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The addition of the ATR inhibitor, AZD6738, did not show synergistic cytotoxicity with SG3199 or ADCs in parental MDA-MB-361 cells

Characterization of 361-PBDr resistant line

Differentially expressed proteins identified in the 361-PBDr cell line

Table S1. Summary of samples used for this study. Table S2. Antibodies used in the study. Figure S1. Mass spectra of all peptides referenced in figures 2 and 3. Figure S2. ETD Mass spectrum of RPPItQSSL containing an asymmetrically dimethylated Arg residue. Figure S3. ICS flow cytometry plots for HD5. Figure S4. Gating strategy for phenotyping shown in Fig 2E. Figure S5. Healthy donor immunity to O-GlcNAc peptides measured using IFNgamma ELISpot. Figure S6. ICS flow cytometry plots for HD5. Figure S7. Positional analysis of O-GlcNAc peptides.

Knockdown of 5T4 expression do not confer SG3199 resistance to parental MDA-MB-361 cells.

DNA damage checkpoint activation mediated by SLFN11 and ATR pathways in response to SG3199.

Leukemias are highly immunogenic, but they have a low mutational load, providing few mutated peptide targets. Thus, the identification of alternative neoantigens is a pressing need. Here, we identify 36 MHC class I–associated peptide antigens with O-linked β-N-acetylglucosamine (O-GlcNAc) modifications as candidate neoantigens, using three experimental approaches. Thirteen of these peptides were also detected with disaccharide units on the same residues and two contain either mono- and/or di-methylated arginine residues. A subset were linked with key cancer pathways, and these peptides were shared across all of the leukemia patient samples tested (5/5). Seven of the O-GlcNAc peptides were synthesized and five (71%) were shown to be associated with multifunctional memory T-cell responses in healthy donors. An O-GlcNAc-specific T-cell line specifically killed autologous cells pulsed with the modified peptide, but not the equivalent unmodified peptide. Therefore, these posttranslationally modified neoantigens provide logical targets for cancer immunotherapy. Cancer Immunol Res; 5(5); 376–84. ©2017 AACR.

Supplementary Data from Antibody-Peptide Epitope Conjugates for Personalized Cancer Therapy

Antibody–peptide epitope conjugates (APEC) are a new class of modified antibody–drug conjugates that redirect T-cell viral immunity against tumor cells. APECs contain a tumor-specific protease cleavage site linked to a patient-specific viral epitope, resulting in presentation of viral epitopes on cancer cells and subsequent recruitment and killing by CD8+ T cells. Here we developed an experimental pipeline to create patient-specific APECs and identified new preclinical therapies for ovarian carcinoma. Using functional assessment of viral peptide antigen responses to common viruses like cytomegalovirus (CMV) in patients with ovarian cancer, a library of 192 APECs with distinct protease cleavage sequences was created using the anti-epithelial cell adhesion molecule (EpCAM) antibody. Each APEC was tested for in vitro cancer cell killing, and top candidates were screened for killing xenograft tumors grown in zebrafish and mice. These preclinical modeling studies identified EpCAM-MMP7-CMV APEC (EpCAM-MC) as a potential new immunotherapy for ovarian carcinoma. Importantly, EpCAM-MC also demonstrated robust T-cell responses in primary ovarian carcinoma patient ascites samples. This work highlights a robust, customizable platform to rapidly develop patient-specific APECs.Significance:This study develops a high-throughput preclinical platform to identify patient-specific antibody–peptide epitope conjugates that target cancer cells and demonstrates the potential of this immunotherapy approach for treating ovarian carcinoma.

