Identification and Characterization of Presented Tumor Neo-epitopes from Metastatic Colon Cancer

Transcription

1 Identification and Characterization of Presented Tumor Neo-epitopes from Metastatic Colon Cancer Eustache Paramithiotis, PhD Vice President, Discovery NeoAg Summit 15 November 2018

2 CAPRION BIOSCIENCES Specialty contract research lab Biomarkers, personalized medicine & biologics Clinical & pre-clinical Deep expertise in both mass spectrometry & immune monitoring Well positioned to impact immune oncology Founded 2000 Team 200 employees including 160 scientists Accreditation & Rigorous QA/QC CAP accreditation, CLIA lab, GLP/GCLP environment Expert Scientists Assigned to each project 2

3 ANTIGEN PRESENTATION IN CANCER IMMUNE THERAPY MHC I POLYMORPHIC AMINO ACIDS Tumors create immunosuppressive environments that can decrease normal lymphocyte effector functions Vaccines are one strategy to induce host immunity against malignant cells MHC II A key element in this strategy is the incorporation effectively presented tumorassociated antigens PRESENTED PEPTIDE 3

4 DIRECT OBSERVATION OF PEPTIDES PRESENTED BY MHC Most physiologically relevant method for neo-epitope discovery Profiles what is actually presented by tumors Canonical, PTM-modified, or peptides resulting from multiple types of mutation Too much starting material used to be required Epitope prediction by algorithm Exome sequencing Models limited by incomplete understanding of antigen presentation regulation High rate of candidate attrition Instrumentation improvements Higher sensitivity Better mass accuracy Improved bioinformatics Epitope discovery directly from tissue now practical 4

5 APPLICATIONS OF DIRECT ANTIGEN PRESENTATION MEASUREMENT NEO-EPITOPE DISCOVERY AG PRESENTATION ALTERATIONS PRODUCT IMMUNOGENICITY PEPTIDE QUANTITATION Primary tumor tissue Dendritic cell Ag presentation Ex vivo In vitro In vitro In vitro or ex vivo Tumor vs non-tumor Tumor specific targets Condition & process specific changes to presentation Peptide presentation from biologics & HCP Quantitation of specific presented peptides Image by Scray from Wikimedia Commons 5

6 CAPRION PROCESS FOR DIRECT IDENTIFICATION OF NEO-EPITOPES DISCOVERY QUALIFICATION CHARACTERIZATION Multi-parametric flow cytometry Primary tissue Normal body atlas screen ELISPOT Presented peptides Tumor neo-epitopes Presented peptides Off target presentation Epitope prioritization Immune activation Response type Phenotype Best in class mass spectrometry & immune monitoring practices improves quality of epitope candidates 6

7 CAPRION S METHODOLOGY: Directly identifies naturally presented tumor antigens relative to adjacent normal tissue Focuses on naturally-processed peptides that are presented by MHC complexes in vivo Improves coverage of the presentome Reduces reliance on epitope modeling Fewer, better characterized candidates for follow-up 7

8 ISOLATION OF PEPTIDES NATURALLY PRESENTED BY MHC Lysate preparation Non-ionic detergents Protease inhibitors MHC complex isolation Ab affinity chromatography Pan- or allele specific MHC complex elution Disruption of complex with mild acid Release of peptide MHC I MHC II a & b Affinity chromatography b2m MHC I MHC II Recovery of presented peptides MCX chromatography Mass spectrometry analysis MHC-bound peptides are a fraction of the peptides produced by a cell Isolation of the MHC-peptide complex a critical step for quality sample preparation 8

9 IDENTIFICATION OF NATURALLY PRESENTED MHC PEPTIDES Mass spectrometry analysis Raw data acquisition Comprehensive or targeted acquisition Catalogue of presented peptides Peptide sequence Relative intensity Parent protein ID Binding affinity predictions Differential expression analysis Condition specific comparisons 9

10 M/Z HIGH QUALITY DATASETS FROM TISSUE & CELL CULTURES 1400 Lung tumor MHC I (0.8g) Tumor adjacent lung MHC I (1.4g) 50.0% MHC I % % % % % 7aa 9aa 11aa 13aa 15aa 17aa 19aa 21aa 23aa 25aa Tumor adjacent lung MHC II (2.5g) Dendritic cell MHC II (3.6M) 20.0% 16.0% MHC II % % % RT (min) 0.0% 7aa 9aa 11aa 13aa 15aa 17aa 19aa 21aa 23aa 25aa 10

