(GSK2857916) Drives Immunogenic Cell Death and Immune-Mediated 2 Anti-Tumor Responses in Vivo 3

Author Manuscript Published OnlineFirst on July 12, 2021; DOI: 10.1158/1535-7163.MCT-21-0035 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

1 Belantamab Mafodotin (GSK2857916) Drives Immunogenic Cell Death and Immune-Mediated 2 Anti-Tumor Responses In Vivo 3

4 Rocio Montes de Oca1*, Alireza S. Alavi2, Nick Vitali2, Sabyasachi Bhattacharya2, Christina 5 Blackwell2, Krupa Patel2, Laura Seestaller-Wehr2, Heather Kaczynski2, Hong Shi2, Eric 6 Dobrzynski3, Leslie Obert4, Lyuben Tsvetkov2, David C. Cooper5, Heather Jackson2, Paul 7 Bojczuk2, Sabrina Forveille6,7, Oliver Kepp6,7, Allan Sauvat6,7, Guido Kroemer6-10, Mark 8 Creighton-Gutteridge11, Jingsong Yang2, Chris Hopson2, Niranjan Yanamandra2, Christopher 9 Shelton2, Patrick Mayes2, Joanna Opalinska12, Mary Barnette2, Roopa Srinivasan1, James 10 Smothers2, Axel Hoos12

12 Affiliations 13 1 Experimental Medicine Unit, Oncology R&D, GlaxoSmithKline, 1250 S. Collegeville Rd, 14 Collegeville PA 19426. 15 2 Immuno-Oncology and Combinations RU, Oncology R&D, GlaxoSmithKline, 1250 S. 16 Collegeville Rd, Collegeville PA 19426. 17 3 Bioanalysis, Immunogenicity and Biomarkers, GlaxoSmithKline, 1250 S. Collegeville Rd, 18 Collegeville PA 19426. 19 4 Translational Medicine and Comparative Pathobiology, GlaxoSmithKline, 1250 S. Collegeville 20 Rd, Collegeville PA 19426. 21 5 Research Statistics, GlaxoSmithKline, 1250 S. Collegeville Rd, Collegeville PA 19426 22 6 Equipe labellisée par la Ligue contre le cancer, Université de Paris, Sorbonne Université, 23 INSERM U1138, Centre de Recherche des Cordeliers, Paris, France. 24 7 Metabolomics and Cell Biology Platforms, Gustave Roussy Cancer Center, Villejuif, France. 25 8 Pôle de Biologie, Hôpital Européen Georges Pompidou, AP-HP, Paris, France. 26 9 Suzhou Institute for Systems Medicine, Chinese Academy of Medical Sciences, Suzhou, China. 27 10 Karolinska Institute, Department of Women's and Children's Health, Karolinska University 28 Hospital, Stockholm, Sweden. 29 11 Cell Therapy RU, Oncology R&D, GlaxoSmithKline, Gunnels Wood Road, Stevenage, 30 Hertfordshire UK, SG1 2NY.

31 12 Oncology R&D, GlaxoSmithKline, 1250 S. Collegeville Rd, Collegeville PA 19426. 32 33 * To whom correspondence should be addressed: 34 Rocio Montes de Oca 35 1250 S. Collegeville Road, Collegeville, Pennsylvania, 19426-0989, United States 36 Email: [email protected] 37 Tel: +1 (610) 917-5746 38 39 Running title (60 characters with spaces): GSK2857916 drives ICD and adaptive anti-tumor 40 responses. 41 42 Keywords: Belantamab mafodotin; B-cell maturation antigen; immunogenic cell death; 43 immune-mediated anti-tumor responses; auristatins. 44 45 Financial support: This study was funded by GlaxoSmithKline. 46 47 Abbreviations 48 ADC, antibody-drug-conjugate; ADCC, antibody-dependent cellular cytotoxicity; ADCP, 49 antibody-dependent cellular phagocytosis; ANOVA, analysis of variance; APCs, antigen 50 presenting cells; APRIL, a proliferation-inducing ligand; ATCC, American Type Culture 51 Collection; ATP, adenosine triphosphate; AUC, area under the curve; BAFF/BlyS, B-cell 52 activating factor; BAP31, B-cell receptor-associated protein 31; BCMA, B-cell maturation 53 antigen; DAMPs, danger-associated molecular patterns; DCs, dendritic cells; DREAMM, 54 DRiving Excellence in Approaches to Multiple Myeloma; ELISA, enzyme-linked 55 immunosorbent assay; ER, endoplasmic reticulum; GrzB, granzyme B; HMGB1, high mobility

56 group box 1 protein; HSPs, heat-shock proteins; IC50, half maximal inhibitory concentration; 57 ICD, immunogenic cell death; IFNs, interferons; IHC, immunohistochemical; L, length; mAbs, 58 monoclonal antibodies; mc, maleimidocaproyl; MFI, median fluorescence intensity; MM,

59 multiple myeloma; MMAE, monomethyl auristatin-E; MMAF, monomethyl auristatin-F; NF-B

60 nuclear factor kappa B; PI, propidium iodide; PBD, pyrrolobenzodiazepines; RRMM, relapsed 61 refractory multiple myeloma; S.D., standard deviation; SEM, standard error of the mean;

62 SLAMF, signaling lymphocyte activation molecule family; TILs, tumor-infiltrating 63 lymphocytes; Tub, tubulysin; W, width. 64 65 Conflict of interest disclosures: The authors declare the following conflicts of interest: RM, 66 AA, NV, SB, CB, KP, LS-W, HK, HS, ED, LO, LT, DC, HJ, PB, MC-G, JY, CH, NY, CS, PM, 67 JO, MB, RS and JS were employees of GlaxoSmithKline at the time the work was performed 68 and own stocks/shares. AH is an employee of and holds stocks and shares in GlaxoSmithKline 69 and is a non-executive Director of TCR2 Therapeutics Inc. GK and OK received funds from 70 GlaxoSmithKline for this research. OK holds patent US8828944B2, broadly relevant to the 71 work. 72 73

74 Abstract 75 B-cell maturation antigen (BCMA) is an attractive therapeutic target highly expressed on 76 differentiated plasma cells in multiple myeloma (MM) and other B-cell malignancies. 77 GSK2857916 (belantamab mafodotin, BLENREP) is a BCMA-targeting antibody-drug- 78 conjugate approved for the treatment of relapsed/refractory MM. We report that GSK2857916 79 induces immunogenic cell death in BCMA-expressing cancer cells and promotes dendritic cell 80 activation in vitro and in vivo. GSK2857916 treatment enhances intra-tumor immune cell 81 infiltration and activation, delays tumor growth and promotes durable complete regressions in 82 immune-competent mice bearing EL4 lymphoma tumors expressing human BCMA (EL4- 83 hBCMA). Responding mice are immune to re-challenge with EL4 parental and EL4-hBCMA 84 cells, suggesting engagement of an adaptive immune response, immunologic memory, and tumor 85 antigen spreading, which are abrogated upon depletion of endogenous CD8+ T-cells. 86 Combinations with OX40/OX86, an immune agonist antibody, significantly enhance anti-tumor 87 activity and increase durable complete responses, providing a strong rationale for clinical 88 evaluation of GSK2857916 combinations with immunotherapies targeting adaptive immune 89 responses, including T-cell-directed checkpoint modulators.

92 Introduction 93 While many neoplasms are eliminated by the immune system, some tumors can develop when 94 malignant cells evade immune surveillance mechanisms. Tumor immune escape can be mediated 95 by multiple factors including immunosuppressive factors within the tumor microenvironment 96 and negative regulators that inhibit T-cell activity and promote tolerance (1). Modulation of such 97 checkpoints by inhibitory monoclonal antibodies (mAbs) against CTLA-4 and PD-1 has been 98 associated with clinically significant responses in a variety of cancers (2). Moreover, agonism of 99 co-stimulatory receptors such as OX40, upregulated on naïve and memory T cells following T- 100 cell receptor ligation, significantly enhances T-cell proliferation and survival and differentiation 101 to a memory phenotype, which may provide additional therapeutic benefit (3). Current efforts are 102 focused on improving the clinical efficacy of immunotherapies through combinations with other 103 checkpoint modulators, immunogenic chemotherapeutics, and radiotherapy (4-6).

104 A subset of chemotherapeutics have been shown to contribute to immune stimulation through 105 antigen release secondary to cytotoxic cell death (7). While normal apoptosis is non- 106 immunogenic, treatment with selected chemotherapeutics, such as anthracyclines and oxaliplatin, 107 induces a unique form of cell death termed immunogenic cell death (ICD). ICD is characterized 108 by the induction of the endoplasmic reticulum (ER) stress response and the exposure and/or 109 release of danger-associated molecular patterns (DAMPs), many of which are Toll-like receptor 110 ligands (8). After ICD induction, calreticulin, a DAMP molecule normally found in the ER 111 lumen, is translocated to the surface of the dying cell, where it functions as an “eat me” signal for 112 immature dendritic cells (DCs) (9). Other important DAMPs are heat-shock proteins (HSPs), 113 namely HSP70 and HSP90, the secreted amphoterin high mobility group box 1 (HMGB1) 114 protein, and extracellular adenosine triphosphate (ATP). ATP functions as a chemoattractant 115 recruiting antigen presenting cells (APCs) by interacting with purinergic receptors, while 116 HMGB1 is considered a late apoptotic marker and its release to the extracellular space seems to 117 be required for the optimal release and presentation of tumor antigens to DCs, DC maturation, 118 and synthesis of pro-inflammatory molecules including type I interferons (IFNs) (10,11). 119 Further, exposure of DCs to tumor cells undergoing ICD evokes an inflammatory phenotype 120 (innate response), including an increase in co-stimulatory markers CD86 and MHC Class II

121 antigens and the activation of nuclear factor (NF)-B, a pro-inflammatory transcription factor

122 complex (10). Mature DCs play a key role in priming robust immune responses in tumor-bearing

123 hosts, and ICD can induce an effective anti-tumor immune response through DC activation and 124 subsequent activation of tumor antigen-specific T-cell responses. Therapeutic approaches that 125 activate DCs and promote tumor antigen-specific T-cell priming are needed to enhance the 126 immunogenicity of tumors. In this regard, several chemotherapeutic agents and potent tubulin 127 inhibitors, such as vinblastine and the auristatin analogues dolastatin 10 and monomethyl 128 auristatin-E and -F (MMAE and MMAF, respectively), have been recently identified as potent 129 activators of DC maturation and antigenic responses (12-14).

