Purpose: 4-1BB (CD137) is a key costimulatory immunoreceptor and promising therapeutic target in cancer. To overcome limitations of current 4-1BB-targeting antibodies, we have developed PRS-343, a 4-1BB/HER2 bispecific molecule. PRS-343 is designed to facilitate T-cell costimulation by tumorlocalized, HER2-dependent 4-1BB clustering and activation. Experimental Design: PRS-343 was generated by the genetic fusion of 4-1BB-specific Anticalin proteins to a variant of trastuzumab with an engineered IgG4 isotype. Its activity was characterized using a panel of in vitro assays and humanized mouse models. The safety was assessed using ex vivo human cell assays and a toxicity study in cynomolgus monkeys. Results: PRS-343 targets 4-1BB and HER2 with high affinity and binds both targets simultaneously. 4-1BB-expressing T cells are efficiently costimulated when incubated with PRS-343 in the presence of cancer cells expressing HER2, as evidenced by increased production of proinflammatory cytokines (IL2, GM-CSF, TNFa, and IFNg). In a humanized mouse model engrafted with HER2-positive SK-OV-3 tumor cells and human peripheral blood mononuclear cells, PRS-343 leads to tumor growth inhibition and a dose-dependent increase of tumor-infiltrating lymphocytes. In IND-enabling studies, PRS-343 was found to be well tolerated, with no overt toxicity and no relevant drug-related toxicologic findings. Conclusions: PRS-343 facilitates tumor-localized targeting of T cells by bispecific engagement of HER2 and 4-1BB. This approach has the potential to provide a more localized activation of the immune system with higher efficacy and reduced peripheral toxicity compared with current monospecific approaches. The reported data led to initiation of a phase I clinical trial with this first-in-class molecule. See related commentary by Su et al., p. 5732

Purpose: Cathepsin S is a cysteine protease that promotes the invasion of tumor and endothelial cells during cancer progression. Here we investigated the potential to target cathepsin S using an antagonistic antibody, Fsn0503, to block these tumorigenic effects. Experimental Design: A panel of monoclonal antibodies was raised to human cathepsin S. The effects of a selected antibody were subsequently determined using invasion and proteolysis assays. Endothelial cell tube formation and aorta sprouting assays were done to examine antiangiogenic effects. In vivo effects were also evaluated using HCT116 xenograft studies. Results: A selected cathepsin S antibody, Fsn0503, significantly blocked invasion of a range of tumor cell lines, most significantly HCT116 colorectal carcinoma cells, through inhibition of extracellular cathepsin S-mediated proteolysis. We subsequently found enhanced expression of cathepsin S in colorectal adenocarcinoma biopsies when compared with normal colon tissue. Moreover, Fsn0503 blocked endothelial cell capillary tube formation and aortic microvascular sprouting. We further showed that administration of Fsn0503 resulted in inhibition of tumor growth and neovascularization of HCT116 xenograft tumors. The lysosomal cysteine cathepsins encompass a family of closely related cysteine proteases, mediating a diverse range of proteolytic effects (1-4). However, an increasing body of evidence has shown the overexpression of a number of cysteine cathepsins in cancer (5-7). Significantly, these proteases are secreted into the tumor extracellular milieu, producing potent degradative effects on a broad range of extracellular matrix (ECM) components, including collagen and laminins (8-10). Further confirmation of these effects were provided in a murine model of sporadic pancreatic carcinogenesis (RIP1-Tag2), in which the genetic ablation of either cathepsin B or cathepsin S severally attenuated tumor invasion and angiogenesis, and cathepsin L or cathepsin B deficiency inhibited tumor proliferation (11). These observations highlight their potential as therapeutic targets in cancer treatment. Indeed, the application of synthetic broad-spectrum probes and combination therapies has successfully shown efficacy in vivo using various tumor models (12-15). However, given the roles that these proteases play in normal cellular homeostasis, an approach that selectively targets a specific cathepsin with limited normal tissue distribution may be more therapeutically attractive.Cathepsin S, unlike the ubiquitous cathepsin B and cathepsin L, exhibits a restricted tissue expression. It is found predominantly in lymphatic tissue, macrophages, and other professional antigenpresenting cells (16); mediating key steps in antigen presentation through cleavage of the invariant chain (17,18). However, the inappropriate expression of cathepsin S has also been observed in a range of tumors such as astrocytomas (19-21), prostate (22), hepatocellular (23), and pancreatic carcinomas (11). Crucially, evidence from the RIP1-T...

