BAFF and APRIL are TNF superfamily members that bind both TACI and BCMA on B cells; BAFF also binds BAFF-R. Together, BAFF and APRIL support B cell development, differentiation, and survival. Their co-neutralization dramatically reduces B cell function, including antibody production, whereas inhibition of either BAFF or APRIL alone mediates relatively modest effects.While CTLA-4-based therapeutics that block T cell costimulation provide safe and moderately effective T cell inhibition in many disease settings, and while B cell targeting therapies have demonstrated promising therapeutic potential, we postulate that improved, combined BAFF and APRIL inhibition, either alone or coupled with inhibition of T cell costimulation, will provide more effective and durable relief from severe B cell-related autoimmune diseases like SLE.We used our directed evolution platform to identify variant domains of the TNF family receptors TACI or BCMA that exhibit enhanced affinity for BAFF and APRIL as compared to their wild-type (WT) counterparts. These variant TACI or BCMA domains (vTD), alone or together with platform-derived CTLA-4 domains (vIgD), were fused to a modified human IgG1 Fc lacking effector function, yielding a panel of immunomodulatory molecules: TACI vTD-Fc, BCMA vTD-Fc, TACI vTD/CTLA-4 vIgD-Fc, & BCMA vTD/CTLA-4 vIgD-Fc. All were evaluated for functional activity: 1)in vitroin primary human B cell & MLR assays and in a Jurkat/NF-kB reporter cell line expressing TACI, and 2)in vivoin standard immunization models, and in the bm12-induced and NZB/NZW spontaneous mouse models of lupus.The novel engineered TACI vTD-Fc or BCMA vTD-Fc fusion proteins significantly inhibited BAFF- and APRIL-mediated signalingin vitroin TACI+Jurkat cells. TACI (or BCMA) vTD/CTLA-4 vIgD-Fc proteins also attenuated T cell activation in primary human lymphocyte assays. When administered to mice, these molecules rapidly and potently reduced key B and T cell subsets, including plasma cells, follicular T helper cells, germinal center cells, & memory T cells. Treatment with TACI vTD-Fc or TACI vTD/CTLA-4 vIgD-Fc proteins also significantly reduced titers of antigen-specific antibodies in immunized mice more so than abatacept or WT TACI-Fc, and potently suppressed anti-dsDNA autoantibodies, blood urea nitrogen levels, proteinuria, and renal immune complex deposition in the bm12 & NZB/W lupus models.Directed evolution of TNFR and IgSF domains has successfully facilitated the development of Fc fusion proteins containing TACI or BCMA vTDs, with or without fusion to CTLA-4 vIgDs. These novel immunomodulators consistently demonstrate potent immunosuppressive activity and efficacyin vitroandin vivo, appearing superior to existing and/or approved immunomodulators like belimumab, abatacept, or atacicept. Such biologics may therefore be attractive candidates for the treatment of serious autoimmune diseases, particularly B cell-related diseases such as SLE, Sjogren’s syndrome, etc.Stacey R. Dillon Shareholder of: Shareholder of Alpine Immune Sciences, Inc., Employee of: Employee of Alpine Immune Sciences, Inc., Lawrence S. Evans Shareholder of: Shareholder of Alpine Immune Sciences, Inc., Employee of: Employee of Alpine Immune Sciences, Inc., Mark W. Rixon Shareholder of: Shareholder of Alpine Immune Sciences, Inc., Employee of: Employee of Alpine Immune Sciences, Inc., Joe Kuijper Shareholder of: Shareholder of Alpine Immune Sciences, Inc., Employee of: Employee of Alpine Immune Sciences, Inc., Dan Demonte Shareholder of: Shareholder of Alpine Immune Sciences, Inc., Employee of: Employee of Alpine Immune Sciences, Inc., Katherine E. Lewis Shareholder of: Shareholder of Alpine Immune Sciences, Inc., Employee of: Employee of Alpine Immune Sciences, Inc., Steve Levin Shareholder of: Shareholder of Alpine Immune Sciences, Inc., Employee of: Employee of Alpine Immune Sciences, Inc., Kayla Kleist Shareholder of: Shareholder of Alpine Immune Sciences, Inc., Employee of: Employee of Alpine Immune Sciences, Inc., Sherri Mudri Shareholder of: Shareholder of Alpine Immune Sciences, Inc., Employee of: Employee of Alpine Immune Sciences, Inc., Susan Bort Shareholder of: Shareholder of Alpine Immune Sciences, Inc., Employee of: Employee of Alpine Immune Sciences, Inc., Janhavi Bhandari Shareholder of: Shareholder of Alpine Immune Sciences, Inc., Employee of: Employee of Alpine Immune Sciences, Inc., Fariha Ahmed-Qadri Shareholder of: Shareholder of Alpine Immune Sciences, Inc., Employee of: Employee of Alpine Immune Sciences, Inc., Jing Yang Shareholder of: Alpine Immune Sciences, Inc., Employee of: Alpine Immune Sciences, Inc., Michelle A. Seaberg Shareholder of: Shareholder of Alpine Immune Sciences, Inc., Employee of: Employee of Alpine Immune Sciences, Inc., Rachel Wang Shareholder of: Shareholder of Alpine Immune Sciences, Inc., Employee of: Employee of Alpine Immune Sciences, Inc., Russell Sanderson Shareholder of: Alpine Immune Sciences, Inc., Employee of: Alpine Immune Sciences, Inc., Martin F. Wolfson Shareholder of: Shareholder of Alpine Immune Sciences, Inc., Employee of: Employee of Alpine Immune Sciences, Inc., Jan Hillson Shareholder of: Alpine Immune Sciences, Inc., Employee of: Alpine Immune Sciences, Inc., Stanford L. Peng Shareholder of: Alpine Immune Sciences, Inc., Employee of: CMO and President of Alpine Immune Sciences, Inc., Kristine M. Swiderek Shareholder of: Shareholder of Alpine Immune Sciences, Inc., Employee of: Employee of Alpine Immune Sciences, Inc.

