Predictive Biomarkers for Pembrolizumab. Eric H. Rubin, M.D.

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1 Predictive Biomarkers for Pembrolizumab Eric H. Rubin, M.D.

2 PD-1 and PD-L1/L2 Pathway PD-1 is an immune checkpoint receptor Binding of PD-1 by its ligands PD-L1 or PD-L2 leads to downregulation of T-cell function This mechanism is usurped by many tumors PD-1 blockade through mab therapy can restore and reveal effective anti-tumor immunity Topalian et al. N Engl J Med Garon et al. N Engl J Med Robert et al. Lancet

3 Using Predictive Biomarkers (Companion Diagnostics) to Select Patients for Treatment No test is perfect, but some tests are useful Histology is an imperfect biomarker that is used to select cancer patients for treatment Imperfect HER2 IHC test allowed rapid development of an effective treatment for breast cancer patients Companion diagnostics may be used to select among treatment options, vs excluding patients from an immunotherapy treatment Companion diagnostic development typically lags behind therapeutics, creating scientific and regulatory complexity Several biomarkers for PD-1 targeting agents have been identified that are predictive for efficacy, including PD-L1 protein expression, RNA signatures, and MSI/DNA mutation burden

4 Clinical Utility of PD-L1 Expression in Lung Cancer PD-L1 expression among the earliest biomarkers evaluated as potentially predictive for response to PD-1 pathway inhibitors Current data indicate that PD-L1 expression predicts survival outcome in lung cancer patients treated with PD-1 antibodies In a randomized study of pembrolizumab in 2L NSCLC, a survival benefit vs docetaxel was observed in patients with 1% PD-L1 tumor staining (Herbst, et al, Lancet 215) In a randomized study in 2L non-squamous NSCLC, survival was similar in patients with PD-L1-negative tumors treated with nivolumab vs docetaxel (Borghaei, et al, NEJM 215)

5 Pembrolizumab vs Docetaxel in Previously Treated NSCLC Patients O v e r a l l S u r v i v a l, % OS, PD-L1 TPS 1% (Total Population) Treatment Arm Median (95% CI), mo Rate at 1 y HR a (95% CI) P Pembro 2 mg/kg 1.4 ( ) 43.2%.71 ( ).8 Pembro 1 mg/kg 12.7 ( ) 52.3%.61 ( ) <.1 Docetaxel 8.5 ( ) 34.6% Time, months a Comparison of pembrolizumab vs docetaxel. Analysis cut-off date: September 3,

6 Nivolumab vs Taxotere in Previously Treated Non- Squamous NSCLC Patients: OS by PD--L1 Status

7 Multiple PD-L1 IHC Assays and Cutoffs Dako 22-C3 and 28-8 Assays are FDA-Approved Agent Pembrolizumab Nivolumab Durvalumab Atezolizumab Diagnostic Platform Dako Ventana Antibody 22-C SP 263 SP 142 Cut-off(s) being tested TC 1 1%, 5% 1%, 5% or 1% (TC 1 ) TC 1 25% TC 1 or IC 2 1%, 5%,1% 1) TC = tumor cell staining. 2) IC = infiltrating immune cell staining AACR-sponsored Blueprint project designed to compare the 4 assays

8 What About the Other Ligand, PD-L2? Health PD-L2 - one of two known binding partners for PD-1 PD-L2 negatively regulates T cells in immune responses, and plays an important role in immune tolerance 1-6 Disease Documented PD-L2 expression by tumor and/or infiltrating immune cells in some tumors 7 Roles in mediating severity of disease in murine models of autoimmunity, hypersensitivity, and infection with various agents 3-5, Latchman et al. Nature Immunology. 21;2: Rodig et al. Eur J Immunol.23; 33: Zhang et al. PNAS. 26;13: Zhu et al. J Immunol; 26;176: Matsumoto et al. J Immunol;24;172: Keir et al. J Exp Med. 26;23: Taube et al. Clin Cancer Res. 214;2: Fukushima et al. Br J Ophthalmol. 26;9: Liang et al. Eur J Immunol : Terrazas et al. Int J Parasitol.25;35:

9 PD-L2 in Normal Human Tissues PD-L2 immunohistochemistry (IHC) Assay Assay developed de novo 449 antibodies/hybridomas screened clone 3G2 selected Normal tissue distribution - limited Intense labeling within lymphoid tissues, most prominent in germinal centers Intense labeling of extravillous trophoblast cells in placenta and sinusoidal endothelium in spleen Tonsil Lymph node Placenta Spleen

10 PD-L2 in Human Tumors To determine potential relevance of PD-L2 expression in multiple cancers currently being treated with pembrolizumab in the clinic Seven histologies initially evaluated using archival samples Renal cell carcinoma (n = 71) Bladder carcinoma (n = 34) Melanoma (n = 83) Non-small cell lung cancer (NSCLC) (n = 94) Head and neck squamous cell carcinoma (n = 4) Triple negative breast carcinoma (n = 22) Gastric carcinoma (n = 73)

11 PD-L2 in Human Tumors: Distribution Patterns renal melanoma PD-L2 IHC H&E gastric STROMAL CELLS ENDOTHELIUM TUMOR CELLS

12 PD-L2 Expression by Cell Type Stromal Cell Expression Tumor Cell Expression Endothelial Expression Percent of samples* R C C M e la n o m a G a s tr ic N S C L C T N B r e a s t B la d d e r H e a d a n d N e c k R C C M e la n o m a G a s tr ic N S C L C T N B r e a s t B la d d e r H e a d a n d N e c k *Percent of samples with PD-L2 expression score of 1 on -5 scale Endothelial PD-L2 expression of special interest due to potential for PD-1+ T cell binding on trafficking into tumor tissue Endothelial PD-L2 expression has been shown to downregulate CD8 T cell activation and cytolysis 1 1. Rodig et al. Eur J Immunol.23;33:

13 Discordance vs Concordance Patterns by Tumor Type 5 Melanoma NSCLC HNSCC (n = 83) (n = 94) (n = 4) 5 5 PD-L1 IHC PD-L1 IHC RCC PD-L2 IHC R 2 =.3431 P < PD-L1 IHC Bladder PD-L2 IHC (n = 71) (n = 34) R 2 =.2324 P <.1 PD-L1 IHC R 2 =.393 P <.1 R 2 =.2915 P = Gastric PD-L2 IHC PD-L2 IHC PD-L2 IHC PD-L2 IHC PD-L1 IHC PD-L1 IHC R 2 =.3858 P <.1 (n = 73) R 2 =.3491 P <.1 TN Breast (n = 22) 5 PD-L1 IHC PD-L2 IHC R 2 =.6238 P <.1

14 PD-L2 in Human Tumors: Clinical Relevance in Head & Neck Cancer 144 HNSCC patients (KEYNOTE-12) Recurrent or metastatic disease, regardless of PD-L1 or HPV status Measurable disease per RECIST 1.1 ECOG PS of or 1 Received pembrolizumab 2 mg every 3 weeks Both PD-L1 (22C3) and PD-L2 (3G2) scoring available Central question Do levels of PD-L2 predict outcome?

