Synthesis and characterization of a thermally-responsive tumor necrosis factor antagonist

Abstract

Numerous antagonists of tumor necrosis factor alpha (TNFalpha) have been developed to attenuate inflammation and accompanying pain in many disease processes. Soluble TNF receptor type II (sTNFRII) is one such antagonist that sequesters TNFalpha away from target receptors and attenuates its activity. Systemic delivery of soluble TNF receptors or other antagonists may have deleterious side effects associated with immune suppression, so that strategies for locally targeted drug delivery are of interest. Elastin-like polypeptides (ELPs) are biopolymers capable of in situ drug depot formation through thermally-driven supramolecular complexes at physiological temperatures. A recombinant fusion protein between ELP and sTNFRII was designed and evaluated for retention of bivalent functionality. Thermal sensitivity was observed by formation of supramolecular submicron-sized particles at 32 degrees C, with gradual resolubilization from the depot observed at physiological temperatures. In vitro refolding of the sTNFRII domain was required and the purified product exhibited an equilibrium dissociation constant for interacting with TNFalpha that was seven-fold higher than free sTNFRII. Furthermore, anti-TNF activity was observed in inhibiting TNFalpha-mediated cytotoxicity in the murine L929 fibrosarcoma assay. Potential advantages of this ELP-sTNFRII fusion protein as an anti-TNFa drug depot include facility of injection, in situ depot formation, low endotoxin content, and functionality against TNFalpha.

Figure 1

Dynamic light scattering data (mean ± standard deviation) for ELP-sTNFRII at 25 µM shows a transition from monomers to larger, micron-sized complexes at subphysiological temperatures.

Figure 2

ELP (♦) and ELP-sTNFRII (■) proteins resolubilize into warm PBS supernatant reaching a steady-state thermodynamic equilibrium. Data were fit to an increasing monoexponential (Equation 1) with coefficients of determination, r², exceeding 0.98 for both datasets. The extent of protein release into the supernatant was greater for the fusion protein than for the free ELP (p < 0.05). Furthermore, ELP-sTNFRII resolubilizes more rapidly than the non-fusion ELP (p < 0.05).

Figure 3

The temperature at which ELP-sTNFRII refolding was performed impacted upon yield, with the optimal temperature observed to be 4°C (ANOVA, p < 0.05). No difference was noted in the absence (black) or presence (grey) of protein disulfide isomerase (ANOVA, p = 0.86).

Figure 4

SDS-PAGE of successful ELP-sTNFRII immunoprecipitation (left) and TNFα-affinity precipitation (right) yielding a 44 kDa protein. Depicted for the immunoprecipitation are starting material (SM), washing steps (W_1–5), elutions (E_1–3), and molecular weight ladder (λ). Silver staining reveals several contaminant proteins in the starting material that are effectively eliminated during the immunoprecipitation. Depicted for the TNFα-affinity precipitation are the molecular weight ladder (λ), starting material (SM), the fifth final wash step, and the first two eluted fractions. The lower sensitivity SimplyBlue™ staining does not reveal the same contaminant proteins as evident from silver staining.

Figure 5

Thermal purification of both ELP and ELP-sTNFRII by inverse thermal cycling effectively removes endotoxin from these recombinant proteins. The endotoxin content of ELP-sTNFRII is 0.35 ± 0.13 EU/mg and that of non-fusion ELP is 0.27 ± 0.16 EU/mg.

Figure 6

In vitro bioactivity of ELP-sTNFRII (♦) against TNFα-mediated L929 cytotoxicity has an IC50 of 1200 ± 500 nM. This is significantly reduced (p < 0.01) compared with commercial sTNFRII () with an IC50 of 12 ± 4 nM. Coefficients of determination, r₂, for fitting to a dose-response curve (Equation 2) ranged from 0.94 to 0.99.

Figure 7

The supernatant ELP-sTNFRII fractions obtained from the fusion protein resolubilization experiment demonstrate in vitro bioactivity (#, p < 0.01) against TNFα-mediated L929 cytotoxicity (0.25 ng/mL TNFα, *, p < 0.01). The extent of cytoprotection was equivalent to that of ELP-sTNFRII that was not put through the depot-formation and resolubilization experiment.