1837 Serum Proteomic Analysis of Multiple Myeloma Subjects Treated With Daratumumab Monotherapy

Tineke Casneuf, 1 Andrew Lysaght, 2 Clare LeFave, 3 Jaime Bald, 4 Brendan Weiss, 5 Niels W.C.J. van de Donk, 6 Henk M. Lokhorst, 6 Tahamtan Ahmadi, 4 A. Kate Sasser 4, * 1Janssen Research & Development, Beerse, Belgium; 2Immuneering Corporation, Cambridge, MA, USA; 3LabConnect, LLC, Seattle, WA, USA; 4Janssen Research & Development, LLC, Spring House, PA, USA; 5Division of Hematology/Oncology, Department of Medicine, Abramson Cancer Center and Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA; 6Department of Hematology, VU University Medical Center, Amsterdam, The Netherlands. *Presenting author.

– Site ID was determined to be significantly associated with ✦ Expression of interleukin (IL)-1ß was significantly lower in – PD-L1/CD274 is the ligand for the receptor PD-1; the ✦ Two proteins, VCAM1 and GSN, were identified by all TLR2 ICOS CD163 INTRODUCTION METHODS the variables of interest (eg, demographics, response responders compared to non-responders ( P = 0.046; –7 engagement of PD-1 on T cells leads to reduced T-cell 3 applied statistical tests (shown in red text in Table 1 ) P = 0.001 14 P = 0.007 P = 2.53!10 11.0 17 class, and sample time point; ≥7.37% variability explained; Figure 4B ) 13.0 function and increased regulatory T-cell development Daratumumab Study Designs Wald test P = 3.71×10 –9 ), and ComBat 7 was utilized to Table 1. Proteins Exhibiting Differential Expression – In the bone marrow microenvironment, high 13 12.5

correct for site ID effects to reduce the impact of site- n 10.5 PD-L1 Between Baseline Versus On-Treatment Samples by o i

✦ Daratumumab is a first-in-class, human anti-CD38 IgG1 ✦ GEN501 was a phase 1/2, open-label, multicenter study concentrations of IL-1ß elicit the release of IL-6, which s related effects within the data s 12.0 Statistical Test e 14 r 12 Responders Non-responders monoclonal antibody in clinical development across the conducted in 2 parts ( Figure 3 ) promotes myeloma cell survival and expansion p x multiple myeloma (MM) disease spectrum ✦ For GEN501 Part 2, paired baseline and on-study samples E 10.0 11.5 ● Wilcoxon ANOVA:Visit Friedman – Part 1 was a dose-escalation phase ✦ E-cadherin (CDH1) and P-cadherin (CDH3) were shown to 14 from 16 patients were run in 1 batch and the data were VCAM1 VCAM1 VCAM1 1,2 11 11.0 ✦ High levels of CD38 are expressed on myeloma cells be increased in responders ( P = 0.007 and 0.012, GSN GSN GSN – Part 2 was a dose-expansion phase pre-processed with the SomaLogic standardization 9.5 ● ● respectively; Figure 4B ) 10.5 ● CXCL12 THBS4 CXCL12 ✦ The antimyeloma activity of daratumumab is mediated workflow 13 ● Baseline On BaselineOn Baseline On ● ● IGHM MMP3 ✦ SIRIUS was a phase 2, open-label, international, multicenter ● ● treatment treatment treatment ● ● through a number of mechanisms of action, including – Soluble serum E-cadherin is an independent prognostic ● PRTN3 ● study of Simon 2-stage design ( Figure 3 ) ● ● ● n ● ● ● PLG Non-responder Responder Stable o

complement-dependent cytotoxicity, antibody-dependent Statistical Analyses marker of poor survival for newly diagnosed patients i ● ● s ●

