Background/Purpose: Despite many recent advances in immunotherapy, graft versus host disease (GvHD), which reflects immune-mediated attack of recipient tissue by donor T cells, remains the major cause of morbidity and nonrelapse mortality after hematopoietic stem cell transplantation (HCT). The immunoglobulin superfamily (IgSF) is a large, diverse family of proteins that includes several key T cell-expressed members such as CD28, inducible T cell costimulator (ICOS), PD-1, and CTLA-4. CD28 and ICOS in particular play critical roles in T cell activation and adaptive immunity. Blockade of either pathway suppresses GvHD in syngeneic and xenograft mouse models; however, these two pathways are nonredundant and monotherapies directed against either pathway alone do not appear to be completely efficacious. Therefore, novel molecules that co-antagonize both CD28 and ICOS may provide more effective therapies for the prevention or treatment of GvHD. Methods: We used our proprietary variant Ig domain (vIgDTM) platform, which consists of directed evolution of select IgSF proteins, to create tailored ICOSL-Fc fusion proteins that can bind both ICOS and CD28 with high affinity, and co-inhibit both pathways. Mutant ICOSL-Fc molecules were evaluated in vitro with allogeneic mixed lymphocyte reactions (MLR) by co-culturing human pan T cells with activated human monocyte-derived dendritic cells. Lead candidate molecules were subsequently tested in multiple in vivo mouse models, including ovalbumin-induced delayed type hypersensitivity (DTH) and human xenograft PBMC-NSGTM GvHD. Results: ICOSL-Fc fusion proteins containing variant ICOSL domains significantly attenuate T cell activation in vitro when compared to wild-type (WT) ICOSL or belatacept, a clinically available CTLA4-Ig therapeutic, as assessed by suppressed proliferation and cytokine production in MLR, and ear swelling in DTH. In the human PBMC-NSG GvHD model, treatment with ICOSL-Fc significantly protected mice from the effects of xenogeneic T cell activation in vivo, with treated animals exhibiting greatly enhanced survival and reduced disease scores compared to WT ICOSL and belatacept. The level of protection in this model roughly correlated with the potency of the molecules in the in vitroMLR assay. Conclusion: The vIgD therapeutic platform has broad potential to generate potent biologics for the treatment of serious inflammatory conditions. Efficacy in vitro and in vivo of ICOSL-Fc vIgDs exceeds that of WT ICOSL-Fc or belatacept, correlating with their engineered increased affinities for cognate ligand (ICOS) and CD28. ICOSL-Fc vIgDs are therefore promising, novel therapeutic candidates for the prevention and/or treatment of GvHD. Preclinical development to enable clinical studies is underway.

Abstract Introduction: PD-1 pathway antagonists have revealed the importance of checkpoint pathways in regulating antitumor immunity, but an existing immune response is generally required for clinical efficacy. Specific T cell costimulation through CD28 is central to this process, but the CD28 ligands CD80 and CD86 are often poorly expressed in the tumor microenvironment, accounting for a second important mechanism of immune evasion by tumors. In contrast, PD-L1 expression has been found extensively in multiple tumor cell types. Therapeutics that combine PD-L1/PD-1 antagonism coupled with PD-L1 dependent CD28 agonism may therefore provide a more potent, yet safe immunotherapeutic approach. Experimental Procedures: The variant Ig Domain (vIgD)TM platform has generated a diversity of human CD80 variants using yeast display affinity maturation and selections against all three CD80 counterstructures CD28, CTLA-4, and PD-L1. CD80 vIgDs were produced in a mammalian expression system as recombinant Fc fusion proteins (CD80 vIgD-Fc proteins) and their binding properties were quantified by flow cytometry. Functional activity was determined in vitro by assessing responses from human primary T cells or an IL-2-luciferase Jurkat T cell reporter line stimulated with PD-L1-expressing artificial antigen presenting cells (aAPCs). In vitro human T cell cytotoxicity assays with a human PD-L1-expressing tumor line were also performed. Antitumor activity was assessed in vivo with mice implanted with human PD-L1 transduced MC38 tumors. Data Summary: The human CD80 IgV fragment was found to be optimal for high affinity PD-L1 and CD28 binding. A large panel of CD80 vIgD-Fc proteins demonstrated a range of binding towards CD28, PD-L1, and/or CTLA-4, and CD80 vIgD-Fc proteins with high affinity for PD-L1 antagonized the PD-L1/PD-1 interaction. Some CD80 vIgD-Fc proteins agonized CD28 in a PD-L1 dependent fashion with increased luciferase activity in the Jurkat reporter assay as well as increased cytokine production by primary human T cells when stimulated with a PD-L1 expressing aAPC in vitro. The same candidates also showed specific killing of human PD-L1 expressing tumor cells in vitro compared to the parental tumor line lacking PD-L1 expression. Importantly, selected CD80 vIgD-Fc proteins caused significant tumor reduction in the MC38 in vivo tumor model. Conclusion: Engineered CD80 vIgD-Fc proteins that deliver a localized CD28 costimulatory signal to T cells while simultaneously antagonizing the inhibitory PD-L1/PD-1 pathway may provide a transformative mechanism of action to drive potent, tolerable antitumor immunity. Preclinical development of therapeutic candidates is under way. Citation Format: Ryan Swanson, Mark F. Maurer, Chris L. Navas, Chelsea J. Gudgeon, Joseph L. Kuijper, Martin Wolfson, Katherine E. Lewis, Stacey R. Dillon, Steve D. Levin, Michael G. Kornacker. CD80 vIgD-Fc proteins combine checkpoint antagonism and costimulatory signaling for potent antitumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4550.