Prostate cancer is traditionally considered an immunologically “cold” tumor type rendering patients insensitive to immunotherapy. Targeting surface antigens on tumors through cellular therapy can induce a potent anti tumor immune response to “heat up” the tumor microenvironment. However, many antigens expressed on prostate tumors are also found on normal tissues, potentially causing on-target, off-tumor toxicities and a sub-optimal therapeutic index. Our target discovery and validation efforts identified STEAP2 as a superior prostate antigen for therapeutic targeting. Importantly, STEAP2 is a highly prevalent prostate cancer antigen displaying high, homogeneous cell surface expression across all stages of disease. A novel lead generation approach facilitated the development of a potent and specific armored STEAP2 CAR-T therapeutic candidate, AZD0754. This second generation CAR-T product is armored with a dominant-negative TGFβRII, thereby bolstering activity in the TGFβ-rich immunosuppressive environment of prostate cancer. Armored STEAP2 CAR-T cells demonstrate favorable in vitro properties, robust dose dependent in vivo efficacy in STEAP2 expressing cell line- and patient derived- mouse xenograft models and encouraging preclinical safety. Taken together, this data builds confidence in the specificity and potency of this potential first in class STEAP2 targeted CAR-T therapy and supports future clinical development. Citation Format: Dewald van Dyk, Peter Zanvit, Christine Fazenbaker, Kelly McGlinchey, Weichuan Luo, Jessica Pezold, John Meekin, Chien-ying Chang, Benjamin Clark, Philip L. Martin, Clare Hoover, Eric Tu, Ryan Gilbreth, Mark Cobbold, Gordon Moody, Emily E. Bosco. Antitumor activity of AZD0754, a dnTGFbRII armored STEAP2 targeted CAR-T therapy, in preclinical models of prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB085.

Introduction: Chimeric antigen receptor (CAR) T cells can mediate deep and durable responses in hematologic malignancies, however, achieving success in solid tumors has been so far limited largely by lack of suitable solid tumor-associated antigens and the immunosuppressive tumor microenvironment (TME). GPC3 is a surface antigen overexpressed in hepatocellular cancer (HCC) and virtually absent on healthy tissues. In this first-in-human (FIH) study, we investigated the feasibility, safety and initial anti- HCC efficacy of C-CAR031. C-CAR031 is an autologous, GPC3-directed armored CAR-T with affinity-tuned scFv to enhance the safety profile, and a 4-1BB and CD3ζ signaling domain. The C-CAR031 transgene includes a T2A viral self-cleaving peptide and a dominant negative TGF-β receptor II (TGFβRIIDN). The expression of TGFβRIIDN protects the cells against the immunosuppressive HCC TME and the T2A peptide allows for equimolar expression of the two transgene products. Methods: This FIH, open-label dose escalation trial employs an accelerated titration plus i3+3 design. Histologically confirmed GPC3+ advanced HCC patients (pts) who failed systemic treatments received a single-dose i.v. infusion of C-CAR031 following standard lymphodepletion. The primary objective was to assess the safety and tolerability. Adverse events (AEs) were graded using CTCAE 5.0, and cytokine release syndrome (CRS)/immune effector cell-associated neurotoxicity syndrome (ICANS) were graded according to ASTCT 2019 criteria. Results: As of Dec. 31st 2022, 7 pts received two dose levels (DL1, n=1; DL2, n=6) of C-CAR031. The median number of prior lines of therapies was 4 (range 1-6). The median follow-up was 77 (40-213) days. Six pts with ≥28 days’ follow-up were eligible for safety evaluation. The only ≥Gr3 non-hematologic product-related AE observed was transient Gr3 AST elevation in two pts. Five of 6 pts experienced Gr1/2 CRS, with median time to onset and duration of 3 (range 2-7) and 4 (4-6) days. No DLT or ICANS was observed. Of the 5 pts evaluable for preliminary efficacy, 4 pts had unconfirmed PR, which are currently pending confirmation. AFP was also stabilized or reduced in all 4 patients with uPR. All 5 pts had reduction in tumor burden, with a median change of -31.2% (range -3.4- -60.6%)/-41.4% (-3.4- -56.6%) per RECIST1.1/mRECIST. C-CAR031showed a robust cellular kinetic profile. In DL2, the median Tmax, Cmax and AUC0-28Day were 15 days, 772,014 copies/μg gDNA and 7,747,054 days*copies/μg gDNA, respectively. CAR-T cells were detectable in blood of all pts in the last follow-up. Conclusions: In this FIH study, C-CAR031 is well tolerated and shows promising anti- tumor activity. Enrollment is ongoing to confirm initial results. Citation Format: Qi Zhang, Qihan Fu, Wanyue Cao, Xingyuan Xu, Ao Xia, Jiaqi Huang, Andy Zou, Judy Zhu, Fei Wang, Yi Hong, Hui Wan, Yihong Yao, Nina Chu, Ryan Gilbreth, Maria Letizia Giardino Torchia, John Stone, Attilio Bondanza, Benny Farsaci, Gordon Moody, Mark Cobbold, Tingbo Liang. First report of preliminary safety, efficacy, and pharmacokinetics of C-CAR031 (GPC3-specific TGFβRIIDN CAR-T) in patients with advanced HCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT097.

