A novel format for recombinant antibody-interleukin-2 fusion proteins exhibits superior tumor-targeting properties in vivo

Abstract

The targeted delivery of interleukin-2 to the tumor is gaining attention as an avenue to potentiate the action of T and NK cells at the site of disease. We have previously described the fusion of the L19 antibody, specific to the EDB domain of fibronectin, with human interleukin-2, using a non-covalent homodimeric diabody format. Here, we describe four novel formats for the L19-IL2 fusion, featuring different arrangements of antibody and IL2. A comparative quantitative biodistribution analysis in tumor-bearing mice using radioiodinated proteins revealed that the novel format (L19L19-IL2, with the antibody in single-chain diabody format) exhibited the best biodistribution results. In vitro assays on peripheral blood mononuclear cells showed a decrease activation of regulatory T cells when single IL2 domain was used. In vivo, both L19-IL2 and L19L19-IL2 inhibited tumor growth in immunocompetent mouse models of cancer. T-cell analysis revealed similar levels of CD4 + and FoxP3 + cells, with an expansion of the CD8 + T cell in mice treated with L19-IL2 and L19L19-IL2. The percentage of CD4 + regulatory T cells was markedly decreased with L19L19-IL2 combined with a mouse-specific PD-1 blocker. Collectively, these data indicate that the new L19L19-IL2 format exhibits favorable tumor-homing properties and mediates a potent anti-cancer activity in vivo.

Engineered human cytokine/antibody fusion proteins expand regulatory T cells and confer autoimmune disease protection

“…F5111 IC biases the activity of IL-2 while also extending its serum half-life; however, it does not target IL-2 toward specific tissues. Several recent approaches have been taken to target IL-2 and other cytokines to specific locations, such as the tumor microenvironment ( Hutmacher et al, 2019 ; Mortara et al, 2018 ; Ongaro et al, 2020 ; Silver et al, 2021 ). Our approach could be integrated with these emerging technologies to bias the local immune microenvironment and enhance therapy.…”

Effect of non-repetitive linker on in vitro and in vivo properties of an anti-VEGF scFv

“…They showed that structural stability and functionality were significantly affected by linker content and a natural linker composed of IgG3 upper hinge region had the highest functionality and stability. In one study, three different GS-bearing linkers with neutral, positive, and negative charges were employed and no significant difference was observed for their biodistribution patterns in tumor-bearing mice 32 . In another study, GS and only glycine-containing linkers varying from 1 to 25 amino acids were screened and GS linkers showed better biophysical characteristics and pharmacokinetic properties 14 .…”

Targeted Association and Intracellular Delivery of Nanocargoes into Primary T Lymphocytes via Interleukin-2 Receptor-Mediated Endocytosis

“…Yet, this random biotinylation approach was not as efficient as enzymatic C-terminal conjugation, for C-terminal conjugation guarantees to avoid sterical hindrance while keeping the central core untouched for effective engagement with IL2R. Based on the crystal structure of IL2-IL2R, we presume the conjugation to the other terminus (N-terminus) of IL2 might be as efficient, − which needs to be confirmed by further investigation.…”