(W1230-01-03) Elastin-Like Polypeptide (ELP) Antibody Fusions Targeting IL-6: Pharmacology and Pharmacokinetics

Purpose: Interleukin 6 (IL-6) is a pleiotropic cytokine involved in inflammation, immune response, hematopoiesis, and autoimmune disease. Dysregulated, constitutive activation of the IL-6 signaling pathway is implicated in chronic inflammatory disorders, such as Sjögren’s disease (SjD) and rheumatoid arthritis. An early and extensive symptom of SjD is the lymphocytic infiltration of the tear-producing lacrimal gland, which is highly enriched in IL-6 signaling. Anti-IL-6 single-chain antibody fragments (scFv) are immunomodulatory components that may inhibit IL-6-mediated signaling; however, these have yet to be developed to alleviate SjD. Partly due to glomerular filtration, scFvs have short plasma half-lives. One method to extend their pharmacokinetic (PK) properties and enhance therapeutic efficacy is to attach scFvs to high molecular weight recombinant polypeptides, such as elastin-like polypeptides (ELPs). Derived from human tropoelastin, ELPs are genetically engineered biopolymers that self-assemble into multivalent nanoparticles. ELPs consist of repeated amino acid sequences (Val-Pro-Gly-X_aa-Gly)_n, where the guest residue, X_aa, controls the hydrophobicity, and n determines the ELPs’ molecular weight. This study reports a novel, temperature-sensitive anti-IL-6 therapeutic that consists of the anti-mouse IL-6 scFv and a thermally responsive ELP, A192. These fusions (aIL6-A192) self-assemble into elongated, colloidally stable ‘nanoworms’ that sequester IL-6. We report herein on its biophysical properties, inhibition of IL-6 signaling, and PK properties.

Methods: The aIL6-A192 plasmid was generated by ligating the encoding sequence of the anti-mouse IL-6 scFv sequence to the A192 sequence (VPGAG)₁₉₂. For protein expression and purification, the aIL6-A192 plasmid was expressed in Escherichia coli. UV-visible spectrophotometry was used to quantify the ELP’s thermo-responsive phase separation, in which the optical density at 350 nm (OD₃₅₀) was measured over a range of temperatures. The ELP transition temperature (T_t) was obtained from the following equation: Tt=b-mlog₁₀(C_ELP), where b is the y-intercept representing the extrapolated T_t at a concentration of 1 μM and m is the slope representing the change in °C per 10-fold change in the ELP concentration (C_ELP). Dynamic light scattering (DLS) was used to measure the hydrodynamic radius (R_h), colloidal stability, and polydispersity of aIL6-A192 nanoparticles. Size exclusion chromatography-multiangle light scattering (SEC-MALS) was performed to measure the absolute molar mass of the nanoworms, as well as the degree of oligomerization. Negative-staining transmission electron microscopy (TEM) was used to visualize nanoparticle morphology. Lastly, surface plasmon resonance (SPR) was used to measure the binding affinity of aIL6-A192 for recombinant mouse IL-6. After biophysical characterization, effects of aIL6-A192 on the binding interaction between mouse IL-6 and IL-6Rα were quantified using an ELISA. To investigate whether aIL6-A192 can block IL-6-IL-6R signaling, RAW 264.7 mouse macrophages were co-incubated with IL-6 and aIL6-A192 at 37 °C for 30 min. Subsequent cell lysates were examined by western blot to determine the phosphorylation of the key IL-6-targeted signal transducer, pSTAT3. To quantify the nanoworms’ PK, aIL6-A192 was fluorescently labeled with rhodamine and then injected into BALB/c mice intravenously (IV) or subcutaneously (SC) beneath the skin overlying the lacrimal gland (supra-LG injection). Diluted plasma was collected at various time points up to 2 days. fluorescence intensity in the plasma was used to estimate the plasma concentrations, which were evaluated by both compartmental and model-independent PK analyses.

Results: While A192 phase separates above 56 °C at 50 μM, the T_t of aIL6-A192 is only 44.3 °C. The T_t is inversely correlated with the ELP concentration; however, the T_t of aIL6-A192 (m = 2.0±1.07 °C/Log₁₀ (μM)) is less sensitive to concentration than A192 (m = 8.4±0.8 °C/Log₁₀ (μM)). Both ELPs remain soluble at physiological temperatures. The R_h of aIL6-A192 (25 μM) is 40.7±5.6 nm, representative of monodisperse nanoworms. This does not change significantly over 72 hrs, suggesting colloidal stability at 37 °C. The absolute molar mass of the aIL6-A192 nanoparticles is 1.2x107 Da, suggesting that ~120 aIL6-A192 monomers oligomerize to form a nanoworm, which were observed using negative TEM. Despite their apparent assembly, these nanoworms achieve a nanomolar binding affinity for mouse IL-6 (K_D=52.2 nM). Using a customized ELISA, aIL6-A192 inhibited binding of the recombinant IL-6 to IL-6Rα with an IC₅₀ of 1.58 μM. In mouse RAW 264.7 cells shown to express ­IL-6Rα, aIL6-A192 inhibited the IL-6-mediated phosphorylation of STAT3 with an IC₅₀ of 582.2 nM. In mice, the terminal half-life of aIL6-A192 following IV and supra-LG injection was 7.6±0.4 hrs and ± 17.5±5.0 hrs, respectively.

Conclusion: The anti-mouse IL-6 scFv can be conjugated to an ELP to generate stable, multivalent nanoworms (aIL6-A192). These nanoparticles effectively disrupt the binding between IL-6 and IL-6R and inhibit IL-6-mediated signaling in mouse macrophages. Lastly, the nanoworms exhibit favorable PK properties following supra-LG and IV delivery, which are ideal for treating mouse models of autoimmune diseases like SjD. Their in vivo evaluation as inhibitors for L-6/IL-6R signaling may promote development of novel, antibody-based modalities to treat autoimmune and inflammatory diseases.

Acknowledgements: This work was made possible by the University of Southern California (USC) to AP, the Gavin S. Herbert Professorship to JAM, National Institutes of Health R01EY026635 to JAM and SHA, P30 CA014089 to the USC Norris Comprehensive Cancer Center, and P30 EY029220 to the USC Ophthalmology Center Core Grant for Vision Research. The authors have no conflicts to report.

An ELP fusion protein expressing the anti-mouse IL-6 scFv is designed to sequester IL-6

aIL6-A192 monomers oligomerize to form stable, rod-like nanostructures that inhibit IL-6-IL6R⍺ interaction and exhibit favorable PK properties

aIL6-A192 inhibits IL-6 signaling in murine RAW 264.7 macrophages expressing endogenous IL-6R