130 B-cell maturation antigen is a transmembrane receptor whose normal function is to promote the 131 survival of cells in the B-cell lineage, by transduction of signals from two known ligands from 132 the tumor necrosis factor family: B-cell activating factor (BAFF/BLyS) and a proliferation- 133 inducing ligand (APRIL). BCMA expression is restricted to B-cells at later stages of 134 differentiation, including germinal center B-cells in tonsil, blood plasmablasts, and long-lived 135 plasma cells (15). BCMA is also expressed in various B-cell malignancies including multiple 136 myeloma, diffuse large B-cell lymphoma, large B-cell lymphoma, chronic lymphocytic 137 leukemia, and Waldenström’s macroglobulinemia, at varying frequencies (16-19). BCMA’s 138 restricted normal tissue expression profile and up-regulation and survival function in MM and 139 other B-cell malignancies, make it an attractive target for a therapeutic antibody with direct cell 140 killing activity.

141 Belantamab mafodotin also known as GSK2857916 is a humanized, IgG1 antibody-drug- 142 conjugate (ADC) that binds specifically to BCMA (20). The parent anti-BCMA antibody 143 (GSK2857914) is produced in an afucosylated form and is conjugated to the microtubule 144 polymerization inhibitor MMAF, via a protease resistant maleimidocaproyl (mc) linker, to 145 produce the GSK2857916 ADC molecule. Upon binding to the cell surface, GSK2857916 is 146 rapidly internalized and active cytotoxic drug (cys-mcMMAF) is released inside the cell causing 147 cell cycle arrest and apoptosis. Further, the afucosylation of GSK2857916 leads to enhanced

148 binding to FcRIIIa receptors on the surface of immune effector cells, recruitment of immune

149 cells, and antibody-dependent cellular cytotoxicity and phagocytosis (ADCC and ADCP, 150 respectively). This dual mechanism of action enables improved efficacy by targeting both 151 dividing (via ADC) and non-dividing (via ADCC/ADCP) tumor cells (21). Recently, belantamab

152 mafodotin was approved under the name BLENREP in the US and EU for the treatment of 153 patients with relapsed or refractory MM.

154 Previous pre-clinical studies linked the direct cytotoxic activity of auristatin-class ADCs (e.g., 155 dolastatins and brentuximab vedotin) to substantial therapeutic efficacy, by augmenting host 156 immunity through direct effects on DC maturation and activation of antigen specific T-cells, in 157 addition to direct tumor cell killing (22,23). In this study, we provide evidence that GSK2857916 158 potently induces prominent ICD markers and DC maturation and activation in vitro. In a 159 syngeneic mouse model, GSK2857916 induced ICD, promoted intratumoral enrichment of 160 tumor-infiltrating lymphocytes (TILs), and T-cell-dependent anti-tumor activity. Our data reveal 161 a novel immune-mediated mechanism of action for GSK2857916, which is significantly 162 enhanced in combination with an agonistic OX86 (mouse OX40) antibody, and provides 163 rationale for the investigation and development of clinical combinations with GSK2857916 and 164 immune-checkpoint modulators.

165

166 Materials and Methods

167 Belantamab Mafodotin or GSK2857916 168 GSK2857916 is a humanized, IgG1 monoclonal antibody known as GSK2857914 (clone J6M0), 169 which is derived from the murine monoclonal antibody clone CA8, conjugated to MMAF. The 170 CA8 murine heavy and light chain variable region amino acid sequences are provided as SEQ ID 171 NO. 7 and SEQ ID NO. 9, respectively, in the published United States patent 9,273,141 B2.

172 GSK2857916 is comprised of the CA8 humanized heavy (CA8 Humanized VH J6) and light

173 (CA8 Humanized VL M0) chain variable region amino acid sequences provided as SEQ ID NO. 174 23 and SEQ ID NO. 31, respectively, of the same patent.

175 Cell Lines 176 NCI-H929 cells were purchased and grown as per American Type Culture Collection (ATCC). 177 The EL4-Luc-2-hBCMA cell line was generated by electroporating a plasmid encoding the 178 human BCMA receptor (residues 1-184) into the Bioware Ultra Cell Line EL4-luc2 (Caliper Life 179 Sciences, Inc., cat #124088, derived from (TIB-39)). The EL4 parental (ATCC, cat#TIB-39) and 180 EL4-hBCMA cells were grown in DMEM High Glucose (1X) + GlutaMAX (Gibco; cat#10564-

181 011 or 11965-092) with 10% FBS (Sigma Aldrich; cat#12176C) ± 200 µg/mL G418/Geneticin

182 (Gibco; cat#10131-027) at 37°C with 5% CO2. Cells were used within <5 passages from day of 183 thaw for experiments. Cells were authenticated by short tandem repeat profiling and were 184 negative for mycoplasma.

185 In vitro Evaluation of Cell Viability, Calreticulin Exposure, and HMGB1 and ATP Release 186 in NCI-H929 MM Cells 187 NCI-H929 cells were left untreated or were exposed to GSK2857916 as indicated for 48 hours.

188 Mitoxantrone (1 M; Sigma Aldrich; cat#M6545), a known ICD inducer (9), was the positive

189 control. Cell viability was assessed by automated flow cytometry. Cell surface calreticulin was 190 analyzed in non-permeabilized cells by flow cytometry (Abcam, cat#ab2907; diluted 1:100) with 191 the fluorescence intensity of stained cells gated on propidium iodide (PI) negative cells. 192 Extracellular HMGB1 concentrations were analyzed using an enzyme-linked immunosorbent 193 assay (ELISA; Shino Test Corp ELISA kit II). Analysis of extracellular ATP content was by the 194 ENLITEN ATP Assay system (Promega Corp, cat#FF2000). Secreted ATP was stabilized using 195 an ectoATPase inhibitor (3 µM ARL 67156 trisodium salt hydrate; Sigma Aldrich, cat#A265). 196 For all measurements, mean ± standard deviation (S.D.) of triplicates from a representative 197 experiment shown.

198 In vitro Evaluation of Additional Hallmarks of ICD 199 Cells (5×105 cells/mL) were left untreated or exposed to the indicated treatments for the 200 indicated time. Cells were counted on a Vi-Cell-XR Cell Viability Analyzer (Beckman Coulter).

201 For immunoblot analyses, cells were lysed using Cell Lysis Buffer (Cell Signaling Technology; 202 cat#9803) with a protease inhibitor cocktail (Roche; cat#11873580001), and probed with the 203 following antibodies: Phospho-PERK (Abcam; cat#ab192591; 1:1,000), PERK (R&D Systems; 204 cat#AF3999; 1:200), phospho-eIF2α (Cell Signaling Technology; cat#3398S; 1:1,000), eIF2α 205 (Cell Signaling Technology; cat#2103S; 1:1,000), BAP31 (Cell Signaling Technology; 206 cat#4043S; 1:1,000), and GAPDH (Bethyl Laboratories; cat#A300-641A; 1:10,000).

207 ICD markers were quantified from cell supernatants by the following ELISAs: HSP70 (R&D 208 Systems; cat#DYC1663-2; 1:2 sample dilution), HSP90α (Invitrogen; cat#BMS2090; 1:25 209 sample dilution), IL-8 (R&D Systems; cat#D8000C; undiluted sample), mouse calreticulin 210 (LifeSpan BioSciences; cat#LS-F6663; 1:10 or 1:15 sample dilution), HMGB1 (IBL

211 International Corp; cat#ST51011; 1:4 sample dilution) and mouse HSPD1/HSP60 (LifeSpan 212 BioSciences; cat#LS-F4239; undiluted sample).

213 Cell surface ICD markers were analyzed in non-permeabilized cells by flow cytometry with the 214 following antibodies or isotypes (at 0.7 mg/mL per reaction): anti-Calreticulin mouse IgG2a 215 (Novus; cat#NBP1-47518AF488), mouse IgG2a (R&D systems; cat#IC003G), anti-HSP90 216 mouse IgG1a (Novus; cat#NB100-1972AF488), mouse IgG1a (Novus; cat#DDXCM01A488), 217 anti-HSP70/HSPA1A rabbit IgG (Novus; cat#NBP1-77455AF488), anti-HMGB1/HMG-1 rabbit 218 IgG (Novus; cat#NB100-2322AF488) or rabbit IgG (Novus; cat#NBP2-24982), per 219 manufacturer’s instructions. Fluorescence intensity of stained cells was gated on live cells using 220 Aqua Live Dead stain (ThermoFischer Scientific; cat#L34957). Cells were acquired on a BD 221 LSR Fortessa X-20 and analyzed by FlowJo software (v10.1r5; FlowJo, LLC).

