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Background V-domain Immunoglobulin Suppressor of T cell Activation (VISTA/PD-1H) is a B7 family ligand expressed on circulating and intratumoural myeloid cells as well as Treg and NK cells. It has been shown to inhibit T cell responses in vitro and in preclinical models. In patients, VISTA is also a potential mediator of resistance to anti-CTLA-4 and anti-PD1 therapies and therefore is a valuable new target for cancer immunotherapy. Methods Kineta has analyzed 107 fully human ScFv antibodies directed against VISTA. Results Our lead candidates exhibit high potencies in the subnanomolar range and are also characterized by a long kDis. They specifically target human and cynomolgus monkey VISTA on a singular unique epitope. In a Staphylococcus Enterotoxin B T-cell activation assay, Kineta’s anti-VISTA antibodies efficiently induce IFNg secretion. They also promote strong maturation of Antigen Presenting Cells with an increase of CD80 and HLA-DR surface expression as well as CXCL10 secretion. The mechanism of action is mediated in part by NK cells. We demonstrated that myeloid cells acquire a high level of VISTA expression during MDSC or M2 differentiation in vitro and that Kineta’s anti-VISTA antibodies prevent the differentiation of MDSC as well as their immunosuppressive activity against T cells. Anti-VISTA antibodies mediate single-agent antitumor effects in syngeneic tumor models in wild-type mice and show enhanced activity in combination with anti-PD1 and anti-CTLA-4 treatment. Candidate anti-VISTA antibodies have also been evaluated in exploratory tolerability and PK studies in cynomolgus monkey. These studies demonstrated that multiple weekly doses of antibodies are well-tolerated with appropriate PK for lead selection and optimization. Conclusions Our results strongly favor further characterization and continued development of selected lead antibodies for the potential treatment of colder, less immunogenic tumors. Ethics Approval Study approved by the Institutional Animal Care and Use Committee PHS Assurance # D16-00885 and D16-00114

CD27 is a member of the TNF receptor superfamily and plays a critical role in T-cell activation by providing a costimulatory signal. CD27 signaling enhances T-cell proliferation, activation and differentiation of effector and memory T cells and therefore promotes cytotoxic T cell (CTL)-based anti-tumor immunity. Agonistic stimulation of CD27 is a promising cancer immunotherapy approach to boost specific T cell driven anti-tumor responses. In this study, we generated a series of 147 fully human monoclonal anti-CD27 antibodies and tested their agonist properties to stimulate T cell activation. Using a NF-κB reporter Jurkat cell line, we evaluated in vitro the ability of anti-CD27 antibodies to induce CD27 receptor activation. With this assay, five antibodies have been selected for their agonist properties. When combined with suboptimal T cell receptor (TCR) stimulation, agonist antibodies induced CD27 receptor activation with an EC50 of 1-5 ug/mL. We also used human peripheral blood T cells to characterize the CD27-mediated costimulatory effects of agonist antibodies in combination with TCR stimulation. Our anti-CD27 monoclonal antibodies boosted T cell proliferation and induced IL-2 and TNFalpha secretion only in a presence of TCR engagement. Moreover, CD27 agonists induce strong T cell proliferation in a Mixed Lymphocyte Reaction. CD27 antibodies were shown to bind human and cynomolgus monkey CD27 with a KD value of 5-20 nM as determined by BioLayer Interferometry, but do not bind to mouse CD27. In vivo experiments are currently ongoing to demonstrate the efficient anti-tumor activity of the selected CD27 agonist antibodies in different mice tumor models. In conclusion, we have developed and successfully selected efficient fully human immunostimulatory agonist CD27 mAbs as a promising cancer immunotherapy. Citation Format: Yulia Ovechkina, Shaarwari Sridhar, David Jurchen, David Peckham, Eric Tarcha, Shawn Iadonato, Thierry Guillaudeux. A promising cancer immunotherapy target: Novel fully human agonist antibodies against the human T-cell costimulatory receptor CD27 [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P035.

The Potassium Kv1.3 channel on effector memory T cells is a target to treat autoimmune diseases. ShK-186, a specific peptide inhibitor of Kv1.3, has shown promise in animal models of multiple sclerosis and rheumatoid arthritis. Here, we describe the development of methods to establish a pharmacokinetic (PK) - pharmacodynamic (PD) relationship for ShK-186, and test the hypothesis that the drug has a sustained PD effect in autoimmune disease models. The PK of ShK-186 was evaluated in rats and cynomolgus monkeys using HPLC-MS/MS and ELISA methods to measure the free fraction in plasma. These results were compared with SPECT/CT data collected with an 111In-DOTA-conjugate of ShK to assess whole blood PK parameters as well as the drug’s absorption, distribution and excretion. Our data suggest ShK-186 is absorbed slowly from the injection site resulting in blood concentrations above the Kv1.3 channel-blocking Kd for up to 7 days in monkey. PD studies on human peripheral blood mononuclear cells show that transient ShK-186 treatment results in a sustained suppression of cytokine responses and may contribute to prolonged drug effects. Moreover, in rat delayed-type hypersensitivity and chronic relapsing experimental autoimmune encephalomyelitis, a single dose of drug every 3 - 5 days is as effective as daily administration. These data suggest the PD effects of ShK-186 are governed primarily by slow distribution from peripheral tissues and long residence time on the Kv1.3 channel.