11 CANDIDATE PRIORITIZATION USING A PRESENTOME BODY ATLAS Body atlas database v1 Liver Heart Lung Kidney Colon CNS (Cerebral Cortex) Adjacent normal tissues from all projects also included in the database Database contains peptides presented by MHC I from major organs Selected tissues are important for toxicity studies and/or have local immune system presence All tissues haplotyped for MHC I alleles Core tissue samples derived from healthy subjects, N=20 per tissue type All tumor-adjacent tissue screens also included 11

12 Tissue (wet weight, g) HLA-A 01:01 HLA-A 02:01 HLA-A 02:05 HLA-A 02:06 HLA-A 03:01 HLA-A 11:01 HLA-A 23:01 HLA-A 24:02 HLA-A 25:01 HLA-A 26:01 HLA-A 30:01 HLA-A 30:04 HLA-A 31:01 HLA-A 32:01 HLA-A 66:01 HLA-A 68:01 HLA-B 07:02 HLA-B 08:01 HLA-B 13:02 HLA-B 14:02 HLA-B 15:01 HLA-B 15:24 HLA-B 18:01 HLA-B 27:02 HLA-B 27:05 HLA-B 35:01 HLA-B 35:03 HLA-B 37:01 HLA-B 38:01 HLA-B 39:01 HLA-B 40:01 HLA-B 40:02 HLA-B 41:01 HLA-B 41:02 HLA-B 44:02 HLA-B 44:03 HLA-B 44:05 HLA-B 49:01 HLA-B 51:01 HLA-B 52:01 HLA-B 57:01 HLA-B 73:01 HLA-C 01:02 HLA-C 02:02 HLA-C 03:03 HLA-C 03:04 HLA-C 04:01 HLA-C 05:01 HLA-C 06:02 HLA-C 07:01 HLA-C 07:02 HLA-C 07:04 HLA-C 08:02 HLA-C 12:02 HLA-C 12:03 HLA-C 15:02 HLA-C 15:05 HLA-C 16:01 HLA-C 16:02 HLA-C 17:01 Allele frequency CORE BODY ATLAS PRESENTOME CHARACTERSTICS 10.0% 8.0% 6.0% 4.0% 2.0% 60 different HLA alleles in core body atlas 0.0% Allele distribution consistent with population expectations 1.2 CNS R² = Presented peptide yield varied between tissues Heart R² = Kidney R² = Colon R² = Lung R² = Liver R² = Epithelia-rich tissues had the most MHC I presentation per gram tissue Peptides sequenced 12

13 METASTATIC COLON CANCER Collaboration with Simon Turcotte, Dept. of Surgery, University of Montreal UNMET NEED Colorectal cancer is the 3rd leading cause of cancer-related deaths in N. America Near-universal development of resistance to chemotherapy in metastatic disease Relatively low mutational burden, although neo-epitope reactive TIL have been identified MATERIALS Liver is the most common site of initial metastases Surgery is a viable option for liver-confined metastases Biospecimens can thus be readily available for neoantigen research PROJECT Two phase project Small scale proof of concept n=5 tumor & matched adjacent liver Larger 2nd phase Initial objective was to describe the metastatic CRC presentome & demonstrate if we can broaden the tumor neo-antigen repertoire 13

14 STUDY SAMPLES CHUM Hepatopancreatobiliary and Colorectal Cancer Prospective Database & Biobank; Simon Turcotte, Director Biobank has patient plasma, snap frozen tissues, tumor homogenates, PBMC Complies with Canadian Tumour Repository Network SOP ( Subject 243 Tumor biopsy H&E Materials for both neo-epitope discovery & follow up functional characterization Subject Primary Pre surgery met-liver leukocytes Intratumor leucocytes 475 T3N0M0 NONE 1 1 Post surgery Folfox and DeGramont HLA-A HLA-B HLA-C 01:01 02:01 37:01 44:03 04:01 06: T3N0M0/T2N0 NONE 2 2 Folfox 24:02 31:01 40:01 44:03 03:04 04: T3N0Mx Folfox + avegf 1 1 Folfox 11:01 33:01 44:03 44:03 02:02 04: T3N0 Folfox + avegf 1 1 Folfox 24:02 25:01 13:02 18:01 06:02 12: T3N3M1 Folfox folforinox + avegf 1 1 Folfirinox 11:01 68:01 15:01 55:01 03:03 03:03 1 portal space 2 portal space & met interface 1 stroma only 2 contact with tumor cells 14

15 Identified peptides SAMPLE PREPARATION Tissue weight (g) Limited starting material Pooled 6-8 biopsy plugs per subject Variable presented peptide yield; perhaps a reflection of the variable epithelial content of the biopsies Identified peptide length distribution matched known properties of MHC I-presented peptides Pooled frozen biopsies provided enough material for analysis 15