222 In vitro Activation of Immature DCs by GSK2857916-Treated NCI-H929 Cells 223 Human blood was obtained from three healthy donors from the GSK Blood Donation Unit using 224 heparin coated syringes. Monocytes were isolated from this blood using the RosetteSep 225 Monocyte Enrichment kit (STEMCELL Technologies; cat#15068) and were assessed for CD14 226 cell surface expression (BD Bioscience; cat#562698) by flow cytometry. Isolated monocytes 227 (1.5x106 cells/well) were cultured in 2 mL of X-Vivo-15 Media (Lonza; cat#BE02-054Q) 228 supplemented with 1% autologous plasma, 250 ng/mL recombinant human GM-CSF (R&D 229 Systems; cat#215-GM-050) and 100 ng/mL recombinant human IL-4 (R&D Systems; cat#204-

230 IL-050) for 7 days at 37°C and 5% CO2. Cultures received a half media change on day 3 or 4 231 while maintaining stimulation factor concentrations. In parallel, NCI-H929 cells were plated at a 232 density of 7.5x105 cells/mL in 2 mL, in 12-well plates, and were left untreated or were treated

233 with GSK2857916 (0.1, 1 or 10 g/mL) or hIgG1-MMAF (10 g/mL) for 48 hours. On day 7,

234 immature dendritic cells derived from the monocytes were co-cultured with the treated NCI- 235 H929 cells at a 1:1 ratio for 24 hours, in 96-well plates, 7.5x104 cells were seeded per well (100

236 L). A flow cytometry panel consisting of CD1a (BD Bioscience; cat#563938), CD11c (BD

237 Bioscience; cat#561355), CD40 (BD Bioscience; cat#555751), CD80 (BD Bioscience; 238 cat#561135), CD83 (BD Bioscience; cat#562630), CD86 (BD Bioscience; cat#555657), HLA- 239 DR (BD Bioscience; cat#340591) and CD14 (BD Bioscience; cat#562698) was used to assess

240 DC differentiation and maturation on a BD FACS Canto II. The data were analyzed by FlowJo 241 software (v7.6.5; FlowJo, LLC).

242 In vitro Growth Death Assays 243 Cells were plated (1x104 cells/mL) in a 96-well format. On assay day 0, cells were treated in 244 quadruplicate with a 12-point, three- or four-fold dilution series of GSK2857916, GSK2857914, 245 MMAE (SAFC; cat#SGD-1010), vinorelbine (Sigma Aldrich; cat#V2264) or colchicine (Sigma 246 Aldrich; cat#C9754), as indicated, for 3 days. Cell growth was evaluated on days 0 and 3 by 247 CellTiter-Glo reagent (Promega; cat#G8462) and luminescence analysis. Dose response curves,

248 IC50 values and sensitivity between cell lines (% growth [mean ± S.D. of biological 249 quadruplicates]) were calculated.

250 In vivo Efficacy Studies 251 Female C57/BL6 mice (8-10 weeks old from Charles River Laboratories) were injected 252 subcutaneously in the right flank with EL4 parental or EL4-hBCMA cells harvested during 253 exponential growth (1 or 3 x 105 cells in 0.1 mL). Tumor growth was monitored and tumor 254 length (L) and width (W) were measured (in mm) to determine tumor volume (L x W2/2). Mice 255 were randomized into groups and treatment began when average tumor volume was ~150-250 256 mm3, or as indicated. Antibodies were administered by intraperitoneal (i.p.) injection twice 257 weekly for three weeks for a total of six doses (or as indicated) and were well tolerated at all 258 doses. Tumor volumes >2000 mm3 for an individual mouse resulted in euthanization.

259 Therapeutic efficacy was determined by calculating tumor burden for all mice over the course of 260 the study by taking the area under the curve (AUC) of tumor volume over time using the 261 trapezoidal method. Adjusted AUC values for all mice in each group were analyzed by a 262 nonparametric analysis of variance (ANOVA on the ranks). Further, incidence, magnitude, and 263 durability of regression responses were also evaluated. An animal with a complete regression 264 response (CR=TV < 13.5 mm3 for three consecutive measurements) at the termination of a study 265 was classified as a tumor-free survivor. For re-challenge studies, tumor-free survivors were re- 266 challenged on the flank opposite to the original implant side. Survival was analyzed by the 267 Kaplan-Meier method.

268 Immunophenotyping by Flow Cytometric Analysis 269 Isolated tumor cells were incubated with human or mouse FcR Blocking Reagent (Miltenyi 270 Biotec; cat#130-059-901 or 130-092-575, respectively) and Live/Dead stain (ThermoFischer 271 Scientific), then with antibodies directed against surface antigens for 30 minutes at 4°C as 272 follows: anti-CD3 (145-2C11; cat#553062), anti-CD4 (RM4-5; cat#550954), anti-CD8 (53-6.7; 273 cat#B560182), anti-CD25 (PC61; cat#552880), anti-CD11c (HL3; cat#560584), and anti-CD86 274 (GL1; cat#553691), all from BD Biosciences. Anti-Granzyme B (GB11; cat#12-8899-41) and 275 anti-FoxP3 (FJK-16s; cat#17-5773-82), from eBiosciences. Anti-human CD269 BCMA (19F2; 276 cat#357504), anti-Ki67 (16A8; cat#652411), anti-PD-1 (RMP1-30; cat#109110), anti-ICOS 277 (C398.4A; cat#313524), anti-CD69 (H1.2F3; cat#104528), anti-CD49b (DX5; cat#108908), anti- 278 CD3 (17A2; cat#100228), anti-CD4 (RM4-4; cat#116004), anti-CD8 (53-5.8; cat#140410), from 279 BioLegend. Anti-OX40 (OX86; cat# FAB1256P) from R&D Systems. Anti-Calreticulin 280 (1G6A7; cat#NBP1-47518AF488) from Novus. Cell surface stains were followed by 2% 281 paraformaldehyde for 20 minutes, washed and resuspended in Stain buffer (BD Pharmingen; 282 cat#554657). For intracellular staining, cells were incubated with Fixation/Permeabilization 283 buffer (BD Biosciences; cat#554714) for 30 minutes, followed by incubation with antibodies 284 directed against intracellular antigens for additional 30 minutes, washed and resuspended in 285 Stain buffer. Cells were acquired on a BD LSR Fortessa X-20 and data were analyzed by FlowJo 286 software (v10.1r5; FlowJo, LLC).

287 NanoString Analysis 288 NanoString analysis was performed on RNA (100 ng) extracted from dissociated tumor samples 289 using the NanoString Mouse PanCancer Immune profiling panel (NanoString; cat#XT-CSO- 290 MIP1-12). Data analysis was performed using nSolver 2.6 and the mouse PanCancer Immune 291 advanced analysis software. The type I IFN-related signature was defined by the following 292 genes: Ccl2, Ccl3, Ccl4, Ccl7, Cd274, Cd69, Cxcl1, Cxcl10, Ifit2, Ifnb1, Il12a, Il12b, Irf3, Irf7, 293 Mx1, Mx2, Oas2, Oasl1, Rsad2, Stat4, and Tnfrsf9 (modified from (24)).

294 Immunohistochemical (IHC) Analysis 295 Mice were injected subcutaneously with EL4-hBCMA cells (1 x 106 cells in 0.1 mL). When 296 tumors averaged a volume of ~800 mm3, mice were randomized and dosed i.p. with 15 mg/kg 297 GSK2857916 or GSK2857914, as described. Tumors were harvested 7 days after randomization

298 and three antibody doses and fixed in 10% neutral buffered formalin, transferred to 70% ethanol 299 and processed into paraffin blocks. IHC staining used a Ventana instrument and reagents 300 (Ventana Medical Systems) and was performed with an anti-CD8 mAb (eBioscience; 4SM15; 301 cat#14-0808-82). Subjective histopathology assessment of H&E and/or IHC stained tumor 302 sections was completed by a board-certified veterinary pathologist.

303 Statistical Analyses 304 Statistical significance was evaluated by a one-way or a two-way ANOVA using GraphPad 305 Prism software (v8), as indicated in each figure legend, p-values are only reported for pre- 306 planned treatment comparisons.

307 Ethics declarations

308 Fresh human blood was obtained from GSK’s on-site blood donation unit, and its ethical 309 research use was in accord with the terms of the local informed consent under an Advarra IRB 310 approved protocol. All animal studies were conducted in accordance with the GSK Policy on the 311 Care, Welfare and Treatment of Laboratory Animals and were reviewed and approved by the 312 Institutional Animal Care and Use Committee either at GSK or by the ethical review process at 313 the institution where the work was performed. 314

315 Results

316 Induction of immunogenic cell death and dendritic cell activation by GSK2857916 317 Recent studies have demonstrated that microtubule-disrupting agents, such as dolastatin 10 and 318 MMAE, can induce ICD and promote an anti-tumor response that is mediated by the adaptive 319 immune system (22,23,25). Therefore, we sought to determine whether GSK2857916 induces 320 hallmarks of ICD through induction of ER stress and autophagy and involving calreticulin cell 321 surface exposure and release of DAMPs including ATP and HMGB1 (9,26-30).

322 Treatment with GSK2857916 significantly decreased the viability of BCMA-expressing NCI- 323 H929 human MM cells (Figure 1A) and also led to a significant and dose-dependent 324 externalization and plasma membrane exposure of calreticulin (Figure 1B) and secretion of 325 HMGB1 and ATP into the culture media (Figure 1C, D). Consistently, in NCI-H929 cells 326 GSK2857916 induced an ER stress response by promoting the phosphorylation of the ER stress

327 kinase PERK (pPERK; Figure 1E) and its substrate, the eukaryotic translation initiation factor

328 eIF2 (p-eIF2; Figure 1F), leading to the proteolytic cleavage of the ER protein B-cell

329 receptor-associated protein (BAP) 31 (Figure 1F, arrowhead), which is critical for calreticulin 330 exposure. Moreover, GSK2857916 treatment induced cell surface exposure (Figure 1G) and 331 significant extracellular release of other DAMPs, including HSP70 and HSP90 (Figure 1H, I), 332 and IL-8 (Figure 1J); the latter being an ICD marker indicative of immune response modulation 333 and inflammatory response initiation. Notably, the dose- and time-dependent induction and 334 release of these DAMPs was GSK2857916 treatment-specific and required BCMA binding and 335 toxin release (as no changes were observed with the hIgG1-MMAF isotype or the non- 336 conjugated GSK2857914 antibody). Further, the enhanced DAMP release was observed despite 337 decreases in cell viability (~10-20% less) and cell number (~1.5-2-fold decrease), compared with 338 controls (Table S1A).

