Identifying Major Immune Subsets of GI Tumors for Clinical

The right drug or right trial… For Every Patient 700 Ellicott Street | Buffalo NY, 14203 Identifying major immune subsets of GI tumors for clinical purposes Early A1,#, Pabla S1, Conroy JM1,2, Nesline MK1, Glenn S1,2, Papanicolau-Sengos A1, Burgher B1, Giamo V1, Andreas J1, DePietro P1, Lenzo F1, Wang Y1, Morrison CD1,2* OmniSeq, Inc1, Buffalo, NY; Roswell Park Comprehensive Cancer Center2, Buffalo, NY # [email protected]; *[email protected]

Introduction PD-L1 Concordance Immune Subsets by GI Tumor Type

PD-L1 Expression by IHC and RNA-Seq Myeloid Suppressed Microsatellite stable (MSS) gastrointestinal (GI) MSS Colorectal (n=80) MSS Pancreatic (n=40) in MSS GI Tumors (n=127) Myeloid suppression, defined as overexpression of CD163 tumors, including colorectal and non-colorectal, 50% 47% or CD68 by RNA-Seq, was the most common immune type have demonstrated low response rates to 45% 2, 5% 40% 14, 18% across all MSS GI tumors, and was more common in in immune checkpoint inhibitors (ICIs). 40% 37% 5, 13% 35% 33% 25, 31% pancreatic (55%) versus colorectal (31%) tumors. 4, 10% 30% Currently, there is uncertainty as to how to 25% PD-L1 Positive, Inflamed evaluate MSS GI tumors for evidence of 20% 18% Overall, very few GI tumors overexpress PD-L1 and have 13% 7, 17% checkpoint blockade, as PD-L1 15% 4, 5% 25, 31% 22, 55%

Percent of Patients Percent evidence of moderate to high CD8 TILs by RNA-Seq (6%) immunohistochemistry (IHC) has demonstrated 10% 7% 5% 2% 3% limited utility for these tumors. 0% 12, 15% 0% PD-L1 Negative, Inflamed PD-L1 IHC <1% TPS (n=97) PD-L1 IHC >=1% TPS (n=30) Tumors with evidence of CD8 TILs but no/low expression of Understanding other mechanisms of PD-L1 Overexpression by RNA-Seq MSS Small Bowel (n=7) All MSS GI Tumor Types (n=127) PD-L1 by RNA-Seq are more than twice as common in immunosuppression in GI tumors, such as Very Low Low Moderate High Very High colorectal (31%) versus pancreatic cancers (13%). myeloid or metabolic suppression and the • 76% (n=97) of MSS GI tumors were PD-L1 IHC negative 16, 13% degree of CD8+ T-cell infiltration, may illuminate (TPS < 1%), with corresponding low/very low PD-L1 RNA- 2, 29% 32, 25% Immune Desert immunotherapy selection in this patient Seq expression values for 63/97 (65%). 34/97 (35%) of the 3, 43% Tumors with no overexpression of known population. PD-L1 IHC negative tumors had discordant high/very high immunotherapeutic targets in clinical development more expression for PD-L1 by RNA-Seq. 24/34 (71%) of these 8, 6% than three times as common in colorectal (18%) versus scores were explained by the presence of immune cells 50, 39% pancreatic (5%) cancer. not captured by the IHC TPS scoring method. 21, 17% Methods • 24% (n=30) of MSS GI tumors were PD-L1 IHC positive 2, 28% Other (TPS ≥ 1%), but only 3 (2%) of these cases were TPS ≥ 50%. Tumors that overexpress ≥ 1 other tested immune markers

