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Identification of Shared Phosphopeptide Tumor Targets in Colorectal Cancer for Novel Off-The-Shelf Vaccine Development Paisley T

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Identification of Shared Phosphopeptide Tumor Targets in Colorectal Cancer for Novel Off-The-Shelf Vaccine Development Paisley T Identification of Shared Phosphopeptide Tumor Targets in Colorectal Cancer for Novel Off-the-shelf Vaccine Development Paisley T. Myers1, Erin D. Jeffery1, Benjamin Morin1, Christine Brittsan1, Bishnu Joshi1, Antoine Tanne1, Karen Smith1, Albert Hurwitz1, Kara L. Cummings2, Amanda M. Lulu2, Daniel Levey1, Linh Mach1, Mark Findeis1, Victor H. Engelhard2, Jeffrey Shabanowitz3, Donald F. Hunt3, Arthur A. Hurwitz4, Robert Stein1, Alex Duncan1, Dennis Underwood1 1. Agenus Inc., Lexington, MA 02421, USA, 2. Department of Microbiology, Immunology, and Cancer Biology, Carter Immunology Center, University of Virginia School of Medicine, Charlottesville, VA 22901, USA, 3. Department of Chemistry, University of Virginia, Charlottesville, Virginia 22904, USA, 4. AgenTus Therapeutics, Inc., Lexington, MA, 02421, USA INTRODUCTION PHOSPHOLIGANDOME ANALYSIS OF CRC PATIENT TISSUES Immunoaffinity Lysis Purification Phosphopeptide Length Phosphorylated Site Phosphorylated Residue The high mortality rate of colorectal cancer (CRC) reflects STAGE Tip Sample Resected CRC Tissue Cleanup pTyr (27 Patients) 300 400 limitations of current treatment modalities aimed at targeting the 1% 350 250 pThr disease. Immunotherapy is a promising alternative to traditional 9% Normal 300 200 chemotherapy, yet its benefit has been limited to patients with high 250 mutational burden. 150 200 # PTTs 0.5 g tissue or 500 million # PTTs Tumor 150 cell equivalents 100 Phosphopeptide Tumor Targets (PTTs) represent a novel class 100 pSer 90% 50 of neoantigens which arise from dysregulated cellular signaling, 50 0 0 are presented by HLA class I molecules, and are recognized as 7 8 9 10 11 12 13 14 15 16 1 2 3 4 5 6 7 8 9 10 11 IMAC Phosphopeptide HPLC-ESI-MS/MS Analysis and Number of Residues Position antigenic by circulating cytotoxic T cells. Furthermore, the shared Biological Characterization of T Cell Tissue Comparison Manual Validation Enrichment nature of PTTs among CRC patients illustrates their role as Responses Normal Tumor excellent targets for the development of novel immunotherapeutics. • HLA-presented phosphopeptides are typically 9- to 11-mers with a phosphorylated serine In this study, we analyze the phospholigandome profiles of residue in positions 4-6 of the peptide sequence IFNγ CD107a 27 CRC patient tissues and identify over 500 unique PTTs. TNFα IL-2 10-200 phosphopeptides/sample • Phosphorylation can enhance binding to HLA molecules: Peptides phosphorylated on a <1-100 copies/cell Through stringent selection criteria, we select 30 highly- serine residue in position 4 bind 10x stronger to HLA-A*0201 than their unmodified prevalent, HLA-restricted targets for inclusion in a CRC PTT- • Robust enrichment methodology coupled to highly-sensitive mass spectrometric counterpart based vaccine. instrumentation • A phosphate group on positions 4-6 is oriented upwards and solvent exposed, available for • Workflow allows for routine identification of low-level