339 Once the ER stress response has triggered the exposure and release of calreticulin and other ER 340 chaperones, these DAMPs act as “eat me” signals, and in concert with the chemo-attractant ATP, 341 promote the uptake of dying cells by APCs. Dendritic cells are APCs that can activate resting 342 naïve T lymphocytes and play a key role in priming robust adaptive anti-tumor immune 343 responses (31). To characterize the immune-modulatory effects of GSK2857916, we investigated 344 the effect of GSK2857916-treated NCI-H929 tumor cells on immature dendritic cells (iDCs) 345 from three healthy donors. Markedly lower CD11c and HLA-DR expression on NCI-H929 cells 346 enabled the distinction from high CD11c+ and HLA-DR+ dendritic cells (Figure 2A, S1). 347 Twenty-four hour co-culture of iDCs with GSK2857916-pretreated tumor cells, led to a 348 significant dose-dependent increase in DC activation and cell surface maturation markers 349 including CD40, CD83, and CD86 (Figure 2B). These results show that GSK2857916-induced 350 ICD in tumor cells can modulate immune function by promoting a more active and mature DC 351 phenotype.

352 Development and characterization of the EL4-hBCMA murine syngeneic model 353 To determine whether the in vitro GSK2857916-induced ICD phenotype translates into increased 354 immunogenicity, activation of an adaptive immune response, and enhanced anti-tumor activity in 355 vivo, an immune-competent mouse tumor model was developed. As GSK2857916 is not cross- 356 reactive with murine BCMA, the murine EL4 T-cell lymphoma cell line was engineered to stably

357 express human BCMA (hBCMA) on the cell surface, at levels approaching those of the NCI- 358 H929 MM cells (Figure 3A). GSK2857916 induced cell death in this cell line in a manner that 359 was dependent on both the conjugated toxin and hBCMA cell surface expression (Figure 3B vs 360 C). However, the EL4-hBCMA cell line was less sensitive to the cytotoxic effects of 361 GSK2857916 as compared to NCI-H929 cells (Figure 3B, mean half maximal inhibitory

362 concentration [IC50] 30.6 vs 0.003 g/mL, respectively). Previous studies have shown that rodent 363 cells are less sensitive to microtubule poisons relative to primate cells (32). Consistently, the EL4 364 parental and EL4-hBCMA cells were also less sensitive to microtubule polymerization inhibitors 365 including free MMAE, a cell permeable auristatin derivative similar to MMAF, vinorelbine and 366 colchicine (Figure S2A-C). These results indicate that EL4-hBCMA cells are less sensitive to 367 GSK2857916 compared with NCI-H929 cells, likely due to lower BCMA surface expression 368 levels and lower intrinsic sensitivity to the cytotoxic effects of MMAF.

369 To evaluate if expression of hBCMA leads to specific induction of ICD by GSK2857916, ICD 370 markers were assessed in EL4-hBCMA cells compared with EL4 parental cells. GSK2857916 371 treatment of EL4-hBCMA cells (Figure 3D), but not the hIgG1-MMAF or GSK2857914 controls 372 (Figure 3E), led to a dose- and time-dependent cell surface exposure of murine calreticulin. 373 GSK2857916 had no effect on the parental EL4 cells lacking hBCMA (Figure 3F vs G). 374 GSK2857916 treatment also induced surface exposure of other prominent hallmarks of ICD: 375 HSP90, HSP70 and HMGB1 (Figure S2D). Further, GSK2857916 treatment (at 10 and 100

376 g/mL) led to significantly increased release of calreticulin, HMGB1, and HSP60 into the

377 culture medium over time in EL4-hBCMA (Figure 3H) but not EL4 parental cells (Figure 3I).

378 The significant release of DAMPs in EL4 cells were only seen at the highest dose of 100 g/mL,

379 presumably due to off-target MMAF cytotoxicity (33). Further, the induction of ICD markers in 380 GSK3857916-treated cells occurred despite reduced viability (~10-15% reduction) and cell 381 number (~1.7-2.8-fold reduction) (Table S1B vs S1C).

382 Sustained anti-tumor activity of GSK2857916 is mediated by host innate and adaptive 383 immune responses 384 Having established a murine cell line in which ICD is specifically induced by GSK2857916, we 385 evaluated the anti-tumor activity of GSK2857916 against EL4-hBCMA tumors established in 386 immune-competent mice. Tumor-bearing mice were treated with GSK2857916 doses ranging

387 from 0.001-30 mg/kg. Dose-dependent anti-tumor activity, characterized by decreased tumor 388 burden and significantly increased survival, was observed starting at 0.1 mg/kg of GSK2857916 389 compared to hIgG1-MMAF control (p<0.05). Additionally, higher doses of GSK2857916 led to 390 significant tumor regressions, complete responses in up to 90% of tumor-bearing mice at the 391 highest dose of 30 mg/kg, and significant long-term survival of ≥60 days (Figure 4A, S3A).

392 To assess whether tumor regression was associated with the establishment of an adaptive 393 immune response, re-challenge experiments were conducted. Tumor-free surviving mice from 394 the dose escalation study were re-challenged with either parental EL4 or EL4-hBCMA cells on 395 day 77 (Figure 4B, green triangles vs red diamonds, respectively). Efficacy was compared 396 against naïve age-matched animals challenged with EL4 parental or EL4-hBCMA cells (Figure 397 4B, black squares vs blue triangles, respectively). All tumor-free mice that exhibited a complete 398 response to GSK2857916 treatment rejected the re-challenge with both EL4-hBCMA and EL4 399 parental cells, with no new tumors developing up to 50 days after the secondary inoculation 400 (Figure 4B, S3B). In contrast, all but one naïve animal established new tumors and ultimately 401 succumbed to this challenge (Figure 4B, S3B). The differences between GSK2857916-exposed

402 and naïve mice (re)-challenged with EL4 or EL4-hBCMA cells were significant (p0.001).

403 Rejection of the parental EL4 cell line by the GSK2857916-responding animals suggests that the 404 adaptive immune response against tumors is not directed solely at hBCMA but may target 405 additional neo-epitopes presented by the APCs in this model.

406 To determine the contributions of the different mechanisms of GSK2857916 in vivo activity in 407 the EL4-hBCMA syngeneic model, we examined each mechanism in isolation by comparing 408 several versions of the GSK2857914 antibody and the GSK2857916 ADC (at the 30 mg/kg 409 dose). The unconjugated afucosylated (Fc-enhanced) GSK2857914 antibody showed 410 significantly decreased tumor burden (Figure S3C) and survival advantage (Figure 4C) over the 411 matched afucosylated hIgG1 control antibody (‘hIgG1’; p<0.001 & p<0.001, respectively) and 412 the fucosylated (Fc-disabled) GSK2857914 antibody (‘GSK2857914 disabled’; p<0.001 & 413 p=0.033, respectively). This suggests a significant contribution of the ADCC mechanism of 414 action to in vivo anti-tumor activity. No significant differences in efficacy were observed 415 between GSK2857914 and a GSK2857914 mouse IgG2a chimeric antibody (p>0.05); the latter 416 being the mouse (IgG2a) functional equivalent to human IgG1. GSK2857916 treatment led to

417 both significantly decreased tumor burden (p<0.001) and increased survival (p=0.031), compared 418 to the hIgG1-MMAF control, and 40% durable complete responses (Figure 4C, S3C). We 419 observed slightly decreased tumor burden (p=0.979) and increased survival (p<0.001) after 420 hIgG1-MMAF treatment compared to saline control, at 30 mg/kg (Figure 4C, S3C) but not 10 421 mg/kg (Figure S3A). Previous studies have shown off-target cytotoxicity of ADCs by premature 422 release of cytotoxic drug or uptake of intact ADCs by both receptor-dependent (FcγRs, FcRn and 423 C-type lectin receptors) and -independent (pinocytosis, macropinocytosis) mechanisms (33). 424 Such off-target mechanisms, particularly macropinocytosis, have been proposed to explain the 425 ocular findings observed in patients treated with GSK2857916 (34) and other ADCs (35). 426 Notably, neither complete regressions nor tumor free animals were observed after treatment with 427 the unconjugated antibodies (Figure S3C). Since in this mouse model, complete regressions and 428 resistance to re-challenge were only obtained with GSK2857916, durable anti-tumor responses 429 must require MMAF-induced ICD and an adaptive immune response.

430 To further examine the contribution of GSK2857916 to an adaptive immune response, we 431 performed transient lymphodepletion studies using specific CD4 and CD8 antibodies in 432 GSK2857916 or hIgG1-MMAF treated animals (15 mg/kg; Figure 5A-J). Depletion of CD4+ 433 and CD8+ T-cells was confirmed by flow cytometry on day 15 post-treatment (Figure 5I, J). 434 Depletion of CD8+ T-cells completely abrogated GSK2857916-mediated anti-tumor activity and 435 complete tumor regressions (Figure 5E vs G, p=0.001; S4). In contrast, depletion of CD4+ T- 436 cells did not significantly alter GSK2857916-mediated anti-tumor activity (Figure 5E vs F, 437 p=0.98; S4). The combined depletion of CD4+ and CD8+ T-cell populations did not significantly 438 enhance the effects observed by the single CD8+ T-cell depletion (Figure 5G vs H, p=0.98; S4), 439 further supporting a preponderant role for CD8+ T-cells in mediating anti-tumor activity in this 440 model. We observed a slight growth delay in GSK2857916, CD8 (-) animals (Figure 5G and H 441 vs C and D, S4) potentially attributable to ADCC.

442 Collectively, these results support the hypothesis that GSK2857916 promotes the development of 443 an adaptive tumor-specific immune response, in addition to its previously reported innate 444 immune activity, and highlight the immunogenic potential of GSK2857916 along with 445 GSK2857916-mediated tumor antigen spreading.

446 GSK2857916 enhances intra-tumor immune cell infiltration and activation 447 The pharmacodynamic effects of GSK2857916 were assessed in vivo using the EL4-hBCMA 448 immune-competent model. In the absence of treatment, BCMA expression in this mouse model 449 was maintained on EL4-hBCMA tumor cells (Figure S5A), while there was a significant 450 reduction in dendritic (CD11c) and natural killer (NK; CD49b) cell infiltration during tumor 451 development and progression (Figure S5B).