Formalin fixed paraffin embedded (FFPE) tissue Immunomodulatory Immunotherapeutic Targets in Myeloid Suppressed GI Tumors from 127 MSI stable (MSS) GI tumors, including Not Myeloid Myeloid colorectal (n=80), pancreatic (n=40) and small Overexpression of Immunotherapeutic Targets in MSS GI Tumor Patients Marker Therapies in Clinical Development Suppressed Suppressed bowel (n=7) were evaluated for PD-L1 expression Myeloid Suppressed (n=50) versus Not Myeloid Suppressed (n=81) ADORA2A AZD4635; CPI-444; NIR178; PBF-509 24 43 by immunohistochemistry (IHC) using the 22c3 90% Myeloid Suppressed CCR2 BMS-813160 24 29 CD137 Urelumab; Utomilumab; PRS-343 9 18 No Yes pharmDx antibody on Autostainer Link 48 80% 78% CD27 Varlilumab 1 3 74% (Agilent, Santa Clara, CA). Tumor proportion CD38 Daratumumab; Isatuximab 2 4 70% Selicrelumab; ABBV-428; ABBV-927; ADC-1013; 66% CD40 12 13 score (TPS) was interpreted per published APX005M; CDX-1140; SEA-CD40 60% CD8 Etirinotecan Pegol; ALKS 4230; ALT-803; NKTR-214 48 12 guidelines. Cabiralizumab; Emactuzumab; AMG 820; ARRY-382; 50% 46% 46% 46% CSF1R BLZ945; JNJ-40346527; LY3022855; PD-0360324; SNDX- 14 45 6352 40% RNA was extracted to perform gene expression Ipilimumab; Tremelimumab; AGEN1884; BMS-986218; 40% CTLA4 10 17 34% BMS-986249; MK-1308; REGN4659; XmAb20717

profiling (GEX) by RNA-Sequencing of 54 Patients of Percent 28% 28% BMS-986156; GWN 323; INCAGN01876; MK-4166; 30% 26% GITR 25 15 24% OMP-336B11; TRX518 immune-related genes, including targets of 21% 20% 22% 18% 18% 17% ICOS BMS-986226; GSK3359609; JTX-2011 13 18 20% 15% 16% immunomodulatory immunotherapeutics that 12% 14% 12% 14% 12% Epacadostat; Indoximod; BMS-986205; KHK2455; 10% 11% 10% IDO1 19 13 10% 9% 8% 9% 9% 9% 9% LY3381916; MK-7162; NLG802 are currently FDA approved (i.e., PD-L1, PD-1) or 5% 4% 1% 2% 1% 2% 2% 1% Relatlimab; BI 754111; BMS-986213; FS118; in clinical development (i.e., CSF1R, VISTA), as 0% LAG3 INCAGN02385; LAG525; MGD013; MK-4280; REGN3767; 7 7 PD1 CTLA4 PDL1 PDL2 LAG3 TIM3 VISTA CD137 CD27 CD40 GITR ICOS OX40 CCR2 CSF1R ADORA2A IDO1 TNF TGFB1 CD38 CD8 Sym022; TSR-033 well as subsets of tumor infiltrating lymphocytes ABBV-368; BMS-986178; GSK3174998; INCAGN01949; OX40 14 13 (TILs), including CD8. • All myeloid suppressed tumors overexpressed at least one myeloid-related immunotherapeutic target with therapies in clinical MEDI0562; PF-04518600 Cemiplimab; Nivolumab; Pembrolizumab; ABBV-181; development including CCR2, CSF1R, or TGFB1. The majority of these 50 cases (n=37; 74%) also over expressed VISTA, TIM3, or both, AGEN2034; BGB-A317; BI 754091; BMS-986213; JNJ- PD-1 20 13 indicating that over expression of these two checkpoint blockade receptors are strong indicators of myeloid suppression. PD-L2 was also 63723283; MEDI0680; MGA012; MGD013; PDR001; PF- Normalized GEX values (nRPM) were ranked from 06801591; Sym021; TSR-042; XmAb20717 frequently overexpressed in myeloid suppressed tumors (n=23; 46%). Atezolizumab; Avelumab; Durvalumab; CA-170; CX-072; 0 to 100 and compared to a reference population PD-L1 20 13 • FAZ053; FS118; KN035; LY3300054; M7824 Patients with myeloid suppressed GI tumors may also be more likely to benefit from drugs in clinical development that target alternative Fresolimumab; Galunisertib; M7824; NIS793; SAR- of 167 cases that had a broad range of immune TGFB1 30 41 checkpoint blockade receptors. 439459 gene expression in multiple tumor types. TIM3 BMS-986258; LY3321367; MBG453; Sym023; TSR-022 3 30 • Overall, very few MSS GI tumors overexpressed CD8. Tumors that were not myeloid suppressed however, (38/81; 47%) more commonly TNF Certolizumab; Lenalidomide; Thalidomide 4 2 had low or very low expression of CD8 and can aptly be called cold tumors. VISTA CA-170 23 39 Conclusion

Using a more sophisticated approach to evaluating the tumor microenvironment in GI tumors 3 major groups including PD-L1 positive, myeloid suppression, and immune deserts Figure 1. PD-L1 IHC and RNA-Seq workflow can be identified that has major implications for the application of precision immunotherapy for this patient population.