DIRECT IDENTIFICATION OF NEO-EPITOPES IN TUMOR TISSUE

Transcription

1 DIRECT IDENTIFICATION OF NEO-EPITOPES IN TUMOR TISSUE Eustache Paramithiotis PhD Vice President, Biomarker Discovery & Diagnostics 17 March 2016

2 PEPTIDE PRESENTATION BY MHC MHC I Antigen presentation by MHC is essential for adaptive immunity Polymorphic amino acids Peptides presented by MHC induce T cell activation which initiate & regulate immune responses MHC II MHC I and MHC II have similar peptide binding sites; MHC II can present longer peptides Presented peptide Peptides presented by MHC are attractive vaccine development targets & biomarkers 2

3 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 3

4 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 4

5 STUDY OBJECTIVE Demonstrate the sensitivity & resolution of the ProteoCarta platform for the identification of naturally presented MHC Class I Peptides from tumor samples. Selected Clear Cell Renal Cell Carcinoma samples from the Caprion tissue collection. 5

6 PREPARATION 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 Affinity chromatography MHC I MHC II α & β β2m 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 6

7 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 7

8 SAMPLE DESCRIPTION Matched tumor & adjacent normal tissue resections Same hospital site Same tumor type & grade No treatment prior to surgery Clear cell RCC is known to have a low mutational load compared to other cancers 8

9 SUBJECT HLA HAPLOTYPES Heterogeneous haplotypes expressed Modest allele overlap 9

10 DATA ACQUISITION Processed 1.3 g tumor & 1.6 g adjacent normal tissue wet weight, on average Normalized injection volumes using total protein yields & levels of B2M from the isolated MHC I complexes Standard bioinformatics workflow, using 5% FDR 10

11 CHARACTERISTICS OF IDENTIFIED PEPTIDES Frequency An average of 1837 MHC I presented peptides identified per sample 92% of identified peptides were 8-15 aa long 48% of identified peptides were 9 aa long Peptide lengths & frequencies match known properties of MHC I-presented peptides Peptide length 11

12 PEPTIDE PRESENTATION OVERLAP IS RELATIVE TO THE HLA ALLELES IN COMMON Peptides in common (%) Subjects that did not have HLA alleles in common had ~6% of their presented peptides in common For every allele in common the presented peptide overlap increased by ~7% (p-value <0.001) Suggests that there is a consistent allele-specific component in antigen presentation For this analysis all identified peptides were used HLA alleles in common 12

13 DIFFERENTIALLY EXPRESSED PEPTIDES OKD73 1.4% OKD12 1.3% Approximately 50% of peptides were under-expressed in tumor tissue, and ~40% were modestly over-expressed (1-3X) OKD28 1.1% OKD10 3.8% A small minority of peptides were unique or highly overexpressed in tumor Despite different subject haplotypes a consistent frequency of differentially expressed MHC peptides was observed OKD7 4.1% T:N < 1 T:N 1-3X Calculations were done for unmodified 8-15 aa peptides T:N 3-5X T:N 5-10X Unique in Tumor 13

14 MODIFIED PEPTIDES UNIQUE TO TUMOR Approximately 1% of mass spectra unique to tumor yielded high confidence peptide sequences without matches in the database 94% of these were eventually matched to canonical, unmodified sequences 2% (18) were unmodified peptides with PTM: Dehydration (4) C-terminal amidation (1) Methylation (1) Dioxidation (4) Hydroxylation (8) 3% (27) were modified peptides: Single amino acid substitutions (2) Non-coding RNA (2) Alternate reading frames (4) Splicing variants (2) Remaining peptides in progress Direct profiling of naturally presented peptides revealed small subset of the peptides unique to tumor had been modified by multiple mechanisms 14

15 HLA BINDING AFFINITY PREDICTIONS Binding calculations done for all 8-15aa peptides identified Shown are peptides presented>10x in tumor compared to adjacent normal tissue for 2 subjects Highlighted high binding predictions (<500nM) All alleles expressed by every subject was able to present tumorassociated peptides 15

16 SELECTION OF REPRODUCIBLY PRESENTED TARGET CANDIDATES # MHC I presented peptides # subjects Target protein uniquely presented by tumor Identical peptide presented 7 candidates expressed in 2 of 5 subjects Target protein uniquely presented by tumor Any peptide presented from target protein 15 candidates & increased patient coverage Target protein presented >5X by tumor but not unique Any peptide presented from target protein 27 candidates & further increased patient coverage Selecting the appropriate tumor-specific parent proteins as candidate targets allows flexibility of antigen presentation by multiple HLA alleles 16

17 TUMOR SPECIFIC CHANGES REPRESENTED BY ANTIGEN PRESENTATION Presented proteins Hypoxia Angiogenesis OKD7 OKD10 OKD12 OKD28 OKD73 Clear cell RCC known to induce a strong hypoxia & angiogenesis response Multiple proteins associated with both processes were differentially presented in tumor Hypoxia & angiogenesis proteins were significantly over-represented Antigen presentation reflected known protein expression changes in RCC Subject 17