15 PD-L2 Clinical Relevance in Head & Neck Cancer Overall Response Rate by PD-L1 and PD-L2 Status PD-L2 Status PD-L1 Status % 1% % 1/18 = 5.6% /3 = % 1% 4/34 = 11.7% 22/89 = 24.7% Logistic regression suggests PD-L2 expression is associated with higher ORR after adjusting for PD-L1 expression (P =.72) PD-L2 is positively associated with longer PFS after adjusting for impact of PD-L1 status (P =.31) Data suggest that PD-L2 status is associated with outcome in pembrolizumab treated patients

16 PD-L2 and Hodgkin Lymphoma Classical HL (chl) is characterized pathologically by a failed immune response HL frequently harbors amplification at 9p24.1 leading to overexpression of PD-L1 and PD-L2 HL may have a genetically driven vulnerability to PD-1 blockade chl independent expansion cohort in KEYNOTE-13, a phase 1 study of pembrolizumab in heme malignancies Green MR et al. Blood. 21. Green MR et al. Clin Cancer Res

17 Pembrolizumab Efficacy in chl 1 8 % Change at Best Response % of patients had a reduction in target lesions 5% Decrease

18 PD-L1 and PD-L2 Immunohistochemistry 16 samples were evaluable at screening for PD-L1 15 (94%) were PD-L1 positive in tumor cells PD-L1 1 samples were evaluable at screening for PD-L2 9 (9%) were PD-L2 positive PD-L2 18

19 Beyond PD-L1 and PD-L2 - NanoString Platform for Gene Expression Profiling of FFPE Tissue 8 immune-focused genes on custom platform 19

20 Immune Gene Signatures in Baseline Tumor Samples Correlate With Response to Pembrolizumab in Melanoma E x p r e s s i o n S c o r e Melanoma Discovery Set 19 Patients Formulate Initial Signatures Preliminary IFNγ (1 gene) Preliminary Expanded Immune (28 gene) Melanoma Validation Set 62 Patients Independent Data Set Test Initial Signatures Signature Refinement & Discovery.8 Nonresponder Responder.8 Nonresponder Responder Best Overall Response, RECISTv1.1 Best Overall Response, RECISTv1.1 Presented by A. Ribas, ASCO 215

21 Association of IFNγ Gene Signature with Efficacy in Head & Neck Cancer N=4 evaluable subjects from KEYNOTE 12 Comparable results in melanoma, SSCHN, gastric cancer, TCC Presented by T. Seiwert, ASCO 215

22 Mutational Burden, Neoantigens, and Response to Checkpoint Blockade ** * * MSI subset **Triple-negative subset Schumacher and Screiber, Science

23 KEYNOTE 16: Pembrolizumab in MSS Colorectal and MSI Colorectal and Non-Colorectal Tumors 23 Presented by Dung Le, ASCO 215

24 Pre-treatment tumor mutational burden correlates with response to pembrolizumab in lung cancer patients Rizvi, Chan et al. Mutational Landscape Determines Sensitivity to PD-1 Blockade in Non-Small Cell Lung Cancer (Science 215)

25 Investigation of an Exceptional Responder in Study KN28 Identifies Another Route to Hypermutation 53 yo F with Stage IB, pt1bpn, FIGO grade III endometrial adenocarcinoma, highgrade endometrioid type, with extensive necrosis, lymphovascular invasion, and myometrial invasion Treated with doxorubicin/ cisplatin/paclitaxel and RT Relapse two years later with supraclavicular and retroperitoneal adenopathy Enrolled in KEYNOTE-28 with rapid response Response sustained for 12 months and is ongoing PRE-TREATMENT 8 Weeks Pembrolizumab Mehnert et al, AACR-NCI-EORTC 215

26 Sequencing results: Lymph Node Biopsy Sequencing revealed POLE mutation in proofreading domain Mehnert et al, AACR-NCI-EORTC 215

27 Summary PD-L1 expression as assessed by IHC is the first FDAapproved companion diagnostic for checkpoint inhibitors PD-L2 expression generally correlates with PD-L1 expression but can occur in absence of PD-L1, and is independently associated with clinical outcome in HNSCC RNA-based gene signatures that reflect the tumor immune microenvironment are showing promise in correlations with outcome across multiple cancer types High mutational burden conferred by defects in DNA mismatch repair, DNA polymerases, or other mechanisms are likely to result in sensitivity to pembrolizumab and other checkpoint inhibitors

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