452 Furthermore, changes in pharmacodynamic markers in vivo following treatment with 453 GSK2857916 were also assessed 24 hours after a second dose of GSK2857916 (15 mg/kg). 454 Immunophenotyping of the tumors by flow cytometry revealed a significant increase in 455 infiltrating CD8+ T-cells after GSK2857916 treatment (Figure 6A); which exhibited 456 significantly increased activation markers including CD25, CD69, and inducible T-cell 457 costimulator (ICOS; Figure 6B). We also observed a significant increase in both CD8+PD-1+ 458 cells (Figure 6B) and CD8+ granzyme B (GrzB)+ cytotoxic cells (Figure 6C). GSK2857916 459 treatment also significantly increased CD4+ T-cell infiltration (Figure 6A) and proliferation; and 460 resulted in a significant increase in regulatory T-cells (Tregs: CD4+CD25+FoxP3+; Figure 6A) 461 in the tumor, only when compared to saline. Increase in tumor infiltrating Tregs has been 462 reported with other ADCs (23) and did not seem to interfere with GSK2857916 anti-tumor 463 effects. Moreover, upregulation of Tregs in the tumor microenvironment has been proposed to 464 control autoreactive effector T-cells and prevent autoimmunity and adverse events (23,36,37). 465 GSK2857916 also significantly increased NK cell (CD49+; Figure 6C) and DC infiltration 466 (CD11c+; Figure 6A), and expression of the co-stimulatory molecule CD86 (Figure 6C), 467 indicating increased DC activation. An independent experiment evaluating TILs by 468 immunohistochemistry 7 days after treatment onset confirmed GSK2857916-dependent increases 469 in infiltrating CD8+ T-cells (Figure 6D, S5C). Moreover, consistent with ICD induction, 470 GSK2857916 treatment led to a reproducible increase in calreticulin expression on the surface of 471 hBCMA expressing tumor cells in vivo (Figure 6E). In vivo ICD was further supported by gene 472 expression analysis of tumors by NanoString, where GSK2857916 treatment induced the 473 expression of a type I IFN-related gene signature (Figure 6F) (24).

474 Combining GSK2857916 with an agonist anti-OX86 antibody increases in vivo efficacy 475 GSK2857916 induced ICD and intra-tumoral immune cell infiltration and activation in vivo, and 476 increased expression of the co-stimulatory molecule OX40 in CD8+ and CD4+ infiltrating T- 477 cells (Figure S5D). We therefore hypothesized that GSK2857916 may synergize with immune- 478 modulatory agents. We evaluated combinations with OX86, a mouse anti-OX40 IgG2a mAb, 479 with a sub-optimal dose of GSK2857916 (10 mg/kg) in the EL4-hBCMA syngeneic model. 480 Combining GSK2857916 with OX86 at 0.2, 1, and 5 mg/kg provided additional benefit

481 compared with each single agent by significantly decreasing tumor burden (p0.002) and

482 increasing survival (p0.003; Figure 7, S6A-C), and resulted in 70, 40 and 50% tumor-free mice,

483 at the respective OX86 doses, at the end of the study (day 60; Figure S6B). In contrast, there 484 were no tumor free mice in the groups treated with GSK2857916 or 0.2 mg/kg OX86 alone, and 485 only one tumor free mouse in each of the groups receiving 1 or 5 mg/kg OX86 alone (Figure 486 S6A, B). Further confirming the role of effector T-cells in GSK2857916-mediated anti-tumor 487 activity and immunologic memory, all tumor-free mice at end of study were immune to re- 488 challenge with EL4-hBCMA cells in an independent experiment.

489

490 Discussion

491 Over the last decade, the 5-year survival rates for MM patients have increased to ~50%, fueled 492 by several new regulatory approvals (38). A wide array of treatment options are now available to 493 patients including autologous stem cell transplant, proteasome inhibitors (bortezomib and 494 carfilzomib), and immune-modulatory agents (thalidomide, lenalidomide and pomalidomide). 495 More recently, additional agents with novel mechanisms of action have been developed like 496 panobinostat, a histone deacetylase inhibitor, and selinexor, a selective inhibitor of the nuclear 497 export protein XPO1. Further, monoclonal antibodies targeting cell surface receptors such as 498 CD38 (daratumumab, isatuximab) and CD319/CS1/signaling lymphocyte activation molecule 499 family (SLAMF) 7 (elotuzumab) have shown anti-neoplastic effects on malignant plasma cells 500 by coopting the innate immune system and eliciting NK cell-mediated ADCC. However, the 501 immune system of MM patients is often significantly compromised contributing to tumor cell 502 immune evasion and suppression (39). Therefore, therapeutic approaches aimed at inducing

503 adaptive immune responses that potentiate tumor-specific immunity represent an exciting area in 504 the treatment of MM.

505 GSK2857916 was evaluated in Phase I and II studies in patients with relapsed/refractory MM 506 (RRMM) and other hematologic malignancies (NCT02064387 and NCT03525678, respectively; 507 www.clinicaltrials.gov), demonstrating clinically meaningful efficacy with a manageable safety 508 profile in RRMM patients (40-42). Further, the FDA and the European Commission recently 509 approved BLENREP (GSK2857916, belantamab mafodotin) as a first-in-class, BCMA targeting 510 therapy for adult patients with RRMM, who have received at least four prior therapies. These 511 clinical studies were supported by the nonclinical evidence that GSK2857916 inhibits tumor 512 growth and prolongs survival in disseminated severe combined immunodeficient (SCID) mouse 513 models bearing human MM xenografts (20). The anti-tumor activity of GSK2857916 was 514 attributed to the direct effect of the toxin (MMAF) on proliferating tumor cells and to NK cell 515 and macrophage mediated ADCC and ADCP, respectively. To allow for the engraftment of 516 human tumor cells, the mice used in the previous nonclinical studies did not contain functional 517 T- or B-cells and consequently, the secondary effects of adaptive tumor immunity were not 518 explored or accounted for in the initial characterization of GSK2857916 mechanism of action in 519 these mouse models.

520 Microtubule-disrupting agents belonging to the maytansine and auristatin family of cytotoxic 521 agents (22,25) and ADCs bearing pyrrolobenzodiazepines (PBD) or tubulysin (Tub) payloads 522 (43), have recently been shown to promote functional DC maturation and activation, and greater 523 anti-tumor activity in immune-competent versus immune-deficient mice. Brentuximab vedotin 524 (an anti-CD30 antibody linked to the payload MMAE) and trastuzumab emtansine (an anti- 525 HER2 antibody linked to the maytansine derivative DM1) are approved ADCs with clinical 526 activity in patients with cancer, shown to promote DC maturation resulting in T-cell priming and 527 adaptive immune responses (22,23). In this study, we provide evidence that GSK2857916 elicits 528 similar immune effects and anti-tumor responses. Our in vitro studies with human and mouse 529 tumor cell lines are the first to demonstrate that GSK2857916 induces an ER stress response that 530 ultimately leads to the production of DAMPs (including calreticulin, HMGB1, ATP) and primes 531 an ICD response in tumor cells in a manner that is dependent on MMAF and BCMA cell surface 532 expression. Further, exposure of human primary immature DCs to tumor cells undergoing

533 GSK2857916-mediated ICD results in DC maturation and activation. DCs represent a rare but 534 key immune cell subset central to developing innate and adaptive immune responses, and much 535 interest exists in modulating their function for the betterment of cancer immunotherapies (44). 536 Our results are in line with prior reports in which the ability of cells to undergo immunogenic 537 apoptosis relied on calreticulin exposure to enable the engulfment of dying tumor cells by DCs 538 (45). Furthermore, exposure or release of other DAMPs including HSP70, HSP90 and ATP, may 539 further enhance DC migration and activation, and cross-presentation of tumor antigens to CD8+ 540 cytotoxic T lymphocytes (46). Interestingly, HSPs are also able to interact with and activate NK 541 cells (47), which might further enhance GSK2857916 anti-tumor activity. In this way, ICD offers 542 additional benefits by boosting the host immune system through tumor-specific immunity.

543 As described, ICD is believed to stimulate immune responses subsequent to the recognition of 544 dying tumor cells by DCs and tumor antigen presentation. Engagement of the immune system in 545 this manner can lead to complete tumor regression and long-term adaptive immunity against the 546 inoculated cancer cells. Furthermore, tumor cells succumbing in a manner that is immunogenic 547 can elicit a protective response against a subsequent challenge with live tumor cells (48). Indeed, 548 GSK2857916 treatment of immune-competent mice bearing syngeneic EL4-hBCMA tumors 549 resulted in potent anti-tumor activity and robust activation of adaptive immune responses, which 550 included intra-tumor dendritic, NK and T-cell infiltration and activation, and maturation of 551 immune cell subsets. These immune responses were likely enhanced by the GSK2857916- 552 dependent induced expression of type I IFN gene signatures, which are known to elicit 553 chemotherapy-dependent anti-tumor immunity by several mechanisms including promoting 554 cross-priming, supporting cytotoxic T lymphocytes and immune effector functions, and releasing 555 pro-inflammatory cytokines. Intra-tumoral expression of such gene signatures has been 556 correlated with favorable disease outcomes in patients with cancer (24,49). Importantly, CD8+ 557 T-cells were indispensable for the potent and durable GSK2857916 anti-tumor activity in our 558 immune-competent mouse model. The requirement for CD8+, but not CD4+, T-cells was 559 previously reported for other ADCs such as dolastatins (22), PBD and Tub (43), and underlines 560 the importance of DCs and subsequently T-cells for the full therapeutic potential of ADCs and of 561 GSK2857916. Notably, the GSK2857916 cytotoxic payload, MMAF, is non-cell-permeable, 562 suggesting that ICD and subsequent DC responses are likely mediated by the BCMA-expressing 563 EL4 tumor cells and not as a result of bystander effects on neighboring “non-target” cells.