s ● 12 ● THBS4

15 e ●

cell-mediated toxicity, antibody-dependent cellular with MM r

p ● ✦ Responders versus non-responders ● ●

x MMP3 TLR2, toll-like receptor 2; ICOS, inducible T-cell co-stimulator; CD163, cluster of differentiation 163. ● 3,4 E ● ● ● phagocytosis, and apoptosis via cross-linking GEN501 SIRIUS ● ● 16 ● PIGR – The role of CDH3 is similar to that of CDH1 ● ● – Statistical comparison of protein concentration ● ● ● ● Boldface and underlined text indicates that the SOMAmer was also identified in the comparison of Figure 5. Immune-related proteins increased on ● ● ● 11 ● ● ✦ Two studies, GEN501 (ClinicalTrials.gov Identifier: Dose-escalation Randomization ● ● baseline versus on-treatment samples in SIRIUS. distributions in daratumumab responders (partial ● ● ● treatment in all response classes. ● VCAM1, vascular cell adhesion molecule 1; GSN, gelsolin; CXCL12, C-X-C motif chemokine 12; ● ● NCT00574288) and SIRIUS (NCT01985126), demonstrated A. ● IGHM, immunoglobulin heavy constant mu; PRTN3, proteinase 3; PLG, plasminogen; Color key ● ● response [PR], very good PR [VGPR], complete response ● ● THBS4, thrombospondin 4; MMP3, matrix metalloproteinase 3; PIGR, polymeric immunoglobulin and histogram ● that single-agent daratumumab had a tolerable safety ● receptor.

Dose from 0.005-24 mg/kg 0 16 mg/kg 8 mg/kg [CR], and stringent CR [sCR]) versus non-responders 6 ● (n = 32) 10 0 (n = 16) (n = 18) t Differential Protein Changes in Responders Versus 4

profile and promising efficacy in patients with relapsed or n

u ●

(progressed disease [PD]) was performed at both baseline o 0 C refractory MM ( Figure 1 )5,6 2 Non-responders Due to Treatment and on treatment using 2 complementary methods: 0 Baseline On treatment Baseline On treatment Safety and response −4 −2 0 2 4 8 Row Z−Score ✦ 60 proteins were identified as having significantly evaluated Response evaluated (i) Wilcoxon rank-sum test on each individual SOMAmer Red lines indicate a decrease over time. Green lines indicate an increase over time. CONCLUSIONS 9 differential levels between responders and non-responders and (ii) Limma analysis on all SOMAmers simultaneously DYRK3 HMGB1 PD-L1, programmed cell death ligand 1. 40 PPIB An additional FLT3 ADAMTS13 upon daratumumab treatment CD163 35 TPM2 Dose-expansion 90 patients TNFSF8 – All P values were adjusted using the Benjamini-Hochberg CADM1 Figure 7. Changes in immune checkpoint marker PD-L1. ✦ Several proteins with differential expression CDC42BPB enrolled at DDR2 30 CAMK1 ENTPD5 ✦ Some of these proteins have known roles in myeloma DARA 16 mg/kg 10,11 MRC1 (BH) method for multiple hypothesis correction MST1 between responders and non-responders at baseline 25 BMP10 ADCYAP1 %

TLR4.LY96

, biology (B-cell maturation antigen [BCMA], signaling IL1B 16 mg/kg 8 mg/kg 16 mg/kg CCL11

