An engineered 4-1BBL fusion protein with “activity on demand”

Abstract

Engineered cytokines are gaining importance in cancer therapy, but these products are often limited by toxicity, especially at early time points after intravenous administration. 4-1BB is a member of the tumor necrosis factor receptor superfamily, which has been considered as a target for therapeutic strategies with agonistic antibodies or using its cognate cytokine ligand, 4-1BBL. Here we describe the engineering of an antibody fusion protein, termed F8-4-1BBL, that does not exhibit cytokine activity in solution but regains biological activity on antigen binding. F8-4-1BBL bound specifically to its cognate antigen, the alternatively spliced EDA domain of fibronectin, and selectively localized to tumors in vivo, as evidenced by quantitative biodistribution experiments. The product promoted a potent antitumor activity in various mouse models of cancer without apparent toxicity at the doses used. F8-4-1BBL represents a prototype for antibody-cytokine fusion proteins, which conditionally display “activity on demand” properties at the site of disease on antigen binding and reduce toxicity to normal tissues.

An Attenuated Targeted-TNF Localizes to Tumors In Vivo and Regains Activity at the Site of Disease

“…Some receptors of the TNF superfamily rely on the formation of higher-order clusters for effective signaling. This feature was demonstrated in a recent study, where antibody fusions exploited the ability of 4-1BB ligand to oligomerize upon antigen binding and regain activity at the site of disease [ 31 ]. Multiple research groups have focused their efforts on the development of attenuated versions of cytokines to reduce toxicity.…”

“…The use of cytokine payloads with decreased affinity towards the cognate receptor lowers the biological activity of the molecule in systemic circulation, thus allowing the administration of higher systemic doses. Promising preclinical results have been obtained with certain antibody-cytokine fusions based on a depotentiated version of IFNα, IL12 or 4-1BB [ 31 , 32 , 33 , 34 ]. Moreover, the group of Tavernier have developed Activity-on-Target cytokines (so called Actakines) by fusing inactivated TNF or IFN-γ mutants to a CD13 antibody, leading to selective targeting to the tumor endothelium and complete tumor destruction without side-effects [ 35 ].…”

Identification and Validation of New Interleukin-2 Ligands Using DNA-Encoded Libraries

“…Interestingly, if the small molecule was to be used to mask the IL2 moiety in a tumor targeting antibody-IL2 fusion protein (such as L19-IL2), it should be possible to generate a novel class of biopharmaceutical-small-molecule complexes that do not bind to T regs , selectively localize at the tumor site, and progressively lose the small-molecule inhibitor at the site of disease, thus regaining full activity. The development of cytokine-based pharmaceuticals with “activity on demand” has been postulated as one of the new horizons of cytokine research for cancer therapy. , …”

“…The development of cytokine-based pharmaceuticals with "activity on demand" has been postulated as one of the new horizons of cytokine research for cancer therapy. 47,48 ■ EXPERIMENTAL SECTION Starting materials, Solvents, and Reagents. Unless otherwise noted, all the solvents were used as supplied by VWR (Pennsylvania, United States).…”

Current approaches to assessing the biological activity of immunocytokines <i>in vitro</i>

“…Цитокиновый участок при конструировании противоопухолевого ИЦ выбирают, основываясь на эффективности его использования в клинической Прямое противоопухолевое действие цитокина может заключаться в подавлении пролиферации, индукции апоптоза, стимуляции экспрессии молекул главного комплекса гистосовместимости I и II типа на поверхности опухолевых клеток [11], а иммуномодулирующее действие -в повышении активности эффекторных иммунных клеток (IL-2, IFN 1-го типа), индукции созревания антигенпрезентирующих клеток (IL-2, 6, 7, 13, 15 [12], IFN 1-го типа), индукции повышенной экспрессии белков, в частности цитокинов (IFN 1-го типа) [11]. Помимо этого, некоторые цитокины в случае применения их с полноразмерными противоопухолевыми антителами способны усиливать функции Fc-фрагмента антитела (АЗКЦ, АЗФ) за счет активации эффекторных клеток (натуральных киллеров (NK), макрофагов) (IFN 1-го типа, IL-2, 12,15,21).…”