Background Despite the obvious success of anti-TNFα agents in Rheumatoid Arthritis (RA) treatment, high number of non-responders and systemic adverse effects,1 including reactivation of latent infections, highlight the need of broader efficacy and improved safety profiles. Recently the importance of angiogenesis in the progression of arthritis has been unveiled.2 Angiogenesis can be exploited for tissue selective drug delivery approaches to drive in situ drug accumulation reducing systemic side effects.
Objectives Develop a bispecific antibody prodrug with vascular specificity to deliver an anti-TNFα payload to the arthritic tissue, allowing anti-TNFα activation selectively in the arthritic synovium and avoiding systemic TNFα engagement.
Methods The anti-ICAM1 variable region was cloned in fusion with the anti-TNFα antibody Adalimumab in a dual variable domain (DVD) bispecific format. Linker between the outer and inner variable domain was engineered to be short and bear an MMP1 cleavable site forming an activatable DVD (aDVD) antibody. Reactivity with RA human synovium was determined using immunohistochemistry on tissue sections. MMP1-mediated antibody activation was tested using RA synovial fluids and RA synovial fibroblasts. Activity and biological functionality of the anti-TNFα moiety was assessed in vitro using ELISA, Biacore and L-929 TNFα-cytotoxicity assays. Human TNF transgenic mouse model of arthritis (tg197) was used to assess in vivo therapeutic activity.
Results The aDVD bispecific antibody was able to retain synovial microvascular targeting, conveyed by the anti-ICAM1 variable domain, in RA synovial tissue sections. The intact molecule however, showed a reduced capacity to bind TNFα with 500-fold affinity decrease caused by steric hindrance of the outer variable region. Digestion with physiologically relevant concentrations of MMP1 was sufficient to restore 100% anti-TNFα activity (EC50 53 pM) comparable to the parent Adalimumab antibody (49 pM). The un-processed aDVD antibody was significantly impaired in rescuing the L-929 cell line from TNFα-induced cytotoxicity. Digestion with MMP1 and activation of the anti-TNFα domain restored the anti-TNFα biological activity with an IC50 of 0.1 nM equal to Adalimumab IgG (IC50 0.1 nM). The antibody was also able to localise in the arthritic joint and reduce bone degradation in the tg197 mouse model in vivo.
Conclusions Our results demonstrate the development of a synovium-specific pro-drug delivery system for RA. The impaired TNFα binding capacity of the intact molecule ensures a decreased systemic TNFα engagement. Presence of metalloproteinases in RA synovium3 would activate and deliver the anti-TNFα activity locally in the site of disease. This strategy may allow greater therapeutically-relevant doses to be delivered in the synovium, increasing pharmacological potency, potentially allowing reduction in the dosage and/or administration frequency with the ultimate goal of reducing systemic exposure, achieve better therapeutic index and decreasing healthcare costs.4
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Disclosure of Interest None declared