R were identified, many of which are associated with 20 KYNU GEN501 Part 2 CD84 R (n = 42) (n = 30) (n = 106) – Wilcoxon-adjusted P values are reported here CLEC7A HSD17B1 lymphocyte activation molecule [SLAM] family member 7 O 15 SIGLEC7 MM or CD38 HAPLN1 PSMA2 CSNK2A1 ✦ As in SIRIUS, treatment induced differential protein DNAJC19 [SLAMF7], transmembrane activator and CAML interactor 10 TNFRSF11A DARA, daratumumab. ✦ Baseline versus on treatment ING1 SHH CDC37 expression in GEN501 Part 2 ( Table 1 ) 5 IL11 [TACI], and beta-2-microglobulin [B2M]; Figure 6 ) ✦ 142 proteins were identified as differentially CNTF PES1 TNFSF9 0 CCL8 Figure 3. Study design for GEN501 and SIRIUS. – Baseline versus on treatment protein levels were TGM3 expressed between baseline and on treatment SOD2 CDH3 – Of the proteins identified, all but 3 were also identified in 8 mg/kg 16 mg/kg 8 mg/kg 16 mg/kg EGFR SERPINC1 MAP2K4 Immune and T-cell–related Proteins compared using 3 alternative statistical methods: (i) 2-way SERPINA4 GEN501 SIRIUS CDH1 NOTCH1 the comparison of baseline versus on-treatment samples – Two proteins involved in T-cell stimulation (TLR2 SEMA6A 12 SPARCL1 ✦ In both studies, patients were ≥18 years of age, had repeated-measures analysis of variance (ANOVA), (ii) the SET ✦ On treatment, expression of immune checkpoint marker HFE2 PDGFRB in SIRIUS PR VGPR CR sCR SLAMF7 and ICOS) increased in all patients 8 8 CFP R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R documented myeloma requiring systemic therapy, and Wilcoxon signed-rank test, and (iii) the Friedman test R programmed cell death ligand 1 (PD-L1) decreased in N N N N N N N N N N N N N N N N N had an Eastern Cooperative Oncology Group performance responders and increased in non-responders compared to – These results further validate the observations in the ✦ 60 proteins were identified as differentially ORR, overall response rate; PR, partial response; VGPR, very good partial response; CR, complete – All P values were adjusted to control false discovery rate Centered and scaled protein concentrations are presented in the rows, and patients are presented response; sCR, stringent complete response. status of ≤2 in the columns. The horizontal color-bar located above the heat map indicates response class: baseline ( Figure 7 ) SIRIUS study expressed between daratumumab responders using the BH method for multiple hypothesis correction purple = non-responder, green = responder. Figure 1. Overall response rates with daratumumab versus non-responders over time; these proteins – In GEN501, patients had relapsed from or were refractory monotherapy in GEN501 and SIRIUS. – A 2-way repeated-measures ANOVA 12 was applied to B. include markers of tumor burden, which decrease in to ≥2 prior lines of therapy, including proteasome MST1 TNFSF8 TNFSF9 BCMA SLAMF7 determine if significant time-point:response-class 8.75 Responders Non-responders Responders Non-responders responders and increase in non-responders inhibitors (PIs), immunomodulatory drugs (IMiDs), P = 0.012 P = 0.003 P = 0.019 ● ● ● interaction occurred for each SOMAmer; a modified 13 12.0 ● ● SomaLogic Platform ● chemotherapy, and autologous stem cell transplantation ● ✦ On treatment, PD-L1 expression decreased in ● 18 ● 8.50 ● ● ● Wilcoxon rank-sum test was applied as a post hoc test to ● ● ● ● ● ● ● ● ● ● ● n ● responders and increased in non-responders ● ● ● ● ● ● ✦ The SOMAscan Assay (SomaLogic, Inc., Boulder, CO) utilizes ● ● ● ● ● ● o 12 ● ● ● – In SIRIUS, patients had progressed on their most recent specifically determine if responders and non-responders i 11.5 16 ● ● ●

s ● ● ● ● ● s ● ● ● compared to baseline an aptamer-based technology to measure >1,100 native 8 e 8.25 ● ● ● ● r ● ● ● line of therapy, and had received ≥3 prior lines of therapy showed different treatment effects ● p ● ● ●

x ● ● ● 17 ● ● proteins in complex matrices by transforming each E 11 ● ● ● ● ● ● including a PI and an IMiD or were double refractory to 11.0 ● – The relevance of these findings in the context of ● ● ● ● ● – Significance values were adjusted using the BH method, ● ● ● individual protein concentration into a corresponding ● ● ● 8.00 n ● ● ● n ● the observed immunomodulatory effect of both a PI and an IMiD o ● ● o i ● ● ●

