Concentrations of cytokines in the serum and exudate were measured by a Cytometric Bead Assay Mouse Inflammatory Kit (Becton Dickinson, Franklin Lakes, NJ). (ADC) that selectively targets immune cells through the CD11a antigen. The resulting ADC consisted of a human CD11a antibody (based on efalizumab clone hu1124) conjugated to an analog of the highly potent PDE4 inhibitor GSK256066. Both the human CD11a ADC and a mouse surrogate CD11a ADC (based on the M17 clone) rapidly internalized into immune cells and suppressed lipololysaccharide (LPS)-induced TNF secretion in primary human monocytes and mouse peritoneal cells, respectively. In a carrageenan-induced air pouch inflammation mouse model, treatment with the ADC significantly reduced inflammatory cytokine production in the air pouch exudate. Overall, these results provide compelling evidence for the feasibility of delivering drugs with anti-inflammatory activity selectively to the immune compartment via CD11a and the development of tissue-targeted PDE4 inhibitors as a promising therapeutic modality for treating inflammatory diseases. Significance PDE4 inhibitors are clinically validated molecules with considerable efficacy but relatively low safety profile in treating chronic inflammatory diseases. Therefore, the potential expansion of clinical indications of these molecules is relatively unexplored. Bringing to bear medicinal chemistry and bio-conjugation methods, we generated huCD11a-PDE4 and its mouse equivalent muCD11a-PDE4, which target the pan-immune cell surface antigen CD11a and demonstrated potent suppression of inflammation that is explicitly receptor-dependent. Pharmacokinetic and pharmacodynamic analysis of muCD11a revealed translation of these effects. With antibody-based therapies becoming a mainstay in the treatment of inflammation, this study provides critical validation for a new paradigm which could lead to second generation PDE4 inhibitors with an improved safety and efficacy. Introduction AntibodyCdrug conjugates (ADCs) Levocetirizine Dihydrochloride are an attractive platform for highly potent drugs whose therapeutic potential can be improved by selective delivery to target tissues while avoiding nontarget tissues that drive dose-limiting toxicity. ADCs comprise an antibody that selectively targets a cell surface antigen and has been modified by a cleavable or noncleavable chemical linker that supports stable attachment and intracellular release of a small molecule payload, which is most often a cytotoxin1,2 and in limited examples can be a potent bioactive molecule.3,4,5 In this regard, targeted delivery with ADCs potentially represents a modular therapeutic platform to capture the efficacy of validated small molecules while eliminating their unwanted nontarget tissue side effects to yield a drug with improved safety profile. To date, significant efforts have been made to generate ADCs for oncology, with three ADCs receiving market approval. Comparatively, less emphasis has been placed on the application of ADCs in other disease areas such as inflammation and autoimmunity to deliver noncytotoxic, therapeutic drugs in a cell-specific manner in order to decrease potential side effects due to activity in off-target tissues.3,4,5 Immune responses are fine-tuned processes initiated by various cellular signals and mediated by complex intracellular cascades. Phosphodiesterases (PDEs) are a class of enzymes that control the amplitude and duration of the signal of cAMP, a key second messenger of inflammatory responses. Increases of cAMP dramatically decrease inflammatory responses of leukocytes to stimuli.6 PDE4 is a cAMP phosphodiesterase widely expressed Levocetirizine Dihydrochloride in hematopoietic cells (stability of the conjugate Levocetirizine Dihydrochloride and correspondingly improve the specificity of drug delivery. Noncleavable ADC linkers have been shown to effectively deliver drugs that upon internalization and degradation of the antibody also have minimal extracellular release and reuptake into other tissues.20 Towards this end, we designed and synthesized model compounds containing different linker chemistries with varying hydrophilicity and flexibility at Levocetirizine Dihydrochloride the meta- and para- positions of the arylsulfone moiety (Figure 1a). The activity of these derivatives was evaluated in a biochemical assay for inhibition of PDE4 enzymatic activity (Figure 1b) and a cell-based assay for cAMP accumulation and subsequent cAMP-response element (CRE)-dependent expression of luciferase in THP-1 (CRE-luc) cells (Figure 1c). GSK256066 potently inhibited PDE4B enzymatic activity at sub-picomolar EC50 as previously reported. Among the GSK256066 derivatives, compound 3 with substitution at the meta position was the most potent in both the enzyme and cell-based assays (Figure 1e). While the inhibitory effect of compound 3 against the isolated PDE4B enzyme is comparable to GSK256066 it exhibited a ~300-fold drop in cellular CRE-Luc activity. The attenuated cellular activity of compound 3 was hypothesized to be due to decreased cellular permeability, an attribute that should not adversely affect the efficiency of antibody-mediated uptake. This may favorably contribute to specificity by retention of the molecule inside the target cell, and decrease of recirculation of free drug payload following delivery by ADC. Importantly, compound 3 did not exhibit any cytotoxicity in THP1 cells up to 10 mol/l (Figure 1d). Based on these results, we chose compound 3 Levocetirizine Dihydrochloride as the basis for a linker conjugate to generate a PDE4 inhibitor ADC. Open in a separate window Figure 1 Generation and characterization of PDE4 inhibitor payload drug. (a) GSK256066 and its Rabbit polyclonal to VCAM1 derivative linker analogs. (b) PDE4B enzymatic activity inhibition by a range of concentrations of compound 1 to.