Selective CD28 antagonist prevents Aldara‐induced skin inflammation in non‐human primates (2015)

T-lymphocyte activation is triggered by antigen recognition and the engagement of costimulatory molecules. The CD28-CD80/86 costimulatory interaction is the most important costimulatory pathway for effector T-cell responses, and the therapeutic potential of CD80/86 blockade has reached the market with Abatacept in autoimmune diseases and Belatacept in kidney transplantation. However, the drawback of these molecules is to blunt CD80/86 interactions with CTLA-4 and PD-L1 checkpoints and to interfere with crucial mechanisms of immune tolerance such as CTLA-4-dependent suppressive function of regulatory T cells and self-inhibition of autoreactive and memory T cells 1. We therefore developed a new selective antagonist of CD28 (FR104: a humanized pegylated anti-CD28 Fab' antibody fragment) devoid of agonist activity (S1–4) which spares CTLA-4 and PD-L1 inhibitory signals. We previously described that this novel drug candidate promoted immune tolerance and induced long-term allograft survival in non-human primates 2, 3. It also efficiently prevented brain inflammation in an experimental autoimmune encephalomyelitis (EAE) non-human primates model 4 and, as opposed to CD80/86 antagonists, differentially controlled human effector and regulatory memory T-cell activation in vitro at the immune synapse level 5. Here, we evaluated the therapeutic potential of this novel selective CD28 antagonist to control skin inflammation. Psoriasis is a complex, multifactorial, relapsing inflammatory skin disease characterized by erythematous scaly plaques. Psoriasis lesions are characterized by histological features such as a thickened epidermis caused by keratinocyte proliferation (acanthosis), the retention of nuclei in the stratum corneum (parakeratosis) that arises from an aberrant differentiation of keratinocytes, and inflammatory cell infiltrates by innate and adaptive immune cells in the epidermis and dermis. Since psoriasis does naturally occur only in humans, a large number of rodent models have been set up and characterized to mimic at least some aspects of the human disease 6. However, mouse immune system is significantly different from human and pharmaceutical development of biologics is complicated by a lack of cross-reactivity with rodents. The development of preclinical models of skin inflammation, which closely resemble human diseases, in large animals is lacking to evaluate the therapeutic potential of these novel immunotherapies. Aldara cream contains 5% Imiquimod, a potent immune activator ligand of TLR7 and TLR8. Aldara is used in humans for topical treatment of genital and perianal warts caused by papillomavirus, actinic keratosis and superficial basal cell carcinoma. However, it has been reported that Aldara application induces psoriasis or exacerbates the disease in patient with a well-controlled psoriasis during topical treatment for aforementioned indications and that psoriasis occurs at both the treated area but also at distant skin sites that were unaffected before treatment 7. Later, topical treatment of mouse skin with Aldara was reported as a novel mouse skin inflammatory model which histology closely resembles psoriasis since inducing acanthosis, parakeratosis and a mixed inflammatory infiltrate with a predominance of the IL23/IL-17 axis like humans 8. More recently, the effects of Aldara application to non-lesional psoriatic skin of patients with psoriasis were evaluated 9. Similar to mouse, Aldara induces in human skin redness, induration and scaling. Histological analysis revealed psoriasis-like inflammation with focal parakeratosis, acanthosis, a perivascular mononuclear infiltration and significant expression of IL-17 family cytokines. To evaluate the therapeutic potential of the novel selective CD28 antagonist, we developed for the first time an Aldara-induced skin inflammation model in non-human primates. Daily topical administration of Aldara cream for 2 weeks on depilated-skin in baboons induced strong erythema and skin thickening after few days, which resolved after arrest of Aldara application (Fig. 1). Important scaling of the skin was also observed after 2 weeks of application and resolved after 4–5 weeks. mRNA analysis of skin biopsies confirmed overexpression of the IL-17 pathway after Aldara application as compared to naïve animals (Fig. 1). Similar to the mouse Aldara model, histopathological analysis of the baboon skin treated with Aldara revealed interface dermatitis characterized by a band-like lymphohistiocytic infiltrate in the upper dermis, irregular epidermal thickening (acanthosis), epidermal hyperproliferation (Ki-67 staining), hyperkeratosis, hypergranulosis and change of the epidermal basal layer; the pathological features of lichenoid pattern (Fig. 2). Of note, the presence of some epidermal necrotic bodies suggested lichenoid drug reaction. As a matter of comparison, histology in a conventional Tuberculin-induced delayed-type hypersensitivity (DTH) model in the same species was clearly different from psoriasis with a mild epidermal thickening, without epidermal lesion, and superficial dermal and perivascular mononuclear infiltrates (S6). To assess action of selective CD28 costimulatory inhibition in this novel model, a single administration of FR104 at 10 mg/kg was administered intravenously a day before Aldara application. FR104 significantly reduced skin erythema (although not completely), thickening and desquamation (Fig. 1). Infiltration by T lymphocytes (CD8+ and CD8− T cells) and macrophages was also prevented, in particular at the epidermal interface, epidermal thickening was also reduced while epidermal hyperproliferation was not completely prevented with FR104 treatment (Fig. 2). Indeed, it was reported that Aldara and its vehicle cause TLR7-independent acanthosis as well as hyperproliferation, apoptosis and activation of the inflammasome of keratinocytes (S7). Epidermal necrotic bodies were still present also after FR104 treatment. This suggested that FR104 was able to prevent the inflammatory phase in this model but not the limited epidermal toxicity (lichenoid drug reaction), clarifying small redness scores recorded in treated animals. Two months after FR104 administration, when blood receptor occupancy started to decrease, animals were challenged again with Aldara. Skin inflammation and desquamation in treated baboons remained weaker in comparison with the control group (Fig. 1), although both groups displayed more pronounced scaling during this second challenge. A third challenge performed 5 months after FR104 administration, after the drug had been totally eliminated, demonstrated no visible difference between groups (Fig. 1). Selective CD28 blockade has been previously described to be highly effective to control antigen-specific-induced diseases (allotransplantation or autoimmune disease models induced by immunization). Here, we showed that this strategy also reduces skin inflammation induced by Aldara in non-human primates, as it was previously described in a mouse xenograft model with another anti-CD28 which unfortunately was not only antagonist (S8). This study confirms the promising potential of selective CD28 antagonists to prevent relapse in patients suffering from autoimmune and chronic inflammatory diseases, such as psoriasis. N.P. and M.C. organized and performed experiments, analysed and interpreted the data and prepared the manuscript; C.M., L.B. and V.D. performed some experiments; J.H., D.M. and SLB assisted with animals experiments. E.C. performed histological analyses of skin biopsies. B.V. and G.B funded the research, designed and supervised experiments, interpreted the data and edited the manuscript. Animal studies were approved by the French National Ethical Committee (n° CEEA-2011-51). The authors thank the Nantes SFR MicroPICell imaging platform (Nantes, France) and Sabrina Pengam (INSERM UMR-S 1064, Nantes, France) for cell sorting with Aria Flow Cytometer. The authors received funding from the European project TRIAD (EU-FP7-Health program EC-GA N°281493; www.TRIAD-CD28.eu). This work was also supported by the Progreffe and Centaure foundations (France) and the IHU-Cesti project, supported by Nantes Metropole and the Pays de la Loire Region. N.P., C.M., N.D., L.B. and B.V. are employees of Effimune, a company developing CD28 antagonists. N.P., C.M. and B.V. are shareholders of Effimune. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.