Purpose: To evaluate the mechanisms of how therapeutic upregulation of the transcription factor, CCAAT/enhancer-binding protein alpha (C/EBPα), prevents tumor progression in patients with advanced hepatocellular carcinoma (HCC) and in different mouse tumor models. Experimental Design: We conducted a phase I trial in 36 patients with HCC (NCT02716012) who received sorafenib as part of their standard care, and were given therapeutic C/EBPα small activating RNA (saRNA; MTL-CEBPA) as either neoadjuvant or adjuvant treatment. In the preclinical setting, the effects of MTL-CEBPA were assessed in several mouse models, including BNL-1ME liver cancer, Lewis lung carcinoma (LLC), and colon adenocarcinoma (MC38). Results: MTL-CEBPA treatment caused radiologic regression of tumors in 26.7% of HCC patients with an underlying viral etiology with 3 complete responders. MTL-CEBPA treatment in those patients caused a marked decrease in peripheral blood monocytic myeloid-derived suppressor cell (M-MDSC) numbers and an overall reduction in the numbers of protumoral M2 tumor-associated macrophages (TAM). Gene and protein analysis of patient leukocytes following treatment showed CEBPA activation affected regulation of factors involved in immune-suppressive activity. To corroborate this observation, treatment of all the mouse tumor models with MTL-CEBPA led to a reversal in the suppressive activity of M-MDSCs and TAMs, but not polymorphonuclear MDSCs (PMN-MDSC). The antitumor effects of MTL-CEBPA in these tumor models showed dependency on T cells. This was accentuated when MTL-CEBPA was combined with checkpoint inhibitors or with PMN-MDSC–targeted immunotherapy. Conclusions: This report demonstrates that therapeutic upregulation of the transcription factor C/EBPα causes inactivation of immune-suppressive myeloid cells with potent antitumor responses across different tumor models and in cancer patients. MTL-CEBPA is currently being investigated in combination with pembrolizumab in a phase I/Ib multicenter clinical study (NCT04105335).

Leukemias are highly immunogenic, but they have a low mutational load, providing few mutated peptide targets. Thus, the identification of alternative neoantigens is a pressing need. Here, we identify 36 MHC class I–associated peptide antigens with O-linked β-N-acetylglucosamine (O-GlcNAc) modifications as candidate neoantigens, using three experimental approaches. Thirteen of these peptides were also detected with disaccharide units on the same residues and two contain either mono- and/or di-methylated arginine residues. A subset were linked with key cancer pathways, and these peptides were shared across all of the leukemia patient samples tested (5/5). Seven of the O-GlcNAc peptides were synthesized and five (71%) were shown to be associated with multifunctional memory T-cell responses in healthy donors. An O-GlcNAc-specific T-cell line specifically killed autologous cells pulsed with the modified peptide, but not the equivalent unmodified peptide. Therefore, these posttranslationally modified neoantigens provide logical targets for cancer immunotherapy. Cancer Immunol Res; 5(5); 376–84. ©2017 AACR.

