under neutral pH or acid-activated conditions to determine target engagement by ELISA, bio-layer interferometry. The target-specific bioactivity and therapeutic window was determined by TDCC assays in multiple models. Results ON-BOARD nanoparticles successfully encapsulated bispecific antibodies across a wide range of tumor-associated antigens (TAAs), including HER2, EpCAM, CEACAM5, CD19, and CD20, and structural configurations (tandem scFv and Fc-fusion). ON-BOARD formulations were stable nanoparticles with narrow size distribution (<70 nm), good encapsulation efficiency (up to 98%) and drug loading (up to 8%). Acid-mediated release and target engagement of both TAA-targeting and CD3-targeting arms was demonstrated using in vitro binding assays with >100-fold activation window. Further pH-specific cell killing was confirmed by TDCC assays in multiple in vitro models including Burkitt lymphoma, breast cancer, colorectal cancer, and lung cancer. Conclusions The ON-BOARD pH-sensitive nanoparticle platform demonstrated potential as an effective and universal tool for solid tumor specific activation and delivery of bispecific antibody therapeutics, potentially minimizing systemic side effects. References Goebeler ME, Bargou R. T cell-engaging therapies — BiTEs and beyond. Nat Rev Clin Oncol 2020;17:418–34. Kantarjian H, et al. Blinatumomab versus chemotherapy for advanced acute lymphoblastic leukemia. N Engl J Med 2017;376:836–847. Zhou S, Liu M, Ren F, Meng X, Yu J. The landscape of bispecific T cell engager in cancer treatment. Biomark Res. 2021;9:38. Edeline J, Houot R, Marabelle A, Alcantara M. CAR-T cells and BiTEs in solid tumors: challenges and perspectives. J Hematol Oncol. 2021;14:65. Kebenko M, et al. A multicenter phase 1 study of solitomab (MT110, AMG 110), a bispecific EpCAM/CD3 T-cell engager (BiTE®) antibody construct, in patients with refractory solid tumors. OncoImmunology 2018;7:8. Ding X, Miller J, Su Q, et al. ONM-400, a novel approach for interleukin-2 therapy using a ph-activated nanoparticle targeting metabolic acidosis in solid cancers. J Immunother Cancer 2020;8(Suppl 3) A345. Bharadwaj G, Su Q, Gutowski S, et al. Encapsulating therapeutic antibodies for tumor specific activation and delivery using a clinically validated pH-sensitive nanoparticle platform. Cancer Res 2022;82(12_Supplement):1734. Voskuil FJ, Steinkamp PJ, Zhao T, et al. Exploiting metabolic acidosis in solid cancers using a tumor-agnostic pH-activatable nanoprobe for fluorescence-guided surgery. Nat Commun. 2020;11:3257.