phosphopeptides from CRC patient interaction with a TCR tumor tissues PHOSPHOPEPTIDE TUMOR TARGETS Arc Segment = CRC Chord = individual Chord Width = phosphopeptide Phosphopeptide Tumor Targets are a novel class of neoantigens for Allelic Distribution Patient PTT Variation • PTTs are assigned to patient sample phosphopeptide abundance in specific sample immunotherapeutic development 160 # PTTs │ # CRC Tissues 70 Patient HLA haplotypes 60 Sample Frequency: Frequency: Frequency: 50 through predictive White – 45.6% White – 18.1% White – 21.5% 141│11 Black – 22.3% 1) Cancer-specific dysregulation of 40 HLA-A*02 Black – 15.8% HLA-B*07 HLA-C*07 3) Aberrant 140 30 algorithms and binding Hispanic – 37.1% Black – 18.1% kinase/phosphatase activity leads to PTTS# Hispanic – 13.2% Hispanic – 24.2% phosphopeptides are 20 assays Asian – 18.1% aberrant phosphorylation of self proteins 10 Asian – 33.3% presented at the cell surface 0 ATP 120 ADP HLA-A*02 HLA-B*07 HLA-C*07 • 42/49 alleles Phosphorylation (Kinase) represented present 2) Aberrantly 100 phosphorylated proteins PTTs enter HLA class I 80 78│2 processing pathway 72│12 • PTT presentation varies # PTTs Protein P Protein among alleles and 60 patients 46│4 40 36│14 • HLA-B*07 is Dephosphorylation 31│1 28│5 consistently a strong 25│2 25│1 (Phosphatase) 24│8 23│2 20│1 • Chord diagrams illustrate the connectivity of the phospholigandome among patients 19│7 P 20 │1 presenter of PTTs 16│2 16│8 14│2 13 13│3 11│6 10│2 10│2 9│1 9│1 7│2 7│1 6│1 6│5 5│6 4│1 4│1 4│1 3│1 3│1 3│3 3│2 2│4 2│7 2│2 2│1 2│1 • Many CRC-associated PTTs are common to allele-matched patients within the indication 1│1 1│1 • Present due to dysregulated cell signaling pathways associated with 0 malignant transformation HLA-A HLA-B HLA-C • The shared nature of PTTs allows for development of an off-the-shelf immunotherapeutic for CRC with pan-indication potential • Shared among individuals within and across indications • Recognized by the immune system as antigenic and elicit memory immune responses in healthy individuals PHOSPHOSYNVAX-CRC: PRODUCT DEVELOPMENT Phosphopeptide Identification Target Selection Therapeutic Benefit 6 HLA-A, 14 HLA-B, 10 HLA-C PTTs selected for PSV-CRC AGENUS’ VACCINE PLATFORM Indication CRC Age (Years) Median 66 27 CRC patient Patient Tissues 27 Range 45-87 Haplotype A*02 B*07 C*07 tumors analyzed HLA % CRC Patients HLA % CRC Patients # Patients % Patients # Patients % Patients PTT PTT Type (Allele-Matched) Type (Allele-Matched) % US Agenus’ versatile synthetic vaccine platform delivers robust immune responses Gender Male 12 44% Tissue Type Primary 25 93% 43% 13% 36% Female 9 33% Metastatic 2 7% Population Unknown 6 22% A*02 PTT 1 50-80% B*07 PTT 16 50-80% Recombinant # PTTs in Synthetic peptides A*02 PTT 2 50-80% B*07 PTT 17 50-80% 6 14 10 heat shock protein 70 > 500 MSI Status MSI-H 6 22% Grade High 4 15% PSV-CRC containing T cell MSI-L 1 4% Low 17 63% A*02 PTT 3 14-30% B*07 PTT 18 30-50% (Hsc70) phosphopeptides MSS 10 37% Unknown 6 22% epitopes A*02 PTT 4 14-30% B*07 PTT 19 50-80% % Patients with identified Unknown 10 37% ~85% ~100% ~100% A*02 PTT 5 14-30% B*07 PTT 20 30-50% ≥1 PTT A*02 PTT 6 14-30% C*07 PTT 21 >80% Avg # Peptides / B*07 PTT 7 >80% C*07 PTT 22 >80% Patient with ≥1 2.7 9.2 6.3 ✓ Reflect frequently occurring HLA types (A*02, B*07, C*07) PTT