ACR ANNUAL MEETING » NOVEMBER 8-13 » ATLANTA, GA, USA 0.00 0.25 0.50 0.75 1.00 GC cell #/spleen (x10 6 ) # GC B cells/spleen INTRODUCTION: ALPN-101 is an Fc fusion protein of a human inducible T cell costimulator ligand (ICOSL) variant immunoglobulin domain (vIgD™) designed to inhibit simultaneously the CD28 and ICOS costimulatory pathways. CD28 and ICOS each play a role in T cell activation and adaptive immunity which can contribute to autoimmune disease when dysregulated. ALPN-101 has previously been shown to have potent immunosuppressive activity in various in vitro and in vivo models of disease, including acute graft versus host disease, inflammatory arthritis, and multiple sclerosis. We report here in vitro assays using peripheral blood mononuclear cells (PBMC) from healthy donors vs. patients to analyze human T cell and B cell activation and suppression of antibodies and inflammatory mediators thought to contribute to the pathogenesis of Sjögren’s syndrome (SjS) and other connective tissue diseases. Additionally, the efficacy of ALPN-101 was confirmed in vivo in mouse immunization models, and in mouse models of SjS and systemic lupus erythematosus (SLE). METHODS: Primary cell assays were performed with healthy donor and SjS patient PBMC stimulated with K562 cells expressing CD80, CD86, ICOSL, and anti-CD3 (OKT3) to evaluate the potency of ALPN-101 to suppress cytokine production and alter gene expression. The activity of dual pathway inhibition by ALPN-101 was compared to the CD28-only inhibitor abatacept (CTLA4-Ig; Bristol-Myers Squibb, via Catalent) and to the ICOS pathway inhibitor prezalumab (AMG-557/anti-ICOSL, Creative Biolabs), or a combination of the two. ALPN-101 was compared to abatacept in vivo in standard mouse immunization models (KLH, sheep RBC), in a model of SjS involving anti PD-L1 antibody-mediated acceleration of sialadenitis in non-obese diabetic (NOD) mice, and in the bm12 inducible model of lupus. RESULTS: Compared to abatacept, prezalumab, or combination abatacept + prezalumab, ALPN-101 demonstrated superior suppression of pro-inflammatory cytokine (i.e. TNFα, IFNγ, IL-1β, IL-2, IL-6, IL-17A, GM-CSF, etc.) release from stimulated healthy SjS, or SLE patient PBMCs (Fig. 3). ALPN-101 treatment reduced germinal center (GC) B cells and follicular helper T cells (T FH ) and inhibited antibody production in vivo in mouse immunization models (not shown) and in the bm12 model (Fig. 9), and suppressed proliferation and antibody production in human B cell/T FH cell co-cultures (Fig. 5). In anti PD-L1-treated NOD mice, ALPN-101 suppressed sialadenitis, insulitis, blood glucose levels, and autoantibodies with activity often superior to that of abatacept (Fig. 6-8, and data not shown). CONCLUSION: The efficacy of dual CD28/ICOS antagonist ALPN-101 is superior to CD28 or ICOS costimulatory pathway inhibitors, administered individually or in combination, in human in vitro and/or mouse in vivo translational studies. A Phase 1 clinical trial with ALPN-101 in healthy volunteers is ongoing (NCT03748836), and trials in inflammatory diseases are planned. Stacey R. Dillon, Lawrence S. Evans, Katherine E. Lewis, Susan Bort, Erika Rickel, Jing Yang, Martin F. Wolfson, Kayla