18 IDENTIFICATION OF A PREVIOUSLY DESCRIBED PEPTIDE PRESENTED BY RENAL CANCER HMOX1 expression induced in renal cancer through increased c- Met activity The HMOX1 peptide APLLRWVL presented by HLA-B08 was previously identified in renal cancer tissue (Flad et al, 2006) MERPQPDSMP QDLSEALKEA TKEVHTQAEN AEFMRNFQKG QVTRDGFKLV MASLYHIYVA LEEEIERNKE SPVFAPVYFP EELHRKAALE QDLAFWYGPR WQEVIPYTPA MQRYVKRLHE VGRTEPELLV AHAYTRYLGD LSGGQVLKKI AQKALDLPSS GEGLAFFTFP NIASATKFKQ LYRSRMNSLE MTPAVRQRVI EEAKTAFLLN IQLFEELQEL LTHDTKDQSP SRAPGLRQRA SNKVQDSAPV ETPRGKPPLN TRSQAPLLRW VLTLSFLVAT VAVGLYAM Known epitope (Flad et al, Proteomics, 6: 364, 2006) Novel epitopes Normal kidney, 61yr M Renal cell adenocarcinoma, 61yr M The same peptide and HLA allele was identified in subject OKD12 Multiple additional peptides were identified, overexpressed in tumor (12X) & presented by other HLA alleles Pilot study reproduced and significantly expanded existing literature 18

19 RENAL CELL CANCER URINE BIOMARKER PRESENTED BY MHC I IN CANCER TISSUE PLIN2 involved in development & activity of intracellular lipid droplets Minimal PLIN2 protein expression in normal kidney, but significantly increased in renal cancer MASVAVDPQP SVVTRVVNLP LVSSTYDLMS SAYLSTKDQY PYLKSVCEMA ENGVKTITSV AMTSALPIIQ KLEPQIAVAN TYACKGLDRI EERLPILNQP STQIVANAKG AVTGAKDAVT TTVTGAKDSV ASTITGVMDK TKGAVTGSVE KTKSVVSGSI NTVLGSRMMQ LVSSGVENAL TKSELLVEQY LPLTEEELEK EAKKVEGFDL VQKPSYYVRL GSLSTKLHSR AYQQALSRVK EAKQKSQQTI SQLHSTVHLI EFARKNVYSA NQKIQDAQDK LYLSWVEWKR SIGYDDTDES HCAEHIESRT LAIARNLTQQ LQTTCHTLLS NIQGVPQNIQ DQAKHMGVMA GDIYSVFRNA ASFKEVSDSL LTSSKGQLQK MKESLDDVMD YLVNNTPLNW LVGPFYPQLT ESQNAQDQGA EMDKSSQETQ RSEHKTH Normal kidney, 68yr F Renal cell adenocarcinoma, 64yr F Urine levels of PLIN2 and AQP1 correlate with RCC tumor size & can distinguish RCC from other cancers & kidney masses (Morrissey et al, 2015, 2014) Identified multiple PLIN2 peptides presented by MHC I and substantially (30X) overexpressed in tumor 19

20 PRESENTATION OF A CANCER ASSOCIATED VARIANT PEPTIDE PH4H catalyzes the conversion of L-Phe to L-Tyr; deficiency in this enzyme results in phenylketonuria PH4H protein highly expressed in in normal kidney tubules, significantly reduced in RCC MSTAVLENPG LGRKLSDFGQ ETSYIEDNCN QNGAISLIFS LKEEVGALAK VLRLFEENDV NLTHIESRPS RLKKDEYEFF THLDKRSLPA LTNIIKILRH DIGATVHELS RDKKKDTVPW FPRTIQELDR FANQILSYGA ELDADHPGFK DPVYRARRKQ FADIAYNYRH GQPIPRVEYM EEEKKTWGTV FKTLKSLYKT HACYEYNHIF PLLEKYCGFH EDNIPQLEDV SQFLQTCTGF RLRPVAGLLS SRDFLGGLAF RVFHCTQYIR HGSKPMYTPE PDICHELLGH VPLFSDRSFA QFSQEIGLAS LGAPDEYIEK LATIYWFTVE FGLCKQGDSI KAYGAGLLSS FGELQYCLSE KPKLLPLELE KTAIQNYTVT EFQPLYYVAE SFNDAKEKVR NFAATIPRPF SVRYDPYTQR IEVLDNTQQL KILADSINSE IGILCSALQK Identified several PH4H peptides presented by MHC I; all were overexpressed (12X) in normal tissue Normal kidney, 61yr M Renal cell adenocarcinoma, 63yr M Identified one peptide derived from a known variant that was exclusively expressed in tumor Confirmatory study in progress, results demonstrate capability to directly identify naturally presented protein variants 20

21 ASSESMENT OF CANDIDATE TARGET IMMUNOGENICITY CD3 T cell proliferation Dividing cells CD3 + CD CFSE Nondividing cells Peptide-pentamer Intracellular cytokine staining CD3 + CD CD8 % IL-2 + % TNFα + % IFNγ + Vehicle Peptide PMA Up to 9 markers can be used for characterization of antigenspecific T cell activation Integrated in house capability to identify neo-epitopes & characterize their effect on T cell activity Activation assays can be combined with multi-color flow cytometry for in depth characterization of antigen-specific T cells. 21

22 IDENTIFICATION OF NEO-EPITOPES: SUMMARY Demonstrated high resolution analysis of naturally presented MHC I peptides from human tissue Generated deep datasets that expanded existing literature and provided high quality candidate targets The combination of ProteoCarta and ImmuneCarta enables integrated discovery of naturally presented MHC peptides and subsequent antigen-specific functional characterization, at one site 22