● i s

and the null hypothesis was rejected when the adjusted ● s SOMAmer reagent concentration, which can then be 10 s 14 s ● 10.5 e ● ● e daratumumab (Poster 3037) will be investigated in r ● ● ● ● r ● p 16 ✦ A dosing schedule of 16 mg/kg weekly for 8 weeks, every P value <0.05 p ● quantified using microarrays or quantitative PCR x ● ● ● x E ● ● future studies ● E ● NR R NR R NR R ● other week for 16 weeks, and then monthly thereafter was ● ● ● ● ✦ SOMAmer reagents cover a broad array of proteins that ● ● ● ● ✦ Confirmation of these observations in a larger established as the recommended dose ● ● IL-1ß CDH1 CDH3 ● ● are associated with cellular processes and disease ● ● 15 ● sample size and using other methods of detection is 12 ● ● P = 0.046 P = 0.007 P = 0.012 ● pathophysiology ( Figure 2 ) RESULTS 16.0 ● ● Analysis Methods ● ● underway 15.2 ● ● 11 ● ● ● Hormones ✦ From SIRIUS, paired baseline and on-treatment serum n 15.5 ● ●

o 14

SIRIUS i

3% s Protease Structural samples of 90 patients, plus another 15 patients at baseline s 14.8 e

inhibitors proteins r Baseline On treatment Baseline On treatment Baseline On treatment Baseline On treatment