Susmilch, Sherri Mudri, Steven D. Levin, Janhavi G. Bhandari, Fariha Ahmed-Qadri, Mark W. Rixon, Jan L. Hillson, Stanford L. Peng, and Kristine M. Swiderek Alpine Immune Sciences, Inc. Seattle, WA ALPN-101, a First-in-Class Dual ICOS/CD28 Antagonist, Suppresses Key Effector Mechanisms Associated with Sjögren’s Syndrome and Systemic Lupus Erythematosus Abstract Summary and Conclusions Figure 1: ALPN-101 (ICOSL vlgD-Fc), Generated Using the vIgD™ Directed Evolution Strategy, Blocks Both CD28 and ICOS T Cell Costimulation Pathways Figure 3: ALPN-101 Inhibits Cytokine Production from Human Sjögren’s and SLE Patient PBMC In Vitro More Potently Than Single CD28 or ICOS Pathway Inhibitors Sponsor: Alpine Immune Sciences, Inc. AlpineImmuneSciences.com @AlpineImmuneSci © 2019 Alpine Immune Sciences. All rights reserved. Figure 7: ALPN-101 Reduces the Incidence and Severity of Sialadenitis in NOD Mice Enrolled in the Anti PD-L1 mAb-Induced Model of Sjögren’s Syndrome Figure 8: ALPN-101 Reduces Serum Autoantibody Titers in the Anti PD-L1 mAb-Induced NOD Mouse Model of Sjögren’s (A) Autologous circulating human B and TFH cells isolated from PBMC from healthy donors were mixed with artificial APCs (K562/CD80) and soluble OKT3, and titrations of the test articles indicated. Cultures were assessed on Day 7 for cellular activation/proliferation and IC50 values are summarized in the table. (B) In a separate study using a similar protocol, supernatants were collected on Day 8, diluted 1:10, and assayed for human immunoglobulin (Ig) secretion. Data in (B) are representative of results from 8 donors. (A) In vitro stimulation of healthy donor, Sjögren’s syndrome (SjS), and SLE patient PBMC with artificial APC [fixed K562 expressing cell surface OKT3 (anti CD3)/CD80/ CD86/ICOSL)] at a 20:1 ratio. Test articles were added at 100 nM and supernatants were collected and assayed for cytokine concentrations after 48 hr incubation. (B) IFNγ, IL-6, and IL-17A levels are shown as examples, though similar trends were observed for all cytokines measured, as summarized in C. (C) ALPN-101 demonstrated statistically significant superiority to prezalumab, abatacept, or a combination of prezalumab+ abatacept for the majority of donors and analytes tested. 2416 Figure 2: ALPN-101 Binds CD28 and ICOS and Blocks Binding to Their Ligands, Impacting B Cell/T Cell Collaboration During Antibody Responses Figure 6: The Anti-PD-L1 mAb-Induced NOD Mouse Model of Sjögren’s Syndrome 0 1 2 3 4 Histology Score (0-4) * **** ** * A mouse model of Sjögren’s syndrome induced in female diabetes-prone NOD/ShiLtJ mice using repeat dosing (on Days 0, 2, 4, and 6) with anti-mouse PD-L1 antibody (based on a modified version of a protocol published by Zhou et al., 2016 5 ) was used to evaluate the impact of ALPN-101 and comparator treatments on the development of sialadenitis and insulitis, the levels of blood glucose, serum autoantibodies, and gene expression in the submandibular gland (SMG). 