564 Furthermore, the capacity of immune-competent mice to reject a re-challenge with EL4 parental 565 or EL4-hBCMA tumor cells after surviving the initial inoculation, supports the role of the 566 immune system in protecting against cancer cells and the idea of tumor-specific antigen 567 spreading and immunologic memory; revealing a novel mechanism of action for GSK2857916. 568 Recent nonclinical studies showed that therapeutic approaches that induce ICD, activate DCs, 569 and promote the priming of tumor antigen-specific T-cells can sensitize to immune checkpoint 570 therapy (50). Concordantly, our in vivo combination results suggest that the anti-tumor activity 571 of GSK2857916 may be further enhanced through combinations with T-cell immune checkpoint 572 inhibitors.

573 Finally, GSK2857916 multiple mechanisms of action provide advantages over other MM 574 therapies. First, the targeted cytotoxic ADC activity of GSK2857916 offers a unique advantage 575 over other “ADCC only” agents in MM. Second, the ADCC/ADCP effector activity of 576 GSK2857916 also contributes to the compound’s anti-tumor activity. The enhanced ADCC 577 activity mediated by the afucosylated Fc portion of GSK2857916 may target multiple mediators 578 of the host immune response resulting in potential synergistic anti-tumor immunity via induction 579 of both innate and adaptive immune pathways. Third, GSK2857916 enhanced antigen 580 presentation through ICD and resulting T-cell mediated durable immunity may provide added 581 therapeutic benefit alone or in combination with immune-enhancing agents. Clinically, there is 582 broad interest in combining immune checkpoint modulation with agents that may increase tumor 583 immunogenicity, potentially expanding the proportion of patients that respond to therapy and 584 favoring achieving minimal residual disease.

585 Here, we describe a novel immune-based mechanism of action for GSK2857916 and its potential 586 for combining with immune-therapeutic agents, providing rationale for testing such 587 combinations in clinical trials; some being evaluated as part of the DREAMM (DRiving 588 Excellence in Approaches to Multiple Myeloma) clinical trial program.

589

590 Acknowledgements

591 The study was funded by GlaxoSmithKline Oncology Research & Development. Drug linker 592 technology licensed from Seagen, Inc.; monoclonal antibody produced using POTELLIGENT

593 Technology licensed from BioWa. The NCI-H929 cell line is licensed with the NIH until 594 December 21, 2025. GSK acknowledges the contributions of Drs. Gazdar and Minna for 595 generating this cell line. The Bioware Ultra Cell Line EL4-luc2 was obtained from Caliper Life 596 Sciences, Inc. (cat #124088). The use and transfer of this product and derivatives thereof are 597 limited by the terms and conditions set by Caliper Life Sciences, Inc., which additionally contain 598 limited use statements from Promega Corporation and The Regents of the University of 599 California (see http://www.caliperls.com/assets/017/7513.pdf). 600

601 Author contributions

602 RM, AA, SB, CB, KP, LS-W, LT, OK, JY, CS, PM, designed the experiments. RM, NV, SB, 603 CB, KP, LS-W, HK, HS, ED, PB, SF, OK, AS, MC-G, performed the experiments. RM, AA, 604 NV, SB, CB, KP, LS-W, HK, HS, HJ, LO, LT, DC, HJ, OK, analyzed the results. GK, CH, NY, 605 JO, MB, RS, JS, AH, provided funding and resources. RM and AA wrote the paper. CB, LS-W, 606 HS, ED, DC, HJ, OK, GK, CS contributed writing to individual sections of the manuscript. All 607 authors reviewed and approved the manuscript.

608

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744 49. Zitvogel L, Galluzzi L, Kepp O, Smyth MJ, Kroemer G. Type I interferons in anticancer 745 immunity. Nat Rev Immunol 2015;15(7):405-14. 746 50. Pfirschke C, Engblom C, Rickelt S, Cortez-Retamozo V, Garris C, Pucci F, et al. 747 Immunogenic Chemotherapy Sensitizes Tumors to Checkpoint Blockade Therapy. 748 Immunity 2016;44(2):343-54.

749

750 Figure Legends 751 Figure 1. GSK2857916 induces ICD markers in NCI-H929 cells. (A) Cell viability was 752 measured by automated flow cytometry 48 hours after treatment. (B) Calreticulin translocation to 753 the cell surface was assessed by flow cytometry 48 hours after treatment. Gating on PI staining 754 allowed to exclude dead cells. (C) HMGB1 release into the media was assessed by ELISA. (D) 755 Extracellular ATP concentrations in the media were determined by a luciferase-based assay. For 756 all measurements, mean ± S.D. of triplicates from a representative experiment shown. A one- 757 way ANOVA statistical analysis was performed. (E-F) NCI-H929 cells were treated with 10

758 g/mL (E) or 1 g/mL (F) of hIgG1-MMAF, GSK2857914 (‘914’) or GSK2857916 (‘916’), or

759 were left untreated (‘No Tx’), for 48 or 72 hours. PERK and eIF2 phosphorylation and BAP31

760 cleavage were evaluated by Western blot. Representative results of multiple independent 761 experiments also indicating the cell viability (%) at the time of sample collection. Note the 762 increase in the high molecular weight band with PERK antibody upon GSK2857916 treatment, 763 which coincides with the PERK phosphorylation (p-PERK) antibody signal. The BAF31 764 cleavage product is indicated by an arrowhead. GAPDH (glyceraldehyde 3-phosphate 765 dehydrogenase) was used as loading control. Indicated in black is the predicted molecular weight 766 of the protein and in red the molecular weight of the protein marker. (G) Cell surface expression 767 of calreticulin, HSP90, HSP70 and HMGB1 by flow cytometry in NCI-H929 cells after 72 hours 768 of treatment. Overlaid histograms of non-permeabilized stained cells shown. Gating was 769 performed on live cells and utilized isotype controls. (H-J) HSP70, HSP90, and IL-8 release into 770 the media after the indicated treatments and at the indicated timepoints was assessed by ELISA 771 in NCI-H929 cells. Shown is a representative result of at least two independent experiments, bars 772 indicate the mean ± S.D. of biological duplicates. A two-way ANOVA statistical analysis was

773 performed, ns= not significant, *p<0.05, **p<0.01, ***p<0.001, ****p0.0001.

774 775 Figure 2. GSK2857916 induces DC activation and maturation in vitro. Differential 776 expression of CD11c and HLA-DR allows for a gating strategy that distinguishes isolated iDCs 777 from NCI-H929 tumor cells. (A) Histograms representing CD11c and HLA-DR surface 778 expression in untreated iDCs and NCI-H929 cells. Gray, antibody histogram; white, isotype 779 histogram. (B) CD40, CD83, and CD86 surface expression by flow cytometry in isolated iDCs 780 from 3 healthy donors following a 24 hour co-culture with NCI-H929 cells, which were 781 pretreated as indicated for 48 hours. Shown are the percentage of positive cells per donor and 782 mean. A two-way ANOVA statistical analysis was performed, ns= not significant, *p<0.05. 783 784 Figure 3. GSK2857916 induces ICD markers in EL4-hBCMA cells. (A) BCMA cell surface 785 expression by flow cytometry in EL4-hBCMA, EL4 parental, and NCI-H929 cells. Overlaid 786 histograms depicting BCMA normalized median fluorescence intensity (MFI) in non- 787 permeabilized stained cells shown. Gating was performed on live cells and utilized isotype 788 controls. (B-C) Dose-response curves in EL4-hBCMA, EL4 parental, and NCI-H929 cells, 789 following 3 days of exposure to GSK2857916 (B) or GSK2857914 (C). Shown is representative 790 data normalized to % growth from at least 3 independent experiments with mean ± S.D. of

791 biological quadruplicates. Mean IC50 values from at least three independent experiments also 792 reported. (D) Calreticulin cell surface expression by flow cytometry in EL4-hBCMA cells at the

793 indicated timepoints in untreated cells and after exposure to 1,10, or 100 g/mL of GSK2857916

794 in vitro. Overlaid histograms of non-permeabilized stained cells shown. Gating was performed 795 on live cells and utilized isotype controls. (E) Calreticulin cell surface expression as in (D) 96

796 hours after exposure to 100 g/mL of GSK2857914 or hIgG1-MMAF and in untreated cells in

797 vitro. (F - G) Calreticulin cell surface expression as in (D or E) in EL4 parental cells. (H - I) 798 Calreticulin, HMGB1 and HSP60 release into the media after the indicated treatments and at the 799 indicated timepoints assessed by ELISA in EL4-hBCMA (H) and EL4 parental (I) cells. Shown 800 is a representative result of at least two independent experiments, bars indicate the mean ± S.D. 801 of biological duplicates or triplicates. A two-way ANOVA statistical analysis was performed,

802 ns= not significant, *p<0.05, **p<0.01, ***p<0.001, ****p0.0001.

