GrB-Fc-KS49, an anti-EMP2 granzyme B fusion protein therapeutic alters immune cell infiltration and suppresses breast cancer growth

• DOI: 10.1136/jitc-2024-008891
• Publication Date: 2024-12-22T14:40:41Z
• Authors: Khalid A Mohamedali; Brian Aguirre; Cheng-Hsiang Lu; Anubhav Chandla; Nidhi Kejriwal; Lucia Liu; Ann M Chan; Lawrence H Cheung; SuYin Kok; Sergio Duarte; Ana Alvarez de Ci...; David Casero; Michael G Rosenblum; Madhuri Wadehra
• Keywords: Granzyme

Background Granzyme B (GrB) is a key effector molecule, delivered by cytotoxic T lymphocytes and natural killer cells during immune surveillance to induce cell death. Fusion proteins immunoconjugates represent an innovative therapeutic approach specifically deliver deadly payload target cells. Epithelial membrane protein-2 (EMP2) highly expressed in invasive breast cancer (BC), including triple-negative BC (TNBC), represents attractive target. Methods We designed novel fusion protein (GrB-Fc-KS49) composed of active GrB fused anti-EMP2 single-chain antibody tethered through the immunoglobulin G heavy chain (Fc) domain. assessed construct’s enzymatic activity, binding affinity, cytotoxicity against panel The pharmacokinetics (PK), toxicity profile, vivo efficacy were also evaluated. Results GrB-Fc-KS49 exhibited comparable activity commercial GrB, as well high affinity EMP2 peptide, with dissociation constant picomolar range. rapidly internalized into EMP2+cancer showed vitro lines expressing surface EMP2, half-maximal (IC 50 ) values below 100 nM for most positive lines. Ex stability at 37°C indicated half-life exceeding 96 hours while PK biexponential plasma clearance, moderate initial clearance (t 1/2 α=18.4 hours) much slower terminal rate β=73.1 hours). No was measured Chem16 between control GrB-Fc-KS49. In vivo, TNBC syngeneic (4T1/ FLuc mouse model, reducing tumor volume proliferation increasing death compared controls. Treatment using EMT6 model confirmed these results. addition significant impact on proliferation, treatment resulted dramatic increase tumor-infiltrating CD45+ redistribution tumor-associated macrophages. Transcriptomic analysis tumors post-treatment remodeling microenvironment immunotoxin. Conclusions specificity towards EMP2-positive it reduced burden increased recruitment tumor, suggesting that promising candidate BC.