J, A2058 cells proliferation after incubation with -CSPG4 rIgE or -DNP rIgE (4?days). human therapeutic candidate. We generated a rat IgE against the human tumor-associated antigen chondroitin sulfate proteoglycan 4 (CSPG4) and cross-reactive for the rat antigen. We analyzed CSPG4 distribution in normal rat and human tissues and investigated the safety of the antibody by monitoring clinical indicators and molecular biomarkers after systemic administration to immunocompetent rats. Human and rat CSPG4 expression in normal tissues were comparable. Animals receiving antibody exhibited transient moderate to moderate adverse events accompanied by moderate elevation of serum tryptase, but not of angiotensin II or cytokines implicated in allergic reactions or cytokine storm. In the long term, repeated antibody administration was well tolerated, with no changes in animal body weight, liver and kidney functions or blood cell counts. This model provides preclinical support for the security profiling of IgE therapeutic antibodies. Due to the comparable antigen tissue distribution in human and rat, this model may also comprise an appropriate tool for proof-of-concept security evaluations of different treatment methods targeting CSPG4. KEYWORDS: IgE, rat model, immunotherapy, allergooncology, CSPG4, antibody, species cross-reactivity Introduction Using recombinant monoclonal antibodies (mAb) of the IgE class for immunotherapy of malignancy is an innovative approach that has shown promising results and IgE was injected into a cynomolgus monkey.12 The clinical translation of IgE immunotherapy in sound cancers is now moving forward, and further studies are needed to elucidate its safety profile and applicability across different tumor types and YHO-13177 target antigens. Chondroitin-sulfate proteoglycan 4 (CSPG4), also known as neuronal-glial antigen 2 (NG2), is YHO-13177 considered a promising candidate target for malignancy immunotherapy because of its diffuse and high level expression in a broad range of tumor types, such as melanoma, glioblastoma and subsets of breast carcinomas.25 Here, we designed a rat model to study the safety of a monoclonal IgE antibody directed against CSPG4. The distribution of human CSPG4 and its rat orthologue were evaluated in normal human and rat tissues. Taking advantage of the ability of a murine anti-CSPG4 antibody (clone 225.28) to cross-react with human CSPG4 and its rat orthologue, we generated a surrogate rat IgE mAb, -CSPG4 rIgE, and looked for immediate and long-term adverse effects in immunocompetent rats through analysis of clinical indicators and molecular biomarkers. Results CSPG4 distribution in rat and human normal tissues In order to test the relevance of a rat model in the context of the security of a CSPG4-targeted antibody, we compared the distribution of CSPG4 across a range of human and rat normal tissues. Comparable patterns of CSPG4 expression were observed between the two species (Physique 1ACL). In agreement with studies that have reported the expression of CSPG4 in oligodendrocyte progenitor cells of the central nervous system (CNS),26,27 we detected CSPG4-positive Rabbit polyclonal to AASS cells in rat and human cerebrum (Physique 1A,B). In lung and liver tissues, we observed scattered cells with YHO-13177 a moderate expression of CSPG4, whereas low expression was detected in pneumocytes (Physique 1E, F) and hepatocytes (Physique 1G, H). In line with previous studies that recognized CSPG4 as a marker of angiogenetic vasculature,28C30 we observed CSPG4 expression along blood vessels in rat and human uterus tissues (Physique 1I, J). Human bone marrow, thyroid gland and adenohypophysis showed moderate CSPG4 expression, whereas no expression was detected in human peripheral nerve, cerebellum and esophagus tissues (Supplemental Physique 2, data for the respective rat tissues are not available). Moreover, when comparing CSPG4 gene expression in normal tissues of four different species through interrogation of transcriptomic datasets (EMBL-EBI Expression Atlas), human and rat showed similar expression profiles (Physique 1M). Open in a separate window Physique 1. CSPG4 expression in normal human and rat tissues. (A-L) CSPG4 expression in rat (left) and human (right) tissues was investigated by immunohistochemistry of tissue microarrays using commercial anti-CSPG4 antibodies and developed using DAB chromogen. Hematoxylin was used to counterstain. Level bars symbolize 200 m (lower magnification) and 20 m (higher magnification). M, Histogram (left) summarizing CSPG4 gene expression (Transcripts Per Million, TPM) in the specified tissues of four different species based on the transcriptomic dataset (EMBL-EBI Expression Atlas, https://www.ebi.ac.uk/gxa/). Dataset and respective mRNA expressions are outlined in the table (right); n.a.: data not available. Open in a separate window Physique 2. In vitro characterization of -CSPG4 rIgE antibody. A, SDS-PAGE of.