Intratumoral nanobody–IL-2 fusions that bind the tumor extracellular matrix suppress solid tumor growth in mice

Abstract

Confining cytokine exposure to the tumors would greatly enhance cancer immunotherapy safety and efficacy. Immunocytokines, cytokines fused to tumor-targeting antibodies, have been developed with this intention, but without significant clinical success to date. A critical limitation is uptake by receptor-expressing cells in the blood, that decreases the dose at the tumor and engenders toxicity. Small-format immunocytokines, constructed with antibody fragments, are hypothesized to improve tumor specificity due to rapid systemic clearance. However, effective design criteria for small-format immunocytokines need further examination. Here we engineer small interleukin-2 (IL-2) immunocytokines fused to nanobodies with nanomolar to picomolar affinities for the tumor-specific EIIIB domain of fibronectin (also known as EDB). Upon intravenous delivery into immunocompetent mice, such immunocytokines led to similar tumor growth delay as size-matched untargeted IL-2. Intratumoral delivery imparted improved survival dependent on affinity to EIIIB. Intratumoral administration offers a promising avenue to deliver small-format immunocytokines, given effective affinity for the tumor microenvironment.

Spatiotemporally programming cytokine immunotherapies through protein engineering

“…By fusing wild‐type cytokines to antibodies (referred to as immunocytokines hereafter) directed at tumor‐associated antigens (TAAs), it was hypothesized that systemic exposure and on‐target/off‐tumor toxicity could be reduced. However, we have previously shown that simple immunocytokine targeting does not solve this problem in the context of solid tumors 39,40 . In the case of a systemically administered IL‐2 immunocytokine targeted to the melanoma marker TRP1, Tzeng et al demonstrated that the biodistribution of the fusion protein is entirely governed by cognate cytokine receptor expression patterns 39 .…”

“…Small‐format immunocytokines, such as those designed with nanobody fragments instead of full‐length IgGs, have been proposed to have improved tumor specificity over larger antibody‐based immunocytokines due to their rapid renal clearance 39,40 . However, theoretical analysis of the fundamental rate processes of tumor localization suggests that with few exceptions, this may not be the case 44‐46 .…”

“…Fusing a cytokine to the targeting moiety may only exacerbate this valley of death due to peripheral sinks before reaching the tumor. Lutz et al recently demonstrated that even for a small format IL‐2/domain antibody immunocytokine with picomolar affinity for EIIIB, a tumor‐associated extracellular matrix component, no additional survival benefit was seen over an untargeted size‐matched cytokine when dosed systemically 40 . It should be noted, however, that intratumoral administration of these same agents was efficacious, with higher affinity leading to improved efficacy.…”

“…Lutz et al also reported on IL‐2 targeted to the EIIIB domain of fibronectin, an ECM component that is specific to tumor tissue, unlike collagen 40 . The tumor specificity of EIIIB makes it a compelling target for systemic administration.…”

“…However, we have previously shown that simple immunocytokine targeting does not solve this problem in the context of solid tumors. 39,40 In the case of a systemically administered IL-2 immunocytokine targeted to the melanoma marker TRP1, Tzeng et al demonstrated that the biodistribution of the fusion protein is entirely governed by cognate cytokine receptor expression patterns. 39 For IL-2, this led to an appreciable systemic "sink" from NK and NKT cells, as opposed to the desired stimulation of intratumoral CD8 T cells.…”