0 20 40 60 80 % Incidence of Sialadenitis n=20 n=20 n=20 n=20 n=19 n=3 Fc Control ALPN-101 Abatacept WT ICOSL-Fc Aba + WT Naï ve CD28/CTLA-4 inhibition ICOS-only inhibition CD28+ICOS dual blockade 10 100 1000 10000 1000001000000 0 10000 20000 30000 40000 50000 CD28 [ pM ] Median Fluorescence WT ICOSL ALPN-101 Abatacept Belatacept Prezalumab 100 1000 10000 100000 1000000 0 2500 5000 7500 10000 CD80 CD28 [ pM ] Median Fluorescence Abatacept Belatacept ALPN-101 10 100 1000 10000 1000001000000 0 20000 40000 60000 ICOS [ pM ] Median Fluorescence WT ICOSL ALPN-101 Abatacept Belatacept Prezalumab 100 1000 10000 100000 1000000 0 25000 50000 75000 ICOSL ICOS [ pM ] Median Fluorescence WT ICOSL Prezalumab ALPN-101 Binding to Targets Blockade of Ligand Binding • ALPN-101 (ICOSL vIgD-Fc) is a dual CD28 and ICOS T cell co-stimulation pathway inhibitor targeting both naïve and activated pathogenic T cells, including ICOS+ cells that escape currently available CD28 pathway inhibitors. 7 • ALPN-101 inhibits cytokine production in vitro from human Sjögren’s and SLE patient PBMC, more potently than single CD28 or ICOS pathway inhibitors. • ALPN-101 affects genes involved in B cell differentiation and suppresses proliferation and antibody responses in vitro in human B cell-T FH cell co-cultures. • ALPN-101 suppresses anti-SRBC (and anti-KLH, not shown) antibody responses in vivo in normal mice, and reduces autoantibody titers in mouse models of Sjögren’s syndrome and SLE. • ALPN-101 reduces the incidence and severity of sialadenitis and insulitis, and reduces blood glucose levels (Fig. 7 and data not shown), in NOD mice enrolled in the anti PD-L1-induced model of Sjögren’s syndrome. • ALPN-101 is a novel therapeutic candidate for Sjögren’s syndrome, SLE, and potentially other connective tissue and/or serious autoimmune diseases. A phase 1 clinical trial with ALPN-101 in healthy volunteers is ongoing (NCT03748836), and trials in inflammatory diseases are planned. 1. Panneton et al. (2019) Inducible T cell Costimulator: Signaling Mechanisms in T Follicular Helper Cells and Beyond. Immunol Rev. 291:91-103 2. Paulos et al. (2010) The Inducible Costimulator (ICOS) is Critical for the Development of Human TH17 Cells. Sci Transl Med. 2(55):55ra78. 3. Weber et al. (2015) ICOS Maintains the T Follicular Helper Cell Phenotype by Down-Regulating Krüppel-Like Factor 2. J Exp Med. 212(2):217-33. 4. Evans et al. (2019) ALPN-101, a First-in-Class Dual ICOS/CD28 Antagonist, Suppresses Key Effector Mechanisms Underlying Rheumatoid and Psoriatic Arthritis. Poster #1531, American College of Rheumatology Annual Meeting 2019; Atlanta, GA. 5. Zhou et al. (2016) Endogenous Programmed Death Ligand-1 Restrains the Development of Sjögren’s Syndrome in Non-Obese Diabetic Mice. Sci Rep. 6: 39105. 6. Klarquist & Janssen (2015) The bm12 Inducible Model of Systemic Lupus Erythematosus (SLE) in C57BL/6 Mice. J Vis Exp. (105): e53319. 7. Dillon et al. (2019) ALPN-101, a Dual ICOS/CD28 Antagonist, Demonstrates Potent and Dose-Dependent Suppression of Graft vs. Host Disease (GvHD) in a Human/NSG™ Mouse Xenograft Model, with Activity Superior to CD28 or ICOS Single Pathway Antagonists. Poster #426, Transplantation & Cellular Therapy Meeting of ASBMT and CIBMTR, February 2019, Houston, TX. References IC50 17.2 nM 23.3 nM 4.3 nM Figure 9: ALPN-101 Reduces GC B and T FH Cells and Suppresses Antibody Responses in Normal Mice and in the bm12 Inducible Mouse Model of SLE Anti-mouse PD-L1 mAb (0.1 mg), IP • Dose with anti-mPD-L1 mAb (0.1 mg) • Treat with 0.3 mg Fc control, 0.5 mg ALPN-101, abatacept, or WT ICOSL-Fc, or 0.5 mg aba + 0.5 mg WT ICOSL-Fc NOD/ShiLtJ female, 6 wks of age Day 10 Day 10 Harvest: • SMG (histology, RNA-Seq) • Pancreas (histology) • Blood/serum (glucose, PK) ALPN-101 (0.5 mg), IP Day 0 Day 2 Day 4 Day 6 0 40000 80000 120000 140000 180000 IFN Response Donor Type IFN (pg/mL) SjS SLE HD 0 5000 10000 15000 25000 30000 IL-6 Response Donor Type IL-6 (pg/mL) SjS SLE HD 0 800 1600 2400 3000 6000 IL-17A Response Donor Type IL-17A (pg/mL) SjS SLE HD Fc control Prezalumab Abatacept Abatacept+Prezalumab ALPN-101 Anti ICOSL mAb (prezalumab) ICOS only Abatacept (CTLA4-Fc) CD28 only ALPN-101 Monotherapy ICOS+CD28 Systemic Lupus Erythematosus Sjögren’s Syndrome Healthy Donors % Inhibition vs. Fc (B) Examples of cytokine responses in the PBMC stimulation assay (C) Summary of cytokine responses in the PBMC stimulation assay (A) Diagram of the PBMC stimulation assay Artificial APC K562/OKT3/CD80/CD86/ICOSL T cell CD28 ICOS CD80/86 ICOSL OKT ScFv Target(s) Antagonists ICOSL Prezalumab CD80/ CD86 Abatacept Belatacept ALPN-101 CD28 ICOS (A) BALB/c mice were immunized with SRBC on Day 0 (D0). Half the mice were dosed on D-1 & D7, and half on D14 & D21 with 1.5 mg ALPN-101 or abatacept, or a molar-matched amount of Fc control. The second group was boosted with SRBC on D15 and all mice were euthanized 1 wk after their 2 nd dose (on D14 and D28, respectively). Serum anti-SRBC antibodies on D14 and D28 were measured via flow cytometry by detecting binding to SRBC using isotype-specific anti-mouse Ig Abs. p values were determined using a ordinary one-way ANOVA with Tukey's multiple comparisons test. (B) Spleens were harvested on D14 from the first cohort of mice (treated on D-1 & D7) and analyzed by flow cytometry. (C) Per a published protocol 6 , splenocyte suspensions from female I-A bm12 B6(C)-H2-Ab1 bm12 /KhEgJ (‘bm12’) mice were injected IP into an equivalent number (1:1) of 9 wk old female C57BL/6NJ recipient mice on Day 0. H2-Ab1 bm12 differs from H2-Ab1 b by 3 amino acids in the β-chain of the I-A molecule. Alloactivation of donor CD4+ T cells by recipient APC leads to a cGvHD disease with symptoms resembling SLE. Starting on Day 0, recipient mice were treated 2x/wk for 11 wks with 400 µg ALPN-101 or abatacept, or a molar-matched amount (250 µg) of Fc control protein. At study termination on Day 82, GC B cells and TFH cells were enumerated by flow cytometry. Anti-double stranded (ds) DNA IgG titers were measured on D14 using the Mouse Anti-dsDNA IgG Antibody Assay kit from Chondrex (Redmond WA). Patient or healthy donor peripheral blood mononuclear cells (PBMC) were stimulated with fixed artificial APCs (shown at right) for 48 hrs in the presence of ALPN-101 (100nM) or comparators: o Prezalumab (anti ICOSL mAb) o Abatacept alone (CTLA4-Ig) o Abatacept + prezalumab APC Antigen CD28+ ALPN-101 Inhibition of T cell activation Inhibition of B cell activation and Ab production ALPN-101 ICOS+ Activated TFH cell Existing TFH cell ICOS+ Memory B cells Antibody- secreting plasma cells Naïve T cell B cell o Primary immune responses depend heavily upon the CD28 pathway o Secondary immune responses (e.g. memory, effector) can be CD28-independent. o ICOS is upregulated upon T-cell activation and is the most closely related protein to CD28, yet plays a non-redundant function in many immune responses, such as follicular helper T cell (TFH) differentiation and function. 