B cells contribute to innate and adaptive immunity. In the former, Toll-like receptor (TLR) activation promotes the expansion of inflammatory B cells. In the latter, B cell receptor (BCR) activation results in the production of antibodies or autoantibodies. Antigen processing and presentation are closely associated with major histocompatibility class II (MHC-II) and its companion protein, class II invariant peptide (CLIP). The impact of autophagy on the regulation of these unique mechanisms of B cell activation and subset expansion has not been fully explored. The results from the current study show that activating autophagy with rapamycin (RAPA) or inhibiting autophagy with hydroxycholoroquine (HCQ) differentially influences the TLR9 and BCR activation of B cells. These differences include the selective expansion of B1 and B2 B cell subsets, the regulation of the cell-surface expression of MHC-II and CLIP, and the ability of distinct B cell subsets to present peptide antigens. These novel findings demonstrate that the unique B cell activation mechanisms induced by TLR9 and BCR activation are differentially influenced by RAPA and HCQ, owing to the selective modulation of B cell subset expansion, and antigen processing and presentation by MHC-II proteins.

Study of Bcl-xl and hepatocyte growth factor (HGF) receptor c-Met demonstrates that ETS transcription factors influence the transcriptional control of Bcl-xl in multiple tumors. The phosphorylation of Tel, a transcriptional repressor in the ETS family, by activated c-Met is required to increase Bcl-xl transcription in addition to activation of ETS2, a transcriptional activator. Our data indicate that stimulation by HGF causes the activation of ERK and leads to the nuclear export of Tel. In order to determine whether ERK is responsible for the phosphorylation of Tel after HGF stimulation, physical interaction between Tel and ERK was detected using I.P. (Tel)-western blotting (ERK). To determine if blocking MAP kinase could prevent Tel's phosphorylation after HGF exposure, we again used I.P. Western to compare the phosphorylation of Tel under specific ERK kinase inhibitors, after HGF exposure, to the phosphorylation of Tel in the absence of inhibitors, after HGF exposure. A His-Tel 213mutant (phosphorylation sites mutants) was created. The Tel 213 mutant did not undergo phosphorylation induced by c-Met activation, as demonstrated by I.P. Western blot. Nuclear localization of mutated His-Tel was detected under HGF/EGF exposure via fluorescent microscopy using FITC labeled anti-his antibody, indicating that phosphorylation of Tel is also required for nuclear export. A luciferase reporter assay shows that the transient expression of Tel 213 mutant in cancer cell lines results in further repression of Bcl-xl promoter activity. Indeed, Tel 213's repressive function is independent of c-Met stimulation, as determined by comparing with wild type Tel cDNA transfected cells. Consequently, Tel 213 transfection results in much stronger reduction in Bcl-xl mRNA and protein expression levels in the presence of HGF, as compared to transfection with wild-type Tel cDNAs in the presence of HGF. Furthermore, a CHIP assay demonstrates that the activation of c-Met reduced the binding of wild-type Tel to the Bcl-xl promoter, while the binding of Tel 213 to bcl-xl promoter was not affected. Finally, transfection of Tel 213 leads to increased cell proliferation inhibition and apoptosis. Taken together, these findings are supportive of a role for Tel, through a c-Met aixs, as an inhibitor of Bcl-xl transcription. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4979.

Identification of novel targets in cancer immunotherapy is needed to address the significant number of patients that either do not respond to current therapies or encounter unacceptable toxicities. The first two generations of immuno-oncology drugs have been antagonist antibodies against immune checkpoint proteins, such as cytotoxic T lymphocyte protein 4 (CTLA-4) and programmed cell death protein 1 (PD-1). Moving forward, there has been progress in targeting co-stimulatory receptors like inducible T cell co-stimulator (ICOS), OX40 and CD137 with agonist antibodies. At Oxford BioTherapeutics, in-depth expression profiling of membrane proteins from intact tumors collected in the proprietary OGAP database revealed novel IO targets in primary tumor-derived lymphocytes (TILs). Proteomic and flow cytometry analysis of TILs and PBMCs establish that OX003R is a novel co-stimulatory IO target. It is expressed on naïve T and B cells; however, higher expression is observed in TILs and activated or exhausted T cells. OX003R expression is observed by immunohistochemistry in infiltrating lymphocytes in a variety of solid tumor types. A Fab phage display library was screened by FACS for binding to target on the cell surface. All the Fabs were also profiled by an interferon gamma release assay for T cell activation. Five best binders demonstrating T cell activation were reformatted into full-length chimeric mAbs and expressed in mammalian Expi 293 cells. Recombinant antibodies were extensively screened for T cell activation in an ex vivo 3D tumor culture system developed in-house using fresh non-small cell lung and colorectal carcinomas. Interferon gamma release was assessed by ELISpot assay and expression of the target was confirmed by immunohistochemistry on the corresponding tumor samples. Chimeric antibody 1B3 robustly activated T cells in most of the tumor samples in a dose-dependent manner as compared to isotype control and was chosen as the lead therapeutic antibody for humanization. The lead therapeutic antibody was tested for the propensity to facilitate the undesirable cytokine storm in whole blood and did not induce the release of dangerous levels of cytokines. Conclusion: OX003R is a validated immuno-oncology target and chimeric 1B3 is being developed as a promising therapeutic antibody with agonistic TIL activity, specifically in the tumor microenvironment. Citation Format: Arnima Bisht, Angelo Kaplan, Livija Deban, Chander Sekhar Peddaboina, Murray Cox, Lindsey Hudson, James Ackroyd, Nickolas Attanasio, San Lin Lou, Jason Allen, Martin Barnes, Robert Boyd, Eugene Zhukovsky, Abderrahim Fandi, Christian Rohlff. Identification and validation of a novel immuno-oncology target and selection of a therapeutic antibody candidate with a pharmacologically beneficial activity profile [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5167.