Powerful backbone Novel targets allow for B*07 PTT 8 >80% C*07 PTT 23 50-80% Hsc70-peptide Selection criteria- B*07 PTT 9 >80% C*07 PTT 24 >80% enables optimal peptide personalized and off- based filtering ✓ Prevalence in allele-matched patients complexes B*07 PTT 10 >80% C*07 PTT 25 50-80% delivery the-shelf therapies B*07 PTT 11 50-80% C*07 PTT 26 >80% ✓ Relevance of source protein to malignant transformation B*07 PTT 12 50-80% C*07 PTT 27 >80% QS-21 Stimulon® B*07 PTT 13 50-80% C*07 PTT 28 50-80% • ~70% of US/EU patients are eligible for vaccine based on HLA haplotype 30 specific ✓ Presence in other indications of interest B*07 PTT 14 50-80% C*07 PTT 29 50-80% molecular targets B*07 PTT 15 50-80% C*07 PTT 30 30-50% • 100% of eligible CRC patients’ tumors express ≥ 1 PTT Synthetic Vaccine for PSV-CRC ✓ Expression in tumor vs. normal tissues • >70% of tested PTTs are memory targets % CRC Patients AutoSynVax™: on-demand, defined by 0% 100% • CRC tumors express on average 7-10 PTTs ✓ Memory T cell responses in healthy individuals • 80% of PTTs are presented by ≥50% of allele- tumor NGS, targets patient-specific matched CRC patients Proprietary adjuvant induces neoantigens strong antibody and cell- PhosphoSynVax™: pre-manufactured, • 100% of PTTs are present in other cancers mediated immune responses targets shared phosphorylated epitopes AutoSynVax (ASV™) PhosphoSynVax (PSV™) CLINICAL OPPORTUNITY CONCLUSIONS Phosphopeptide 2 0 0 0 WT Peptide ) ) ) PBS 3 PBS 3 3 Peptides + QS21 m Hsc70-peptides + QS21 • Neoantigen vaccine comprised of 30 cancer-specific phosphopeptide tumor The development of a novel PTT-based vaccine for CRC may improve m 1 5 0 0 Peptides ( Hsc70-peptides e targets prioritized based on: m Product efficacy outcomes compared to standard-of-care treatments. u 1 0 0 0 Hsc70-peptides + QS21 l o v r ➢ Prevalence among patients within CRC o Phospholigandome analysis of a diverse subset of CRC patient tumors 5 0 0 y+ spots/11M splctes spots/11M y+ m - u Tumor Volume (mm Volume Tumor Tumor Volume (mm Volume Tumor ➢ Relevance of source protein to malignant transformation T IFN allowed for selection of novel targets for immunotherapeutic development. 0 0 5 1 0 1 5 2 0 2 5 3 0 ➢ Recognition by memory T cells from healthy individuals – validation of The inclusion of highly-prevalent, multiple allele-associated epitopes from D a y s p o s t t u m o r i n j e c t i o n Days Post Tumor Injection Days Post Tumor Injection safety diverse source proteins expands the eligible patient population and • Agenus proprietary vaccine format – clinical safety and immunogenicity • Experiments conducted in animal models illustrate that all three components increases the likelihood of stimulating an effective anti-tumor immune demonstrated in a viral indication are critical for anti-tumor effect response. Furthermore, the shared frequency observed in the PTT repertoire allows • Agenus proprietary vaccine format demonstrates clinical safety and • PSV is designed to train the patient’s immune system to recognize and kill Opportunity immunogenicity in a viral indication tumor cells, promoting lasting anti-tumor immunity and improved survival for advancement towards the development of an off-the-shelf cancer vaccine with broad therapeutic potential.
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