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A promising strategy in cancer immunotherapy is activation of immune signalling pathways through antibodies that target co-stimulatory receptors. h IgG2, one of four human antibody isotypes, is known to deliver strong agonistic activity, and modification of h IgG2 hinge disulfides can influence immune-stimulating activity. This was shown for antibodies directed against the h CD40 receptor, where cysteine-to-serine exchange mutations caused changes in antibody conformational flexibility. Here we demonstrate that the principles of increasing agonism by restricting antibody conformation through disulfide modification can be translated to the co-stimulatory receptor h 4-1BB, another member of the tumour necrosis factor receptor superfamily. Furthermore, we explore structure-guided design of the anti- h CD40 antibody ChiLob7/4 and show that engineering additional disulfides between opposing F(ab’) arms can elicit conformational restriction, concomitant with enhanced agonism. These results support a mode where subtle increases in rigidity can deliver significant improvements in immunostimulatory activity, thus providing a strategy for the rational design of more powerful antibody therapeutics. Immunostimulatory antibodies have strong potential as anti-cancer therapeutics. Here Elliott et al. show that conformational rigidity determines agonistic activity of antibodies targeting CD40 and 4-1BB and demonstrate further enhancement of agonism through disulfide engineering.

Background Hypoxia is a hallmark of the tumor microenvironment (TME) and in addition to altering metabolism in cancer cells, it transforms tumor-associated stromal cells. Within the tumor stromal cell compartment, tumor-associated macrophages (TAMs) provide potent pro-tumoral support. However, TAMs can also be harnessed to destroy tumor cells by monoclonal antibody (mAb) immunotherapy, through antibody dependent cellular phagocytosis (ADCP). This is mediated via antibody-binding activating Fc gamma receptors (FcγR) and impaired by the single inhibitory FcγR, FcγRIIb. Methods We applied a multi-OMIC approach coupled with in vitro functional assays and murine tumor models to assess the effects of hypoxia inducible factor (HIF) activation on mAb mediated depletion of human and murine cancer cells. For mechanistic assessments, siRNA-mediated gene silencing, Western blotting and chromatin immune precipitation were utilized to assess the impact of identified regulators on FCGR2B gene transcription. Results We report that TAMs are FcγRIIb bright relative to healthy tissue counterparts and under hypoxic conditions , mononuclear phagocytes markedly upregulate FcγRIIb. This enhanced FcγRIIb expression is transcriptionally driven through HIFs and Activator protein 1 (AP-1). Importantly, this phenotype reduces the ability of macrophages to eliminate anti-CD20 monoclonal antibody (mAb) opsonized human chronic lymphocytic leukemia cells in vitro and EL4 lymphoma cells in vivo in human FcγRIIb + / + transgenic mice. Furthermore, post-HIF activation, mAb mediated blockade of FcγRIIb can partially restore phagocytic function in human monocytes. Conclusion Our findings provide a detailed molecular and cellular basis for hypoxia driven resistance to antitumor mAb immunotherapy, unveiling a hitherto unexplored aspect of the TME. These findings provide a mechanistic rationale for the modulation of FcγRIIb expression or its blockade as a promising strategy to enhance approved and novel mAb immunotherapies.

Background Hypoxia is a hallmark of the tumor microenvironment (TME) and in addition to altering metabolism in cancer cells, it transforms tumor-associated stromal cells. Within the tumor stromal cell compartment, tumor-associated macrophages (TAMs) provide potent pro-tumoral support. However, TAMs can also be harnessed to destroy tumor cells by monoclonal antibody (mAb) immunotherapy, through antibody dependent cellular phagocytosis (ADCP). This is mediated via antibody-binding activating Fc gamma receptors (Fc[gamma]R) and impaired by the single inhibitory Fc[gamma]R, Fc[gamma]RIIb. Methods We applied a multi-OMIC approach coupled with in vitro functional assays and murine tumor models to assess the effects of hypoxia inducible factor (HIF) activation on mAb mediated depletion of human and murine cancer cells. For mechanistic assessments, siRNA-mediated gene silencing, Western blotting and chromatin immune precipitation were utilized to assess the impact of identified regulators on FCGR2B gene transcription. Results We report that TAMs are Fc[gamma]RIIb.sup.bright relative to healthy tissue counterparts and under hypoxic conditions, mononuclear phagocytes markedly upregulate Fc[gamma]RIIb. This enhanced Fc[gamma]RIIb expression is transcriptionally driven through HIFs and Activator protein 1 (AP-1). Importantly, this phenotype reduces the ability of macrophages to eliminate anti-CD20 monoclonal antibody (mAb) opsonized human chronic lymphocytic leukemia cells in vitro and EL4 lymphoma cells in vivo in human Fc[gamma]RIIb.sup.+/+ transgenic mice. Furthermore, post-HIF activation, mAb mediated blockade of Fc[gamma]RIIb can partially restore phagocytic function in human monocytes. Conclusion Our findings provide a detailed molecular and cellular basis for hypoxia driven resistance to antitumor mAb immunotherapy, unveiling a hitherto unexplored aspect of the TME. These findings provide a mechanistic rationale for the modulation of Fc[gamma]RIIb expression or its blockade as a promising strategy to enhance approved and novel mAb immunotherapies. Keywords: Hypoxia, Hypoxia inducible factors, Fc[gamma]RIIb, Fc gamma receptors, Tumor-associated macrophages, Monocytes, Monoclonal antibody, Tumor microenvironment, Resistance, Cancer