803

804 Figure 4. GSK2857916 in vivo anti-tumor activity is mediated by host innate and adaptive 805 immune responses and is MMAF-dependent. (A) Therapeutic efficacy (left) and survival 806 (right) in C57BL/6 mice bearing EL4-hBCMA tumors. Animals were treated i.p. when tumors 807 reached ~150-200 mm3 with saline, hIgG1-MMAF or increasing doses of GSK2857916 twice a 808 week for three weeks, as indicated (n=10 mice/group). (B) Animals with complete and sustained 809 tumor regressions from (A; n=9 mice/group) and age matched naïve controls (n=10 mice/group) 810 were (re)-challenged with 1x105 EL4-hBCMA or EL4 parental cells on day 77 (the last dose of 811 GSK2857916 was on day 17). Therapeutic efficacy (left) and survival (right) were monitored for 812 50 days after cell (re)-inoculation. (C) Therapeutic efficacy (left) and survival (right) in EL4- 813 hBCMA tumor-bearing mice dosed with 30 mg/kg as in (A) with several versions of the 814 GSK2857914 antibody or the GSK2857916 ADC, as indicated (n=10 mice/group). Efficacy data 815 expressed as mean tumor volume ± standard error of the mean (SEM), over time. Kaplan-Meier 816 survival in percentage, significant p-values by a standard log-rank test (p<0.05). 817 818 Figure 5. Depletion of CD8+ T-cells abrogates GSK2857916 anti-tumor activity. (A-H) 819 EL4-hBCMA tumors were established (as in Figure 4) and animals were treated on days 1, 3, 6, 820 9, 13, and 16 with GSK2857916 or hIgG1-MMAF (15 mg/kg; red ticks labeled as ‘ADC’). T- 821 cell-depleting doses of anti-CD4, anti-CD8, or both (300 µg/mouse) were administered on days 822 1, 5, 9, and 16 (blue ticks labeled as ‘Depletion’). Efficacy data expressed as individual tumor 823 volumes over time (n=10 mice/group). The number of tumor free/total mice is indicated in the 824 bottom right corner. (I-J) Depletion of CD4+ and/or CD8+ T-cells was verified on day 15 by 825 flow cytometry. 826 827 Figure 6. GSK2857916 enhances intra-tumor immune cell infiltration and activation. 828 Quantification by flow cytometry of immune cells infiltrating C57BL/6 mice bearing EL4- 829 hBCMA tumors following treatment with GSK2857916 or isotype control (at 15 mg/kg), or 830 saline vehicle. (A) CD8+, CD4+, and regulatory (CD4+CD25+FoxP3+) T-cells, and DC 831 (CD11c+). (B) T-cell activation markers CD25, CD69, and ICOS and the exhaustion marker PD- 832 1. (C) Quantification of the DC co-stimulatory molecule CD86, T-cell proliferation (Ki67) and 833 cytotoxicity (Granzyme B, GrzB) in TILs and NK cells (CD49b+). A one-way ANOVA 834 statistical analysis was performed, ns= not significant, *p<0.05, **p<0.01, ***p<0.001,

835 ****p0.0001. (D) Immunohistochemistry for CD8 reactive cells in mice bearing EL4-hBCMA

836 tumors treated with GSK2857914 (top) or GSK2857916 (bottom) at 15 mg/kg (as in Figure 4). 837 Representative images, 20x magnification. (E) Quantification of calreticulin cell surface 838 expression (MFI) on tumor cells in mice. (F) Heat map depicting a type I IFN-related gene 839 signature following treatment with saline, isotype control or GSK2857916 (at 15 mg/kg) from 840 the same experiment as in (A-C, E). Each column represents an individual mouse and each row 841 an individual gene. 842 843 Figure 7. Therapeutic synergy by combining GSK2857916 and an agonist anti-OX86 844 antibody. Therapeutic efficacy (left) and survival (right) in C57BL/6 mice bearing EL4-hBCMA 845 tumors. Animals were treated i.p. when tumors reached ~150-200 mm3 with GSK2857916 or 846 hIgG1-MMAF at 10 mg/kg and/or a mouse anti-OX86 IgG2a or IgG2a isotype antibody at 0.2, 1 847 and 5 mg/kg, plus saline control, all twice a week for three weeks, as indicated. Efficacy data 848 expressed as mean tumor volume ± SEM over time. Kaplan-Meier survival in percentage, 849 significant p-values by a standard log-rank test (p<0.05; n=10 mice/group) provided for the 850 combination against the 1st or 2nd single agent listed, respectively. 851

Downloaded from mct.aacrjournals.org on September 25, 2021. © 2021 American Association for Cancer Research. Author Manuscript Published OnlineFirst on July 12, 2021; DOI: 10.1158/1535-7163.MCT-21-0035 Figure 1. GSK2857916Author manuscripts have induces been peer reviewed ICD andmarkers accepted for inpublication NCI-H929 but have not cells yet been edited.

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GSK2857916 (g/mL) GSK2857916 (g/mL) GSK2857916 (g/mL) GSK2857916 (g/mL) E G No Tx hIgG1-MMAF 914 916

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GSK2857916, 1 g/mL GAPDH 38 kDa 37 kDa GSK2857916, 10 g/mL Viability (%) 92 88 91 91 70

Figure 1. GSK2857916 induces ICD markers in NCI-H929 cells. (A) Cell viability was measured by automated flow cytometry 48 hours after treatment. (B) Calreticulin translocation to the cell surface was assessed by flow cytometry 48 hours after treatment. Gating on PI staining allowed to exclude dead cells. (C) HMGB1 release into the media was assessed by ELISA. (D) Extracellular ATP concentrations in the media were determined by a luciferase-based assay. For all measurements, mean ± S.D. of triplicates from a representative experiment shown. A one-way ANOVA statistical analysis was performed. (E-F) NCI-H929 cells were treated with 10 g/mL (E) or 1 g/mL (F) of hIgG1-MMAF, GSK2857914 (‘914’) or GSK2857916 (‘916’), or were left untreated (‘No Tx’), for 48 or 72 hours. PERK and eIF2 phosphorylation and BAP31 cleavage were evaluated by Western blot. Representative results of multiple independent experiments also indicating the cell viability (%) at the time of sample collection. Note the increase in the high molecular weight band with PERK antibody upon GSK2857916 treatment, which coincides with the PERK phosphorylation (p-PERK) antibody signal. The BAF31 cleavage product is indicated by an arrowhead. GAPDH (glyceraldehyde 3-phosphate dehydrogenase) was used as loading control. Indicated in black is the predicted molecular weight of the protein and in red the molecular weight of the protein marker. (G) Cell surface expression of calreticulin, HSP90, HSP70 and HMGB1 by flow cytometry in NCI-H929 cells after 72 hours of treatment. Overlaid histograms of non- permeabilized stained cells shown. Gating was performed on live cells and utilized isotype controls. (H-J) HSP70, HSP90, and IL-8 release into the media after the indicated treatments and at the indicated timepoints was assessed by ELISA in NCI-H929 cells. Shown is a representative result of at least two independent experiments, bars indicate the mean ± S.D. of biological duplicates. A two-way ANOVA statistical analysis was performed, ns=Downloaded not significant, from *p<0.05, mct.aacrjournals.org **p<0.01, ***p<0.001, on September ****p  25,0.0001. 2021. © 2021 American Association for Cancer Research. Author Manuscript Published OnlineFirst on July 12, 2021; DOI: 10.1158/1535-7163.MCT-21-0035 Figure 1 (continuation).Author manuscripts have GSK2857916 been peer reviewed and inducesaccepted for publication ICD markers but have not yet in been NCI-H929 edited. cells

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Figure 1. GSK2857916 induces ICD markers in NCI-H929 cells. (A) Cell viability was measured by automated flow cytometry 48 hours after treatment. (B) Calreticulin translocation to the cell surface was assessed by flow cytometry 48 hours after treatment. Gating on PI staining allowed to exclude dead cells. (C) HMGB1 release into the media was assessed by ELISA. (D) Extracellular ATP concentrations in the media were determined by a luciferase-based assay. For all measurements, mean ± S.D. of triplicates from a representative experiment shown. A one-way ANOVA statistical analysis was performed. (E-F) NCI-H929 cells were treated with 10 g/mL (E) or 1 g/mL (F) of hIgG1-MMAF, GSK2857914 (‘914’) or GSK2857916 (‘916’), or were left untreated (‘No Tx’), for 48 or 72 hours. PERK and eIF2 phosphorylation and BAP31 cleavage were evaluated by Western blot. Representative results of multiple independent experiments also indicating the cell viability (%) at the time of sample collection. Note the increase in the high molecular weight band with PERK antibody upon GSK2857916 treatment, which coincides with the PERK phosphorylation (p-PERK) antibody signal. The BAF31 cleavage product is indicated by an arrowhead. GAPDH (glyceraldehyde 3-phosphate dehydrogenase) was used as loading control. Indicated in black is the predicted molecular weight of the protein and in red the molecular weight of the protein marker. (G) Cell surface expression of calreticulin, HSP90, HSP70 and HMGB1 by flow cytometry in NCI-H929 cells after 72 hours of treatment. Overlaid histograms of non- permeabilized stained cells shown. Gating was performed on live cells and utilized isotype controls. (H-J) HSP70, HSP90, and IL-8 release into the media after the indicated treatments and at the indicated timepoints was assessed by ELISA in NCI-H929 cells. Shown is a representative result of at least two independent experiments, bars indicate the mean ± S.D. of biological duplicates. A two-way ANOVA statistical analysis was performed, ns=Downloaded not significant, from *p<0.05, mct.aacrjournals.org **p<0.01, ***p<0.001, on September ****p  25,0.0001. 2021. © 2021 American Association for Cancer Research. Author Manuscript Published OnlineFirst on July 12, 2021; DOI: 10.1158/1535-7163.MCT-21-0035 Figure 2. GSK2857916Author manuscripts have induces been peer reviewed DC activation and accepted for publication and maturation but have not yet been in edited. vitro

A Dendritic Cells NCI-H929 cells

CD11c HLA-DR CD11c HLA-DR B % CD40+ cells from % CD83+ cells from % CD86+ cells from CD11c+ DC HLA-DR+ DC HLA-DR+ DC ✱ ✱ ✱ ns ns ns 60 ns 60 ns 60 ns

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Figure 2. GSK2857916 induces DC activation and maturation in vitro. Differential expression of CD11c and HLA-DR allows for a gating strategy that distinguishes isolated iDCs from NCI-H929 tumor cells. (A) Histograms representing CD11c and HLA-DR surface expression in untreated iDCs and NCI-H929 cells. Gray, antibody histogram; white, isotype histogram. (B) CD40, CD83, and CD86 surface expression by flow cytometry in isolated iDCs from 3 healthy donors following a 24 hour co-culture with NCI-H929 cells, which were pretreated as indicated for 48 hours. Shown are the percentage of positive cells per donor and mean. A two-way ANOVA statistical analysis was performed, ns= not significant, *p<0.05.

Downloaded from mct.aacrjournals.org on September 25, 2021. © 2021 American Association for Cancer Research. Author Manuscript Published OnlineFirst on July 12, 2021; DOI: 10.1158/1535-7163.MCT-21-0035 Figure 3. GSK2857916Author manuscripts have induces been peer reviewed ICD andmarkers accepted for publicationin EL4 but-hBCMA have not yet beencells edited.