Tumor associated macrophages (TAM) and myeloid-derived suppressor cells (MDSC) are widely implicated in the suppression of immune responses in cancer and are associated with negative clinical outcomes. We report that in mouse tumor models, therapeutic up-regulation of the transcriptional factor C/EBPα with small activating RNA (MTL-CEBPA) blocks the suppressive activity of monocytic (M) MDSC and TAM without affecting polymorphonuclear (PMN) MDSC. MTL-CEBPA treatment demonstrated antitumor activity that was dependent on T cells. Combination of MTL-CEBPA and anti-PD1 antibody or with PMN-MDSC targeted therapy resulted in marked antitumor effect. A phase I trial was conducted in 36 patients with advanced hepatocellular carcinoma (HCC). Marked decrease in M-MDSC and the expression of genes and proteins involved in immune suppressive activity of these cells were observed. Combined treatment with MTL-CEBPA and sorafenib resulted in a 27% objective radiological response and three complete responses in patients with virally associated HCC. Tissue biopsies from these patients demonstrated decrease in M2 polarized macrophages within the tumors. Thus, therapeutic up-regulation of C/EBPα inhibited suppressive myeloid cells that resulted in potent antitumor effect in mice and with encouraging clinical response in cancer patients. We are currently recruiting to a multi-centre study of MTL-CEBPA in combination with a PD-1 inhibitor (pembrolizumab) in adult patients with advanced solid tumors (TIMEPOINT - ClinicalTrials.gov Identifier: NCT04105335). Citation Format: Ayumi Hashimoto, Debashis Sarker, Vikash Reebye, Sheba Jarvis, Mikael H. Sodergren, Andrew Kossenkov, Nina Raulf, Jenni Vasara, Pinelopi Andrikakou, Tim Meyer, Kai-Wen Huang, Ruth Plummer, Cheng Ean Chee, Duncan Spalding, Madhava Pai, David J. Pinato, Rohini Sharma, Bristi Basu, Daniel Palmer, Yuk-Ting Ma, Robert Habib, Anna Martirosyan, Naouel Elasri, Adeline Reynaud, John Rossi, Mark Cobbold, Nagy Habib, Dmitry Gabrilovich. Up-regulation of C/EBPα inhibits suppressive activity of myeloid cells and potentiates antitumor response in mice and cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1730.

Immunotherapy has revolutionized patient care, however, the majority of patients with cancer still do not benefit from this treatment modality. This is due to many factors, including a complex tumor microenvironment (TME) that has too few functional effector cells and/or the presence of many inhibitory cells. Understanding how to remodel the TME to potentiate productive immunity against cancer cells is an active area of research. Growth and Differentiation Factor 15 (GDF-15) is a member of the TGF-beta superfamily that is overexpressed by many solid tumors and is implicated in dampening immune responses. The present study was aimed at understanding the role of GDF-15 in tumor immune biology. We show that GDF-15 can inhibit the differentiation and activation of human monocyte-derived dendritic cells (DCs) and prevent T cell activation. Furthermore blockade of GDF-15 by AZD8853, an anti-GDF15 monoclonal antibody, can restore these activities in vitro. Tumors derived from GDF15 knock-out syngeneic cell lines, Renca and MC-38, show 2-3 fold increases in T cells and activated DCs in the TME as compared to tumors derived from isogeneic parental cells. Treatment of mice with anti-GDF15 mAb leads to substantial anti-tumor activity in anti-PD-L1 refractory LL/2 and MBT2 syngeneic tumors with 50% of the animals showing complete tumor regressions. Coupled with this anti-tumor activity, we show that after 2 doses of anti-GDF15 mAb there is an increase in both activated T cells and DCs in the TME. Depletion of T cells, using anti-CD4 and anti-CD8, in mice ablates the anti-tumor activity observed with anti-GDF15 mAb treatment. These results suggest that inhibition of GDF15 by AZD8853 may be an attractive therapeutic strategy for patients with solid tumors that have high expression of GDF15. Citation Format: Elaine Hurt, Suneetha Thomas, Kathy Mulgrew, Stephen Blackmore, James Moynihan, Fiona Cusdin, Roger Dodd, Peter Cariuk, Anna Sigurdardottir, Emily Brannigan, Claire Dobson, Rakesh Kumar, Mark Cobbold. AZD8853: A novel antibody targeting GDF15 for immunotherapy refractory tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1828.

