Optical Control of Cytokine Signaling via Bioinspired, Polymer-Induced Latency

Abstract

<jats:title>ABSTRACT</jats:title><jats:p>Cytokine signaling is challenging to study and therapeutically exploit as the effects of these protein are often pleiotropic. A subset of cytokines can, however, exert signal specificity via association with latency-inducing proteins which cage the cytokine until disrupted by discreet biological stimuli. Inspired by this precision, here we describe a strategy for synthetic induction of cytokine latency via modification with photo-labile polymers that mimic latency while attached, then restore protein activity in response to light, thus controlling the magnitude, duration, and location of cytokine signals. We characterize the high dynamic range of latent cytokine activity modulation and find that polymer-induced latency, alone, can prolong <jats:italic>in vivo</jats:italic> circulation and bias receptor subunit binding. We further show that protein de-repression can be achieved with near single-cell resolution and demonstrate the feasibility of transcutaneous photoactivation. Future extensions of this approach could enable multicolor, optical reprogramming of cytokine signaling networks and more precise immunotherapies.</jats:p>

Multicolor Light-Induced Immune Activation via Polymer Photocaged Cytokines

“…IL-2-cNB-PEG). 18 Using this conjugate and the NIR-responsive IL-12 conjugate described here (IL-12-cCy-PEG), we constructed Boolean circuit that combines AND gates in which we digitize the presence or absence of NIR or visible LED light, as well as the presence or absence of discreet concentrations of IL-12-cCy-PEG or IL-2-cNB-PEG (Figure 5a, S6) . Using these components as inputs and blinatumomab-induced cytolysis level as a digitized output, we observed full concordance with the expected AND-AND gate truth table whereby above-threshold B cell lysis was observed only in the presence of both caged cytokines, as well as both visible and NIR LED light (Figure 5b) .…”

“…Cytokines were modified with cCy-PEG using carbodiimide and TCO-tetrazine chemistry, respectively using methods adapted from Perdue et al 18 Briefly, rIL-12 was sequentially reacted overnight with a 10-120-fold molar excess of NHS-TCO followed by a 40-480-fold molar excess of cCy and a 160-1,920-fold molar excess of DBCO-mPEG 20kDa (4°C with 800 rpm rotatory agitation). Excess reactants were removed via 7 kDa size exclusion chromatography (Zeba Spin, Thermo Fisher Scientific) in some experiments.…”

“…rIL-2 was modified with cNB-PEG using carbodiimide and azide-alkyne chemistry, respectively using methods described in Perdue et al 18 Briefly, rIL-2 was diluted in 150 mM sodium phosphate buffer (pH 8.5) containing 0.5 mM SDS and sequentially reacted with 80-fold molar excess of cNB followed by N 3 -mPEG 20kDa at a 400-fold molar excess in PBS (4°C with 800 rpm rotatory agitation, overnight).…”

“…IL-2, IL-12, IL-15) can be blunted via conjugation with a photo-labile polyethylene glycol (PEG) and recovered via brief, in this case blue, LED light exposure. 18 While these studies demonstrated the feasibility of in vivo cytokine photoactivation using tissue phantoms, we concluded that the clinical applications of this approach were limited to cutaneous or subdermal tissues and those accessible by in situ light sources (e.g. catheter-guided fiber lasers) as a result of efficient absorption and scattering of light by tissues and biological fluids at visible wavelengths.…”

“…Leveraging the ability of NIR photons to efficiently penetrate human tissues, here we advance our prior approach 18 to reversibly photocage immunostimulatory cytokines by using photo-labile PEG that de-shields in response to NIR rather than visible light. We show that LED light exposure of NIR-photocaged cytokines can be used to reversibly modulate cognate cytokine receptor signaling and enhance the activity of bispecific cancer immunotherapies that rely on T cell-dependent cytolysis.…”