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.

BACKGROUND:Increased levels of serum IgE are associated with greater asthma prevalence and disease severity. IgE depletion using an anti-IgE monoclonal antibody has met with success in the treatment of moderate-to-severe and severe persistent allergic asthma.OBJECTIVE:To test whether B cell-targeted therapy is a more effective treatment for airway hyperresponsiveness (AHR) in a murine model compared with IgE-depletion.METHODS:We delivered soluble mTACI-Ig, a receptor for the B cell survival factors BLyS (B Lymphocyte Stimulator) and APRIL (A PRoliferation-Inducing Ligand), or anti-IgE to allergen-sensitized mice before airway challenge with allergen.RESULTS:mTACI-Ig treatment reduced circulating mature B cell levels in the blood, while anti-IgE treatment had no effect on B cell counts. Both mTACI-Ig and anti-IgE decreased the levels of total and allergen-specific IgE in the serum. Histopathologic analysis of lungs showed a reduction in disease severity scores for both treatment groups, but results were more pronounced in mTACI-Ig-treated mice. Neutrophil and eosinophil numbers in the bronchoalveolar lavage (BAL) were significantly reduced following mTACI-Ig treatment, but not after anti-IgE delivery. BLyS and APRIL blockade also resulted in a significant decrease in IL-4 and eotaxin mRNA and IL-4 and KC protein levels in total lung homogenates and BAL fluid, respectively. Finally, mTACI-Ig treatment was more effective than anti-IgE treatment in reducing AHR to inhaled antigen.CONCLUSIONS:Our data demonstrate that delivery of mTACI-Ig is a more effective treatment than anti-IgE mAb in a murine model of AHR.

BLyS and APRIL have similar but distinct biological roles, mediated through two known TNF receptor family members, TACI and BCMA. We show that mice treated with TACI-Ig and TACI-Ig transgenic mice have fewer transitional T2 and mature B cells and reduced levels of circulating immunoglobulin. TACI-Ig treatment inhibits both the production of collagen-specific Abs and the progression of disease in a mouse model of rheumatoid arthritis. In BLyS-deficient mice, B cell development is blocked at the transitional T1 stage such that virtually no mature B cells are present, while B-1 cell numbers are relatively normal. These findings further elucidate the roles of BLyS and APRIL in modulating B cell development and suggest that BLyS is required for the development of most but not all mature B cell populations found in the periphery.