1,3 o ICOS+ T cells escape treatment with drugs only blocking the CD28/CTLA-4 pathway, like abatacept. Our Thesis : Blockade of both CD28 and ICOS pathways by ALPN-101 should provide superior efficacy to therapeutics which only interfere with a single pathway ICOS-L+ ICOS+ CD80/CD86+ Figure 5: ALPN-101 Suppresses Proliferation and Antibody Responses in Human B-T FH Cell Co-Cultures and Affects Expression of Genes Involved in B-T Cell Collaboration % Incidence of Sialadenitis 0.0 0.1 0.2 0.3 0.4 Serum anti Ro-52 OD at 1:312.5 dilution vs ALPN-101: *** p=0.0002 **** p<0.0001 **** **** *** 0.0 0.1 0.2 0.3 0.4 Serum anti La OD at 1:312.5 dilution *** ** ^ vs ALPN-101: ^ p=0.0146 ** p=0.0026 *** p=0.0002 Fc Control ALPN-101 Abatacept WT ICOSL-Fc Aba + WT ICOSL-Fc Naïve Figure 4: ALPN-101 Suppresses Expression of Many Genes Associated with SjS/SLE Mechanisms and Phenotypes Individual SMG Histology Scores • CCL20 • CD40LG • CSF2 • ICOS • ID2 • IFNG • IGSF3 • IL17A • IL1R1 • IL2 • IL21 • IL24 • IL3 • IL31 • IL6 • IL9 • LIF • NFKBID • PDCD1 • TNF • BATF3 • CTLA4 • FLT1 • FOSL1 • IL13 • IL17F • IL1R2 • IL23R • IL4 • IL5 • VEGFA • CCL17 • CXCR5 • GATA3 • IL12RB2 • IL18R1 • IL18RAP • KITLG • LTA • PVR • SOCS3 • TFRC • TNFRSF8 • RNA was isolated from patient PBMC samples generated in the assay described in Fig. 3 • Genes significantly down-regulated in response to ALPN-101 across SjS, SLE, and psoriatic arthritis (PsA) stimulated patient populations suggest ALPN-101 can impact pathogenic genes and/or pathways implicated in many autoimmune diseases. Anti Ro-52 and anti La Abs were detected by ELISA (Signosis, Santa Clara, CA) in diluted serum collected on Day 10 from mice in the SjS study described in Figs 6-7. Statistical differences noted between the ALPN-101 group and comparator treatments were determined using an uncorrected non- parametric Dunn’s test. See also: Poster #1531. Evans et al. (2019) ALPN-101, a First-in-Class Dual ICOS/CD28 Antagonist, Suppresses Key Effector Mechanisms Underlying Rheumatoid and Psoriatic Arthritis. ACR Annual Meeting; November 11, 2019. 4 (A) Human T and B Cell Proliferation (B) Human Immunoglobulin Production Better Worse 1 100 10000 1000000 0 20 40 60 80 B cells: % Naive [ pM ] % Positive 1 100 10000 1000000 0 25 50 75 100 B cell: % Divided [ pM ] % Proliferation 1 100 10000 1000000 0 25 50 75 100 CD4+ T cells: % Divided [ pM ] % Proliferation 1 100 10000 1000000 0 500 1000 1500 CD4+ T cells: CD40L+ Cell Count [ pM ] Cell # ICOS WT ICOSL IgV Aba + WT ICOSL-Fc 1 μm Representative H&E Images of SMG (C) RNA expression analysis of key genes involved in B-T cell collaboration (C) RNA isolated from a B/TFH co-culture similar to the one described in A was analyzed for relative gene expression levels by next generation sequencing. Expression values in RPKM are plotted on a relative scale from 0 to 1 for each gene between treatment groups, with degree of red reflecting higher expression