Aging p

5% 1% only, were profiled Responders Versus Non-responders at Baseline x 15.0 Cancer E 10 TACI B2M REFERENCES Cardiovascular Growth ✦ A total of 51 proteins were identified as being significantly 14.4 Responders Non-responders Responders Non-responders 1. Lin P, et al. Am J Clin Pathol . 2004;121(4):482-488. Metabolic/endocrine factors Receptors ✦ For GEN501 Part 2, paired baseline and on-treatment serum Inflammation 13% 14.5 2. Santonocito AM, et al. Leuk Res . 2004;28(5):469-477. 21% different at baseline between responders and non- ● ● Neurologic samples of 16 patients were tested 13 3. de Weers M, et al. J Immunol . 2011;186(3):1840-1848. Pulmonary Proteases Cytokines responders ( P values <0.05; Figure 4A ) 4. Overdijk MB, et al. MAbs . 2015;7(2):311-321. 17% 20% 5. Lokhorst HM, et al. N Engl J Med . 2015;373(13):1207-1219. Renal NR R NR R NR R ● 13 ● Kinases ✦ The assays were performed at SomaLogic, Inc. 6. Lonial S, et al. Lancet . 2015. In press. Critical care ● 20% – Cytokines, cadherins, and tumor necrosis factor (TNF) Non-responder Responder ● 7. Johnson WE, et al. Biostatistics . 2007;8(1):118-127. Signal transduction ● Stress response ● ● 8. Hollander M, et al. Nonparametric Statistical Methods . Hoboken, NJ: John Wiley & Sons, Inc.; 1973:27-75. ● ● ● family members were among the identified proteins ● ● Proteolysis ● ● 9. Ritchie ME, et al. Nucleic Acids Res . 2015;43(7):e47. Data Pre-Processing MST1, macrophage-stimulating 1; TNFSF8, tumor necrosis factor subfamily 8; TNFSF9, tumor necrosis 12 ● ● ● ● 10. Benjamini Y, Hochberg Y. J R Statist Soc B . 1995;57(1):289-300. Phosphorylation factor subfamily 9; NR, non-responder; R, responder; IL-1 ß, interleukin-1 ß; CDH1, cadherin 1; ● ● ● ● ● 12 ● n – Of the 51 identified proteins, 30 were higher in responders CDH3, cadherin 3. n 11. R: A Language and Environment for Statistical Computing. Vienna, Austria: R Foundation for Statistical Intracellular transport ● ● ● ● o Intracellular ✦ For SIRIUS, profiling was performed in 2 batches of ● ● o i i ● ● Computing; 2011. s s ● Immune process ● ● s 25% s ● ● ● Secreted and 21 were lower ● ● ● 12. Chambers JM, Hastie TJ, eds. Statistical Models in S . Boca Raton, FL: Chapman & Hall/CRC; 1992. e e ● ● r Cell differentiation 180 samples (paired baseline and on-treatment samples r ● ● 47% Figure 4. Proteins with different concentration ● ● p p ● ● ● ● 13. Chen L, Flies DB. Nat Rev Immunol . 2013;13(4):227-242. Cell adhesion x ● x ● ● ● ● Extracellular ● from 90 patients) and 50 samples (all at baseline, E ● E ● 14. Lust JA, et al. Mayo Clin Proc . 2009;84(2):114-122. domains distributions between responders and non-responders ● ● ● ✦ Macrophage-stimulating protein (MSP/MST1) expression ● ● ● ● ● ● ● 0100200300400500 11 ● ● 15. Syrigos KN, et al. Anticancer Res . 2004;24(3b):2027-2031. 28% ● 11 ● ● ● ● ● 35 repeated from Batch 1); SomaLogic’s standard workflow ● ● was significantly higher in responders versus non- at baseline. ● ● ● ● 16. van Roy F. Nat Rev Cancer . 2014;14(2):121-134. ● ● Number of SOMAmers ● ● ● ● 17. Armand P. Blood . 2015;125(22):3393-3400. ● ● ● ● controlled for inter-plate variability ● ● ● ● ● ● ● ● ● responders ( P = 0.012; Figure 4B ) ● ● ● ● ● ● ● ● ● ● ● ● ● Figure 2. Proteins measured by the SomaLogic Platform. ● ● ● ● ● ACKNOWLEDGMENTS Treatment-induced Response-independent Changes ● ● – Samples from 35 patients, shared between the 2 batches, ● ✦ TNF family members TNFSF8/CD30L/CD153 ( P = 0.003; ● 10 ● This study was sponsored by Janssen Research & Development, in Protein Expression 10 ● were used to rescale each of the Batch 2 sample ● ● LLC. Editorial and medical writing support were provided by Figure 4B ) and TNFSF9/4-1BBL/CD137L ( P = 0.019; Figure 4B ) ● ● Erica Chevalier-Larsen, PhD, of MedErgy, and was funded by SOMAmer values with the median of the 35 repeated- also had significantly higher expression in responders versus ✦ 142 proteins were identified as having different Janssen Global Services, LLC. Baseline On treatment Baseline On treatment Baseline On treatment Baseline On treatment OBJECTIVES sample Batch 1/Batch 2 ratios for inter-batch variability non-responders concentrations at baseline versus on treatment by ANOVA, correction Wilcoxon rank-sum test, and Friedman test (BH-adjusted Red lines indicate a decrease over time. Green lines indicate an increase over time. DISCLOSURES BCMA, B-cell maturation antigen; SLAMF7 , signaling lymphocyte activation molecule family member 7; TACI, transmembrane activator and CAML interactor; B2M, beta-2-microglobulin. – TNFs co-stimulate T cells 13 T. Casneuf, J. Bald, T. Ahmadi, and A.K. Sasser are employees of P values <0.05) Janssen Research & Development, LLC. A. Lysaght is an ✦ To further investigate the mechanism of action of – SOMAmer sequences that changed between the batches Figure 6. Proteins associated with tumor load that decreased in responders and increased in non-responders. employee of and holds stock in Immuneering Corporation. C. LeFave is an employee of LabConnect, LLC. B. Weiss reports daratumumab in relapsed and refractory MM through and SOMAmers with extremely large or small inter-batch – In all response classes (responders, stable, and consultancy for Janssen and Millennium, and research funding calibration factors were removed from the analysis non-responders), expression of immune-related proteins from Janssen and Onclave. N.W.C.J. van de Donk reports An electronic version of the changes in protein expression research funding from Janssen Pharmaceuticals, Amgen, and poster can be viewed by scanning toll-like receptor 2 (TLR2), inducible T-cell co-stimulator Celgene. H.M. Lokhorst reports honoraria from Amgen, the QR code. The QR code is ✦ To identify predictive and/or pharmacodynamic protein – The data from 35 samples repeated in Batches 1 and 2 were Janssen, and Genmab, and research funding from Janssen and intended to provide scientific (ICOS), and CD163 increased on treatment ( Figure 5 ) Genmab. information for individual markers of clinical response to daratumumab merged by calculating the mean for each protein reference. The PDF should not be altered or reproduced in any way.

POSTER PRESENTED AT THE 57TH AMERICAN SOCIETY OF HEMATOLOGY (ASH) ANNUAL MEETING & EXPOSITION; DECEMBER 5-8, 2015; ORLANDO, FLORIDA.