Introduction: Only a fraction of hepatocellular carcinoma (HCC) patients benefit from programmed death receptor-1 (PD-1) blockade. Combining PD-1 blockade with antiangiogenic treatments has been able to substantially increase the fraction of responsive HCC patients, but the mechanism of this interaction is unknown. We recapitulated these clinical outcomes (response vs. resistance) using orthotopic murine models of HCC. VEGFR2 blockade significantly delayed primary tumor growth but failed to prolong survival, while anti-PD-1 monotherapy conferred a minor survival. However, dual anti-PD-1/VEGFR-2 therapy significantly inhibited primary tumor growth and doubled survival in multiple models. Combination therapy reprogrammed the immune microenvironment by increasing CD8+ cytotoxic T-cell infiltration and activation, improving the ratio of anti- vs. pro-tumor-associated macrophages, and reducing T regulatory cell (Treg) infiltration in HCC tissue. Moreover, we found that VEGFR-2 blockade increased the PD-1 expression in tumor-infiltrating CD4+ cells. Under anti-PD-1 therapy, CD4+ cells promote normalized vessel formation in the face of antiangiogenic therapy. Conclusion: We show that dual anti-PD-1/VEGFR-2 therapy has a durable vessel fortification effect in HCC and can overcome treatment resistance to either treatment alone and increase survival in both anti-PD-1 therapy resistant and responsive HCC models. Citation Format: Meenal Datta, Kohei Shigeta, Tai Hato, Shuji Kitihara, Ivy X. Chen, Aya Matsui, Hiroto Kikuchi, Emilie Mamessier, Shuichi Aoki, Rakesh R. Ramjiawan, Hiroki Ochiai, Nabeel Bardeesy, Peigen Huang, Rakesh K. Jain, Mark Cobbold, Andrew X. Zhu, Dan G. Duda. Dual PD-1 and VEGFR-2 blockade induces vascular normalization and enhances antitumor immune responses in hepatocellular carcinoma [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B89.

Most cancer cells carry mutations unique to the patient’s individual tumor and shared biochemical signatures that are not present in healthy cells. Agenus has three vaccine platforms designed to treat cancers based on the unique needs of a given patient. Our vaccine platforms are designed to educate the patient’s immune system to recognize tumor-specific aberrations, or neo-antigens, and mount an anti-tumor immune response. Agenus’ Prophage™ vaccine platform is an individualized vaccine made from the patient’s own tumor tissue. Heat shock proteins (gp-96) that naturally chaperone and bind tumor-derived peptides are extracted from the patient’s tumor and constitute the vaccine. Some of these peptides are neo-antigens. Agenus has completed Phase 2 clinical trials with Prophage™ vaccine in newly diagnosed glioblastoma (ndGBM), and has previously reported that there was improved progression-free and overall survival with Prophage™ vaccine compared to standard of care. Agenus’ AutoSynVax™ vaccines are uniquely designed and manufactured for each patient based on NGS profiling of the patient’s tumor from a biopsy. Leveraging the Agenus Immunogenic Mutation (AIM™) workflow, we are able to generate a synthesis-ready blueprint for an optimal immunogenic and personalized neo-antigen vaccine. The AIM™ platform provides a robust and efficient approach to computational vaccinology designed to deliver a set of likely immunogenic peptides, agnostic to vaccine format, followed by generation of a format-specific blueprint ready for vaccine synthesis and manufacture. The synthesized neo-epitopes are complexed to recombinant heat shock protein 70 (HSC70) and are administered along with our QS-21 Stimulon® adjuvant. HSC70 is known to transport epitopes and play a role in displaying them to T cells. While the first two of Agenus’ vaccine platforms are highly individualized, our PhosphoSynVax™ vaccine is an off-the-shelf vaccine format targeting a novel class of tumor neo-antigens arising from post translational modifications (PTMs). Due to dysregulated cell signaling pathways in cancer, self-peptides can be aberrantly phosphorylated, a number of which are subsequently presented on HLA molecules. Using mass-spectrometry, we have identified a library of over a thousand HLA phospholigands. Many of these are tumor specific and found in multiple patients across multiple indications, enabling pre-manufacture of PhosphoSynVax™ vaccines for ready use. Upon testing the HSP plus synthetic peptide vaccine format in murine models, we have demonstrated effective tumor control in a therapeutic setting and also effective immune memory in a long-term prophylactic setting. Given Agenus’ diverse portfolio we have the opportunity to combine our immune education strategies with immunomodulatory antibodies to increase therapeutic efficacy. Citation Format: Mohamed Uduman, Mithun Khattar, Bishnu Joshi, Antoine Tanne, Benjamin Morin, Armen Karapetyan, Elise Drouin, Sandra Craig, Paisley Myers, Erin Jeffery, Nicholas Wilson, Amy Yang, Victor H. Engelhard, Mark Cobbold, Donald F. Hunt, Dennis Underwood, Shiwen Lin, Mark Findeis, Jeffrey Raizer, John Goldberg, Jennifer S. Buell, Robert Stein, Daniel L. Levey, John Castle. Agenus’ next generation cancer vaccine platforms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4609. doi:10.1158/1538-7445.AM2017-4609

