Engineered cytokine/antibody fusion proteins improve delivery of IL-2 to pro-inflammatory cells and promote antitumor activity

Abstract

Progress in cytokine engineering is driving therapeutic translation by overcoming the inherent limitations of these proteins as drugs. The interleukin-2 (IL-2) cytokine harbors great promise as an immune stimulant for cancer treatment. However, the cytokines concurrent activation of both pro-inflammatory immune effector cells and anti-inflammatory regulatory T cells, its toxicity at high doses, and its short serum half-life have limited clinical application. One promising approach to improve the selectivity, safety, and longevity of IL-2 is complexation with anti-IL-2 antibodies that bias the cytokine towards the activation of immune effector cells (i.e., effector T cells and natural killer cells). Although this strategy shows therapeutic potential in preclinical cancer models, clinical translation of a cytokine/antibody complex is complicated by challenges in formulating a multi-protein drug and concerns about complex stability. Here, we introduce a versatile approach to designing intramolecularly assembled single-agent fusion proteins (immunocytokines, ICs) comprising IL-2 and a biasing anti-IL-2 antibody that directs the cytokines activities towards immune effector cells. We establish the optimal IC construction and further engineer the cytokine/antibody affinity to improve immune biasing function. We demonstrate that our IC preferentially activates and expands immune effector cells, leading to superior antitumor activity compared to natural IL-2 without inducing toxicities associated with IL-2 administration. Collectively, this work presents a roadmap for the design and translation of immunomodulatory cytokine/antibody fusion proteins.

Cytokine/Antibody Fusion Protein Design and Evaluation

“…Since intramolecular binding is desired for cytokine/anti-cytokine antibody fusion proteins, an N-terminal fusion is more logical to facilitate cytokine/antibody interaction by co-localizing the cytokine with the antibody variable domain. For both F10 IC (Leonard et al, 2023) andF5111 IC (VanDyke et al, 2022), fusion to the N-terminus of the LC was employed. To minimize oligomerization and/or aggregation while also promoting cytokine/antibody binding, various linker lengths should be explored.…”

“…Linker lengths should be optimized for each intramolecular immunocytokine that is developed to minimize oligomerization and/or aggregation and optimize cytokine/antibody (Leonard et al, 2023;VanDyke et al, 2022). One can also vary the composition of the linker to be structured or flexible, as desired based on the orientation of the cytokine and antibody as well as the interface topology.…”

“…To overcome these limitations, our lab developed intramolecularly assembled cytokine/antibody fusion proteins (immunocytokines, ICs) that fuse IL-2 to the N-terminus of the F10 and F5111 antibody light chains. These engineered ICs (denoted F10 IC and F5111 IC) selectively bias the effects of IL-2 and prolong cytokine half-life in serum, and they show encouraging preclinical promise as molecular therapeutics to treat cancer or autoimmune conditions (Leonard et al, 2023;Silver et al, 2023;VanDyke et al, 2022).…”

An engineered immunocytokine with collagen affinity improves the tumor bioavailability, tolerability, and therapeutic efficacy of IL-2

“…To increase the therapeutic potential of hIL-2, we previously engineered an IL-2 cytokine/antibody fusion protein, denoted F10 immunocytokine (IC), that intramolecularly fuses hIL-2 to an engineered anti-IL-2 antibody (F10; derived from MAB602) 9 , 12 , 13 that sterically occludes the IL-2/IL-2Rα interaction while allosterically enhancing the IL-2/IL-2Rβ interaction. 14 This approach extends the circulating half-life of IL-2 (presumably through interaction of the IC fragment crystallizable [Fc] domain with the neonatal Fc receptor [FcRn]), 15 and it eliminates the stimulatory advantage conferred to T Reg cells by IL-2Rα, thereby enhancing the activation of Effs. Indeed, we demonstrated the immunostimulatory bias of F10 IC compared to the natural IL-2 cytokine in both cellular and animal models.…”

“…Indeed, we demonstrated the immunostimulatory bias of F10 IC compared to the natural IL-2 cytokine in both cellular and animal models. 14 However, this molecule is limited in its anti-tumor efficacy and fails to address the dose-limiting toxicity of IL-2, which is thought to be caused by the on-target, off-tumor activity of IL-2 on non-tumor-specific immune cells and pulmonary and hepatic vascular endothelial cells, as well as the indirect effects of IL-2 on vascular permeability exerted through inflammatory cytokines secreted by systemically activated leukocytes. 8 , 9 , 10 , 11 …”