A B C 125 125

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Figure 3. GSK2857916 induces ICD markers in EL4-hBCMA cells. (A) BCMA cell surface expression by flow cytometry in EL4-hBCMA, EL4 parental, and NCI-H929 cells. Overlaid histograms depicting BCMA normalized median fluorescence intensity (MFI) in non-permeabilized stained cells show n. Gating w as performed on live cells and utilized isotype controls. (B-C) Dos e-response curves in EL4-hBCMA, EL4 parental, and NCI-H929 cells, follow ing 3 days of exposure to GSK2857916 (B) or GSK2857914 (C). Show n is representative data normalized to % grow th from at least 3 independent experiments w ith mean ± S.D. of biological quadruplicates. Mean IC50 values from at least three independent experiments also reported. (D) Calreticulin cell surface expression by flow cytometry in EL4-hBCMA cells at the indicated timepoints in untreated cells and after exposure to 1,10, or 100 µg/mL of GSK2857916 in vitro. Overlaid histograms of non-permeabilized stained cells show n. Gating w as performed on live cells and utilized isotype controls. ( E) Calreticulin cell surface expression as in (D) 96 hours after exposure to 100 µg/mL of GSK2857914 or hIgG1-MMAF and in untreated cells in vitro. (F - G) Calreticulin cell surface expression as in (D or E) in EL4 parental cells. (H - I) Calreticulin, HMGB1 and HSP60 release into the media after the indicated treatments and at the indicated timepoints assessed by ELISA in EL4-hBCMA (H) and EL4 parental (I) cells. Show n is a representative result of at least tw o independent experiments, bars indicate the mean ± S.D. of biological duplicates or triplicates. A tw o-w ay ANOVA statistical analysis w as performed, ns= not significant, *p<0.05, **p<0.01, ***p<0.001, ****p≤0.0001.

Downloaded from mct.aacrjournals.org on September 25, 2021. © 2021 American Association for Cancer Research. Author Manuscript Published OnlineFirst on July 12, 2021; DOI: 10.1158/1535-7163.MCT-21-0035 Figure 3 (continuation).Author manuscripts have GSK2857916 been peer reviewed and induces accepted for publication ICD markers but have not yet in been EL4-hBCMA edited. cells

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biological quadruplicates. Mean IC50 values from at least three independent experiments also reported. (D) Calreticulin cell surface expression by flow cytometry in EL4-hBCMA cells at the indicated timepoints in untreated cells and after exposure to 1,10, or 100 g/mL of GSK2857916 in vitro. Overlaid histograms of non-permeabilized stained cells shown. Gating was performed on live cells and utilized isotype controls. (E) Calreticulin cell surface expression as in (D) 96 hours after exposure to 100 g/mL of GSK2857914 or hIgG1-MMAF and in untreated cells in vitro. (F - G) Calreticulin cell surface expression as in (D or E) in EL4 parental cells. (H - I) Calreticulin, HMGB1 and HSP60 release into the media after the indicated treatments and at the indicated timepoints assessed by ELISA in EL4-hBCMA (H) and EL4 parental (I) cells. Shown is a representative result of at least two independent experiments, bars indicate the mean ± S.D. of biological duplicates or triplicates. A two-way ANOVA statistical analysis was performed, ns= not significant, *p<0.05, **p<0.01, ***p<0.001, ****p0.0001.

Downloaded from mct.aacrjournals.org on September 25, 2021. © 2021 American Association for Cancer Research. Author Manuscript Published OnlineFirst on July 12, 2021; DOI: 10.1158/1535-7163.MCT-21-0035 Figure 4. GSK2857916Author manuscripts have in been vivo peer reviewedanti-tumor and accepted activity for publication is but mediated have not yet been by edited. host innate and adaptive immune responses and is MMAF-dependent

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Figure 4. GSK2857916 in vivo anti-tumor activity is mediated by host innate and adaptive immune responses and is MMAF- dependent. (A) Therapeutic efficacy (left) and survival (right) in C57BL/6 mice bearing EL4-hBCMA tumors. Animals were treated i.p. when tumors reached ~150-200 mm3 with saline, hIgG1-MMAF or increasing doses of GSK2857916 twice a week for three weeks, as indicated (n=10 mice/group). (B) Animals with complete and sustained tumor regressions from (A; n=9 mice/group) and age matched naïve controls (n=10 mice/group) were (re)-challenged with 1x105 EL4-hBCMA or EL4 parental cells on day 77 (the last dose of GSK2857916 was on day 17). Therapeutic efficacy (left) and survival (right) were monitored for 50 days after cell (re)-inoculation. (C) Therapeutic efficacy (left) and survival (right) in EL4-hBCMA tumor-bearing mice dosed with 30 mg/kg as in (A) with several versions of the GSK2857914 antibody or the GSK2857916 ADC, as indicated (n=10 mice/group). Efficacy data expressed as mean tumor volume ± standard error of the mean (SEM), over time. Kaplan-Meier survival in percentage, significant p-values by a standard log-rank test (p<0.05).

Downloaded from mct.aacrjournals.org on September 25, 2021. © 2021 American Association for Cancer Research. Author Manuscript Published OnlineFirst on July 12, 2021; DOI: 10.1158/1535-7163.MCT-21-0035 Figure 5. DepletionAuthor manuscripts of have CD8+ been peer T-cells reviewed andabrogates accepted for publication GSK2857916 but have not yet beenanti-tumor edited. activity

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Figure 5. Depletion of CD8+ T-cells abrogates GSK2857916 anti-tumor activity. (A-H) EL4-hBCMA tumors were established (as in Figure 4) and animals were treated on days 1, 3, 6, 9, 13, and 16 with GSK2857916 or hIgG1-MMAF (15 mg/kg; red ticks labeled as ‘ADC’). T-cell-depleting doses of anti-CD4, anti-CD8, or both (300 µg/mouse) were administered on days 1, 5, 9, and 16 (blue ticks labeled as ‘Depletion’). Efficacy data expressed as individual tumor volumes over time (n=10 mice/group). The number of tumor free/total mice is indicated in the bottom right corner. (I-J) Depletion of CD4+ and/or CD8+ T-cells was verified on day 15 by flow cytometry.

Downloaded from mct.aacrjournals.org on September 25, 2021. © 2021 American Association for Cancer Research. Author Manuscript Published OnlineFirst on July 12, 2021; DOI: 10.1158/1535-7163.MCT-21-0035 Figure 6. GSK2857916Author manuscripts enhances have been peer intra-tumor reviewed and accepted immune for publication cell infiltration but have not yet andbeen edited. activation A CD8+ CD4+ CD11c+ CD4+ CD25+ FoxP3+ ✱✱ ✱ ✱✱ ✱

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Figure 6. GSK2857916 enhances intra-tumor immune cell infiltration and activation. Quantification by flow cytometry of immune cells infiltrating C57BL/6 mice bearing EL4-hBCMA tumors following treatment with GSK2857916 or isotype control (at 15 mg/kg), or saline vehicle. (A) CD8+, CD4+, and regulatory (CD4+CD25+FoxP3+) T-cells, and DC (CD11c+). (B) T-cell activation markers CD25, CD69, and ICOS and the exhaustion marker PD-1. (C) Quantification of the DC co-stimulatory molecule CD86, T-cell proliferation (Ki67) and cytotoxicity (Granzyme B, GrzB) in TILs and NK cells (CD49b+). A one-way ANOVA statistical analysis was performed, ns= not significant, *p<0.05, **p<0.01, ***p<0.001, ****p0.0001. (D) Immunohistochemistry for CD8 reactive cells in mice bearing EL4-hBCMA tumors treated with GSK2857914 (top) or GSK2857916 (bottom) at 15 mg/kg (as in Figure 4). Representative images, 20x magnification. (E) Quantification of calreticulin cell surface expression (MFI) on tumor cells in mice. (F) Heat map depicting a type I IFN-related gene signature following treatment with saline, isotype control or GSK2857916 (at 15 mg/kg) from the same experimentDownloaded as in (A-C, from E). Eachmct.aacrjournals.org column represents anon individual September mouse 25, and 2021. each row© 2021 an individual American gene. Association for Cancer Research. Author Manuscript Published OnlineFirst on July 12, 2021; DOI: 10.1158/1535-7163.MCT-21-0035 Figure 7. TherapeuticAuthor manuscripts havesynergy been peer reviewed by combining and accepted for publication GSK2857916 but have not yet and been edited. an agonist anti-OX86 antibody

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Figure 7. Therapeutic synergy by combining GSK2857916 and an agonist anti-OX86 antibody. Therapeutic efficacy (left) and survival (right) in C57BL/6 mice bearing EL4-hBCMA tumors. Animals were treated i.p. when tumors reached ~150-200 mm3 with GSK2857916 or hIgG1-MMAF at 10 mg/kg and/or a mouse anti-OX86 IgG2a or IgG2a isotype antibody at 0.2, 1 and 5 mg/kg, plus saline control, all twice a week for three weeks, as indicated. Efficacy data expressed as mean tumor volume ± SEM over time. Kaplan-Meier survival in percentage, significant p-values by a standard log-rank test (p<0.05; n=10 mice/group) provided for the combination against the 1st or 2nd single agent listed, respectively.

Belantamab Mafodotin (GSK2857916) Drives Immunogenic Cell Death and Immune-Mediated Anti-Tumor Responses In Vivo

Rocio Montes de Oca, Alireza S Alavi, Nick Vitali, et al.

Mol Cancer Ther Published OnlineFirst July 12, 2021.

Updated version Access the most recent version of this article at: doi:10.1158/1535-7163.MCT-21-0035

Supplementary Access the most recent supplemental material at: Material http://mct.aacrjournals.org/content/suppl/2021/07/01/1535-7163.MCT-21-0035.DC1

Author Author manuscripts have been peer reviewed and accepted for publication but have not yet been Manuscript edited.

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