T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological cancer that is treated with intensive multi-agent chemotherapy, often leading to long-term side-effects impacting the quality of life of survivors. Despite the therapeutic success in children, relapses still occur in 10-20% of the cases, and adults face a considerably poorer prognosis. Novel, more selective treatments that contribute to reducing toxicities and improving outcome are thus in need.
Interleukin 7 (IL-7) and its receptor IL-7Rα promote leukemia development in a majority of T-ALL patients and mutational activation of IL-7Rα, which occurs in around 10% of the cases, associates with very high risk in relapsed disease. Using combinatorial scFv phage display libraries and antibody reformatting we have now generated a fully human IgG1 monoclonal antibody (named B12) against human IL-7Rα. B12 does not display cross-reactivity against the mouse receptor and recognizes both wild type and mutant forms of IL-7Rα naturally expressed in T-ALL cell lines and patient samples, as well as in Ba/F3 cells stably transduced with human, but not mouse, IL-7Rα.
Interestingly, molecular dynamics simulations suggest that B12 forms a stable complex with IL-7Rα at a different site from IL-7. Nonetheless, B12 inhibits IL-7/IL-7R-mediated signaling and induces cell death per se in at least some IL-7/IL-7R-reliant T-ALL cell lines (e.g. IL-7-dependent TAIL7 cells and mutant IL7R DND4.1 cells) and patient samples. Using patient-derived xenograft (PDX) samples, HPB-ALL cells and D1 cells overexpressing a mutated gain-of-function form of IL-7Rα, we show that the antibody also promotes antibody-dependent NK-mediated leukemia cytotoxicity in vitro and delays T-cell leukemia development in vivo, reducing tumor burden and promoting mouse survival. Moreover, B12 cooperates with dexamethasone in promoting the death of both dexamethasone-resistant HPB-ALL cells and a dexamethasone-sensitive PDX sample.
Notably, B12 is rapidly internalized via clathrin-coated pits to the early endosome, eventually trafficking to the lysosome - an effect that is slightly accelerated in the presence of IL-7. These characteristics render B12 an attractive vehicle for targeted intracellular delivery of a highly cytotoxic warhead. As such, we engineered a B12-mono-methyl auristatin E (MMAE) antibody-drug conjugate (ADC) in which site-specific conjugation of B12 was carried out by reducing inter-chain disulfide bonds and reacting the thiol group of the free cysteines with a Michael acceptor (carbonyl acrylic derivate) linked to a cleavable linker (valine-citrulline) and the drug (MMAE). Tested against different cell lines, primary patient cells and PDX samples, B12-MMAE ADC demonstrates increased leukemia cell killing ability in vitro as compared to the naked antibody.
Altogether, our studies serve as a stepping stone towards the development of novel targeted therapeutic strategies in T-ALL and other diseases where IL-7Rα was shown to play a pathological role.
Akkapeddi:Instituto de Medicina Molecular João Lobo Antunes: Patents & Royalties: Patents. Neri:Philochem AG: Equity Ownership. Bernardes:Instituto de Medicina Molecular João Lobo Antunes: Patents & Royalties: Patents. Barata:Instituto de Medicina Molecular João Lobo Antunes: Patents & Royalties: Patents.
Asterisk with author names denotes non-ASH members.