and degree of blue representing lower values for each gene. The list of genes shown was sorted by level of inhibition by ALPN-101 (least to most inhibited, from L→R). Statistical significance determined using the Kruskal-Wallis test: * p < 0.05, ** p < 0.01, **** p < 0.0001. The naïve group was not powered for statistical comparisons. IC50 1.0 nM 5.4 nM 2.5 nM 0 1000 2000 3000 4000 5000 IgG anti-dsDNA (ng/mL) Serum anti-dsDNA (IgG) Fc Control ALPN-101 Abatacept Naïve BL/6 -1×10 5 0 1×10 5 2×10 5 3×10 5 4×10 5 5×10 5 GC B cell # / Spleen # GC B Cells Per Spleen 0 1×10 5 2×10 5 3×10 5 4×10 5 # CD4+ T FH Cells Per Spleen T FH cell # / Spleen **** * * * ** **** ALPN-101 inhibits both CD28 and ICOS-mediated costimulation. Comparators such as abatacept / belatacept (CTLA4-Ig, BMS) or FR104 (anti CD28 Fab, OSE Immunotherapeutics) inhibit only the CD28/CTLA-4 pathway, while prezalumab (anti ICOSL mAb, Amgen) inhibits only ICOS costimulation. CD28 costimulation is critical for naïve and memory CD4 + T cell activation. ICOS signaling augments CD28 costimulation but also plays non-redundant roles; ICOS deficiency leads to defective humoral responses in both mice and humans. 1 ICOS signaling is crucial for the development of human T H 17 cells 2 and the function & maintenance of follicular helper T cells. 3 ALPN-101 may thus be uniquely suited to treat diseases driven by these pathogenic cell types. 0.001 0.01 0.1 1 10 100 1000 0 400 800 1200 Human IgG1 [Test Article], nM [Hu IgG1], ng/ml Mean Fc control 0.001 0.01 0.1 1 10 100 1000 0 100 200 300 400 Human IgG3 [Test Article], nM [Hu IgG3], ng/ml Mean Fc control 0.001 0.01 0.1 1 10 100 1000 0 2000 4000 6000 8000 Human IgM [Test Article], nM [Hu IgM], ng/ml Mean Fc control 0.001 0.01 0.1 1 10 100 1000 0 200 400 600 Human IgA [Test Article], nM [Hu IgA], ng/ml Mean Fc control CD80/86 (B7) CD28 T cell prezalumab ICOSL ICOS T cell activation APC/B cell CD80/86 (B7) CD28 CTLA4-lg T cell activation ICOSL ICOS ICOSL ICOS CD80/86 (B7) CD28 T cell Inactivation ALPN-101 D14 D28 0 500 1000 1500 2000 2500 Day Post-Immunization MFI 1:136 Diln 1:45 Diln ** D14 D28 0 5000 10000 15000 20000 Day Post-Immunization MFI 1:409 Diln 1:45 Diln **** **** ** *** (A) Serum Ig titers in BALB/c mice immunized with sheep red blood cells (SRBC) SRBC Day 0 Treated on D-1 and D7 Sac D14 GC B = B220 + CD19 + GL7 + CD95 + TFH = CD4 + CD45 + CD3 + PD1 + CD185 + (B) Numbers of splenic GC B and T FH in BALB/c mice 14 days after immunization with SRBC 0 50 100 150 CD4 + T FH cell #/spleen (x 10 3 ) # CD4 + T FH cells/spleen (C) Numbers of splenic GC B and T FH and serum anti-dsDNA titers in the bm12 inducible model of SLE D14 D28 0 2000 4000 6000 8000 10000 Day Post-Immunization MFI 1:409 Diln 1:45 Diln ** * **** **** D14 D28 0 10000 20000 30000 40000 50000 Day Post-Immunization MFI 1:45 Diln 1:45 Diln *** *** * ** IgM anti-SRBC IgG1 anti-SRBC IgG2a anti-SRBC IgG2b anti-SRBC Statistical significance was determined for GC B and TFH using an unpaired t-test, and for anti dsDNA concentrations using an uncorrected Fisher’s least significant differences test; *p<0.05, **p<0.01, and ****p<0.0001. * p<0.05 ** p<0.005 *** p<0.001 **** p<0.0001 WT ICOSL-Fc Abatacept ALPN-101 Fc control