Abstract Background: The presence of tumor infiltrating lymphocytes (TILs) has been associated with improved prognosis in HER2+ breast cancer patients. Antigen specific TCR and costimulatory receptor signaling drive increases in TIL number, effector function, and tumor cytotoxicity. Improving the number and effector phenotype of tumor localized TILs has curative potential by enhancing the adaptive and memory immune response. Targeting HER2 with the monoclonal anti-HER2 antibody trastuzumabhas improved survival in HER2+ breast cancer patients and is known to increase peripheral type I immunity, which may be reflected by increased TILs. The Immunoglobulin Superfamily (IgSF) includes a large, diverse family of immunotherapy targets expressed on immune cells and tumors. Transmembrane IgSF receptors,CD28 and inducible T cell co-stimulator (ICOS), related costimulatory molecules expressed on T cells, interact with CD80/CD86 and ICOS ligand (ICOSL), respectively, and play critical roles in T cell activation and adaptive immunity.The Alpine Immune Science's VIgDTM platform uses directed evolution to derive novel, therapeutically-applicable IgSF extracellular domains with tailored specificity and affinity. The vIgD platform has generated human ICOSL vIgDs capable of binding both ICOS and CD28, activating both pathways. To promote anti-tumor activity of TILs in HER2+ tumors, we developed trastuzumab-ICOSL “V-mAbs” consisting of trastuzumab fused to activating ICOSL vIgDs. These V-mAbs are designed to localize to HER2+ tumors and activate antigen-specific, resident T-cells through costimulatory receptor agonism. Methods: V-mAbs were generated by fusing ICOSL vIgDs to either the N- or C- termini of the heavy and/or light chains of trastuzumab. V-mAb binding to CD28, ICOS or HER2 was measured by flow cytometric analysis of transfected cells or ForteBio analysis. V-mAb costimulatory activity was confirmed by immobilization in the presence of anti-CD3 in a primary human T cell assay. Finally, V-mAbs were co-cultured with HER2+ target cells and human T cells; T-cell activity was measured by proliferation, cytokine production, and target lysis. Results: V-mAbs were successfully produced and bound to CD28, ICOS and HER2. In a plate bound costimulation assay, the V-mAbs increased the amount of IFN-gamma produced by T-cells stimulated with anti-CD3. When incubated with HER2+ target cells, V-mAbs promoted T-cell proliferation, cytokine secretion, and target cell lysis. Data from in vivo studies, to determine the impact of trastuzumab V-mAbs on HER2+ cancers, will be presented when available. Conclusions: Trastuzumab-ICOSL V-mAbs are novel ICOS- and CD28-activating immunotherapies for HER2-positive tumors, promoting T-cell proliferation, cytokine secretion, and target cell lysis in a HER2 dependent fashion. The V-mAb platform has broad potential to enable tumor-localized immune modulation via the diverse array of IgSF members. Preclinical development of trastuzumab-ICOSL clinical therapeutics is in progress. Citation Format: Rickel E, Evans L, Swanson R, Levin SS, Rixon M, Wolfson M, Bhandari J, MacNeil S, Hoover J, Kornacker M, Capuano I, Peng SL. ICOSL anti-HER2 V-mAbs: Localizing engineered ICOSL costimulatory agonists to HER2+ tumors through trastuzumab [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-09-10.

Immunoglobulin superfamily member (IgSF) proteins play a significant role in regulating immune responses with surface expression on all immune cell subsets, making the IgSF an attractive family of proteins for therapeutic targeting in human diseases. We have developed a directed evolution platform capable of engineering IgSF domains to increase affinities for cognate ligands and/or introduce binding to non-cognate ligands. Using this scientific platform, ICOSL domains have been derived with enhanced binding to ICOS and with additional high-affinity binding to the non-cognate receptor, CD28. Fc-fusion proteins containing these engineered ICOSL domains significantly attenuate T cell activation in vitro and in vivo and can inhibit development of inflammatory diseases in mouse models. We also present evidence that engineered ICOSL domains can be formatted to selectively provide costimulatory signals to augment T cell responses. Our scientific platform thus provides a system for developing therapeutic protein candidates with selective biological impact for treatments of a wide array of human disorders including cancer and autoimmune/inflammatory diseases.

The immunoglobulin superfamily (IgSF) is a large, diverse family of proteins extensively targeted for treatment of cancers and autoimmune diseases. Most of the therapeutic strategies targeting this family have focused on high affinity antibodies binding a single receptor. Wild-type IgSF receptors typically exhibit low affinities for their counter-structures, limiting their utility in therapeutic modulation of immune responses. We have developed a novel variant Ig domain™ (vIgD™) directed evolution platform to affinity mature human IgSF extracellular domains. In this platform, libraries of mutagenized IgSF domains are selected for altered affinity to specific recombinant protein counterstructures. Fc fusion proteins incorporating the resulting engineered IgSF domains are then tested in vitro for their ability to either agonize or antagonize T cell responses.