16 Frequency PRESENTED PEPTIDE COVERAGE 100.0% 10.0% 1.0% 0.1% 0.0% Ezrin peptides Subject Peptides presented per protein HLA Aff (nm) FAIQPNTTGK X A*11: KENPLQFKF X X B*44:03 67 ETAVLLGSY X A*25:01 16 EYTAKIALL X A*24: DIIHNENMR X A*68:01 69 Most proteins were represented by single presented peptides Substantially higher presentation also observed Ezrin is required for microvilli formation in epithelial cells Increased ezrin expression results in typically poor prognosis for CRC metastases Ezrin presentation is typical of the majority 16

17 METASTATIC CANCER PRESENTOME SIMILARITY TO ORIGINAL TISSUE Principal component analysis Rolled up peptide spectral counts to proteins; used 267 most differentiating proteins Compared presentomes of metastatic tumors to normal colon and liver, and to primary colon tumors & adjacent tissue Metastatic colon peptide presentation resembles normal colon 17

18 METASTATIC CANCER PRESENTOME SIMILARITY TO ORIGINAL TISSUE Principal component analysis Rolled up peptide spectral counts to proteins; used 267 most differentiating proteins Compared presentomes of metastatic tumors to normal colon and liver, and to primary colon tumors & adjacent tissue Metastatic colon peptide presentation resembles normal colon Colon mets presentation appears to diverge from that of primary tumors, may be influenced by current tissue of residence 18

19 METASTATIC COLON CANCER CANDIDATE NEO-EPITOPE IDENTIFICATION 50% no hits on body atlas 65% no hits on body atlas Most peptides were under-expressed or modestly over-expressed in the tumor tissues Candidate neo-epitopes (0.2%) and tumorassociated non-mutated peptides (2%) were a small minority Body atlas screening further reduced candidates Candidates were identified by comparing presented peptides from matched tumors to adjacent tissue & body atlas 19

20 PRIMARY KIDNEY CANCER CANDIDATE NEO-EPITOPE IDENTIFICATION 93% no hits on body atlas Frozen tissue resections from subjects taking surgery as first line therapy (n=5) Tumor & adjacent normal comparisons Comparable peptide presentation pattern; neoepitope frequency consistent with genetic mutational load Candidate neo-epitope composition Single aa substitutions: 25% Frameshifts: 19% PTM: 9% De novo (fusions, splice, other): 43% 20

21 SELECTED CANDIDATE CHARACTERISTICS NEO-EPITOPES Cand Mutation Length IEDB CNS Col Kid Liv Lun Hrt 1 SNP 10 No SNP 10 No SNP 9 No SNP 9 No SNP 9 No SNP 10 No SNP 9 No fsx 9 No fsx 9 No fsx 11 No fsx 9 No fsx 9 No fsx 10 No fsx 9 No fsx 9 No fsx 9 No fsx 9 No de novo 9 No de novo 9 No de novo 10 No de novo 9 No de novo 14 No de novo 8 No de novo 9 No de novo 10 No de novo 9 No de novo 9 No CANONICAL PEPTIDES Cand Length IEDB CNS Col Kid Liv Lun Hrt 1 11 No No No No No No No No No No No No No No No No No Yes Yes Yes Yes Yes Selection criteria Tumor presentation >3x background + absent in matched adjacent Predicted affinity <500nM for expressed HLA Candidates mix of zero, minimal, and multiple body atlas hits Selected novel tumor-associated neo-epitopes & non-mutated peptides for functional testing 21

22 CANDIDATE FUNCTIONAL EVALUATION Autologous B cells from PBMC used for antigen presentation Test reactivity of autologous T cell subsets expanded from patient tumors Test reactivity autologous T cell subsets expanded from patient PBMC Evaluate peptide recognition by IFNg elispot & flow cytometry Experiments still in progress 22

23 IDENTIFICATION OF NEO-EPITOPES: SUMMARY Demonstrated high resolution analysis of naturally presented MHC I peptides from metastatic colon cancer biopsies Generated deep datasets that expanded existing literature and provided high quality candidate targets Currently completing functional evaluation of selected candidates 23

24 ACKNOWLEDGMENTS Caprion Mark Watson Marie Christine Doyle Rudolf Guilbaud Anne Jang Daniela Vasilescu Vanessa Laflamme Gloria Giono Chang Michael Schirm Marija Mentinova Christina Bell Maxim Isabelle Laetitia Cortes Jingwei Xie Olivier Gingras University of Montreal Simon Turcotte Melissa Mathieu 24

25 THANK YOU Eustache Paramithiotis PhD Vice President, Discovery LinkedIn

26 26