Immune cell activation by novel hexavalent CD40 agonist APG1233 compared to trimeric formats or agonistic anti-CD40 antibodies

• Published in: European journal of cancer (1990) Vol. 69; p. S99
• Main Authors: Richards, D.M, Merz, C, Sykora, J, Thiemann, M, Beyer, T, Kuehn, S, Fricke, H, Gieffers, C, Hill, O
• Format: Journal Article
• Language: English
• Published: Oxford Elsevier Science Ltd 01.12.2016
• Subjects: Adenomatous polyposis coli; Antibodies; Antigen-presenting cells; Biological activity; CD4 antigen; CD40 antigen; CD40L protein; CD69 antigen; CD86 antigen; Cell activation; Cell culture; Cell proliferation; Clustering; Culture media; Cytokines; Cytometry; Cytotoxicity; Enzyme-linked immunosorbent assay; Exposure; FasL protein; Flow cytometry; Growth media; Hematology, Oncology and Palliative Medicine; Immune system; Immunoglobulins; Immunotherapy; In vivo methods and tests; Inflammation; Interleukin 12; Lymphocytes; Lymphocytes B; Lymphocytes T; Macrophages; Markers; Modular engineering; Molecular chains; Pharmacology; Proteins; Real time; Stability; Surface markers; Tumor cells; Tumors
• ISSN: 0959-8049; 1879-0852
• DOI: 10.1016/S0959-8049(16)32893-3

Introduction: The co-stimulatory receptor CD40 is strongly expressed on B cells, monocytes and antigen-presenting cells (APC). By promoting their maturation, activation and survival, CD40 signaling greatly contributes to anti-tumor responses of the immune system. The HERA-Technology developed by Apogenix is a powerful engineering platform for the production of modular fusion proteins targeting the TNF-receptor super-family. Here we compared the efficacy of different CD40 agonist formats, including the novel hexavalent scCD40L-RBD-Fc (APG1233), and the functional consequences of differential receptor clustering. Materials and Methods: Immune cells were isolated from healthy-donor blood samples and profiled by multicolor flow cytometry (MC-FC). Subsequently, immune cells were cultured in growth media containing various forms of CD40 agonists. Upregulation of activation markers on B cells and monocytes (e.g. CD69, CD86, HLA-DR) and T cell-induced killing of tumor cells in direct co-culture was assessed by MC-FC and employing a real-time cell analysis system (xCELLigence), respectively. Secretion of cytokines in response to CD40 ligation and the pharmacokinetic properties of the fully human APG1233 and the chimeric murine/human APG1274 were determined by ELISA. Results: In vivo stability of APG1233 was demonstrated in a single dose mouse PK study revealing a terminal half-life of 84 hours. The chimeric surrogate molecule APG1274 which binds murine CD40 is eliminated much quicker (ti/2 of 4 hours) demonstrating the specificity of both compounds. In vitro only the hexavalent APG1233 efficiently stimulated B cells, monocytes and PBMCs. In contrast, neither trimeric CD40L nor an agonistic antibody against CD40 were able to upregulate expression of activation markers. Similarly, the secretion of proinflammatory cytokines such as IL-12, CD95L and IFNg by PBMCs was only stimulated after exposure to APG1233 and not in the presence of other CD40 agonists. In functional co-culture assays, after exposure to APG1233, in vitro generated M2-macrophages underwent conversion and acquired M1-type surface markers which strongly enhanced proliferation of naive CD4+ T cells. Consistent with these data, only APG1233 efficiently increased direct cytotoxic activity of immune cells against tumor cells measured by a realtime cell analysis assay. Conclusion: The CD40 agonist APG1233 is a member of a novel class of hexavalent TNFRSF agonists which binds its target with high specificity, exhibits excellent in vivo stability and superior biological activity over other agonistic formats (e.g. agonistic antibodies to CD40).