Antibody targeting of drug substances can improve the efficacy of the active molecule, improving distribution and concentration of the drug at the site of injury/disease. Encapsulation of drug substances into polymeric nanoparticles can also improve the therapeutic effects of such compounds by protecting the molecule until its action is required. In this current study, we have brought together these two rationales to develop a novel immuno-nanoparticle with improved therapeutic effect against colorectal tumor cells. This nanoparticle comprised a layer of peripheral antibodies (Ab) directed toward the Fas receptor (CD95/Apo-1) covalently attached to poly(lactide-co-glycolide) nanoparticles (NP) loaded with camptothecin. Variations in surface carboxyl density permitted up to 48.5 microg coupled Ab per mg of NP and analysis of nanoparticulate cores showed efficient camptothecin loading. Fluorescence visualization studies confirmed internalization of nanoconstructs into endocytic compartments of HCT116 cells, an effect not evident in NP without superficial Ab. Cytotoxicity studies were then carried out against HCT116 cells. After 72 h, camptothecin solution resulted in an IC 50 of 21.8 ng mL (-1). Ab-directed delivery of NP-encapsulated camptothecin was shown to be considerably more effective with an IC 50 of 0.37 ng mL (-1). Calculation of synergistic ratios for these nanoconstructs demonstrated synergy of pharmacological relevance. Indeed, the results in this paper suggest that the attachment of anti-Fas antibodies to camptothecin-loaded nanoparticles may result in a therapeutic strategy that could have potential in the treatment of tumors expressing death receptors.

Efforts to develop a better diagnostic assay for bovine tuberculosis have shown that the sensitivity and specificity of an assay can be improved by the use of two or more antigens. As reported here, we developed a multiplex chemiluminescent immunoassay that can simultaneously detect antibody activity to 25 antigens in a single well in a 96-well plate array format. The chemiluminescent signal is captured with a digital imaging system and analyzed with a macro program that tracks each serum for its pattern of antibody activity for Mycobacterium bovis antigens. The comparison of sera from 522 infected and 1,489 uninfected animals showed that a sensitivity of 93.1% and a specificity of 98.4% can be achieved with a combination of antigens. The assay system is rapid and can be automated for use in a centralized laboratory.Bovine tuberculosis (bTB) continues to be a zoonotic disease affecting multiple species, including humans (3, 9, 21, 26). The disease has been difficult to control in livestock because of the lack of an effective vaccine, the presence of wildlife reservoirs, and the lack of a diagnostic assay with sufficient sensitivity and specificity to detect animals at all stages of infection. Currently, the primary methods used for the detection of TB in humans and ruminants include the measurement of a delayedtype hypersensitivity (skin test) to purified protein derivative (PPD) and an indirect in vitro assay that measures the concentration of gamma interferon (IFN-␥) produced in response to stimulation with PPD (22,30,31). Although the methods have proven useful in controlling bTB, they lack sensitivity and specificity because of a cross-reactive immune response to Tand B-cell epitopes conserved on orthologous molecules present in nonpathogenic mycobacteria and Mycobacterium avium subsp. paratuberculosis (reviewed in references 8, 23, and 27). To obviate this problem, an extensive effort has been under way to identify and characterize antigens unique to Mycobacterium bovis that could be used in a diagnostic assay. To date, studies have shown that the antibody response to M. bovis is not uniform, with no evidence of a dominant persistent response to a single antigen (reviewed in references 4, 7, and 8) at any stage of infection (2,19). This finding has suggested that some type of a multiplex assay is needed to detect animals at different stages of infection (1, 2). However, the necessity of using multiple antigens in an assay has introduced another challenge. The evaluation of the standard type of enzymelinked immunosorbent assay (ELISA) has shown that sensitivity and specificity are reduced when multiple antigens are combined for analysis in a single well, thus limiting the way a conventional ELISA can be used (20). To address this problem, we developed a multiplex assay that can simultaneously detect and analyze the response to multiple antigens spotted in a single well in a 96-well plate array format. We demonstrate the enhanced diagnostic power of a multiplex antigen approach over that of the industry-stand...