Harnessing the power of adaptive immunity to combat cancer has been a long-term goal of translational immunotherapy. Tumor-specific immunity, where present, is typically at low frequency and affinity with compromised effector function. By contrast, immunity against persistent herpesviruses in man is characterised by high affinity cytotoxic T-lymphocytes (CTL) at high frequency with potent effector function. Furthermore, the immunosubversive mechanisms employed by herpesviruses show striking parallels to tumors, yet the associated anti-viral immunity limits these to life-long asymptomatic infections. We reasoned that the delivery of immunodominant viral peptide epitopes to the tumor surface might facilitate passive-loading of peptides into empty MHC class-I molecules, effectively mimicking viral infection, rendering tumors susceptible to lysis by anti-viral immunity. To address this we developed a new class of targeting antibodies: APEC (Antibody Peptide Epitope Complexes) that are able to deliver an antigenic payload at the cell surface through proteolytic release of covalently-coupled peptide antigens. As a proof-of-concept we used clinically-validated antibodies cetuximab (anti-EGFR) and rituximab (anti-CD20) to develop APECs that are able to target human tumors. We screened 15 HLA-A*0201+ EGFR-expressing NCI-60 cell lines, CD20+ lymphoma cell lines, 20 primary CD20+ CLL tumor samples and four healthy B-cells against a library of 190 cetuximab-APECs (cAPEC) or rituximab (rAPEC) incorporating the immunodominant cytomegalovirus (CMV) pp65495-503 epitope and candidate protease cleavage sites following co-incubation with CMV-specific CTL (CMV-CTL). The most effective cAPEC and rAPEC were those incorporating MMP2, MMP9, Cathepsin B and Cathepsin D protease recognition domains. Very few (2/190) rAPEC were able to redirect CMV-CTL against healthy cells. Heterogeneity was observed for primary CLL tumors but a limited number of rAPEC were effective in all cases (5/190). Mechanistic studies demonstrated that: (i) peptide loading occurred at the cell surface, (ii) required the expression of target antigens at the cell surface and (iii) T-cell recognition could be inhibited by unconjugated antibody (92%) or by incubation with protease inhibitors (83%). T-cell specificity was examined using rAPEC treated tumor targets co-incubated with various HLA-matched effector T-cell populations. No activation of CD4+ was observed including CD4+CD25hi regulatory T-cell populations. Incubation with CD8+ T-cells revealed that only pp65495-503-specific CD8+ T-cells engaged with APEC-treated tumor cells. Lastly, xenograft studies using EGFR+ and CD20+ tumor cell lines demonstrated efficacy of both cAPEC and rAPEC to eliminate tumors in vivo by redirecting anti-viral CTL. These data indicate that APECs represent a powerful new approach to combat cancer. Citation Format: David G. Millar, Laura Morton, Manuela Carvalho Gaspar, Punam Mistry, Hugo De La Peña, Ricky Joseph, Sarah Penny, Oliver C. Goodyear, Margaret Goodall, Guy E. Pratt, Mark Cobbold. Engineered release and presentation of antibody-bound viral antigens: A highly specific and novel immunotherapeutic approach to target cancer in vivo. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2893. doi:10.1158/1538-7445.AM2014-2893

Background: There is a pressing need for novel immunotherapeutic targets in colorectal cancer (CRC). Memory CD8+ T cell infiltration is now well established as a key prognostic indicator in CRC, and it is known that these tumor infiltrating lymphocytes (TILs) are specifically targeting and killing tumor cells. However, the epitopes that these TILs use to identify cancer cells have not been determined. This has limited the use of immunotherapies in CRC, despite their efficacy in other cancer types. Recently, phosphopeptides have emerged as strong candidates for tumor-specific epitopes, since dysregulation of signaling in cancers leads to aberrant protein phosphorylation. Here, we identify CRC-associated phosphopeptides and assess the tumor-resident immunity against these novel epitopes. Methods: We compared tumor and healthy tissue from CRC patients, to identify tumor-specific MHC class-I associated phosphopeptides. The tissues were lysed, the MHC class-I complexes affinity purified, and the bound peptides eluted. Phosphopeptides were enriched using immobilized metal affinity chromatography, and characterized using mass spectrometry. TILs, from the same tumors, were extracted and expanded, and their responses to the phosphopeptides assessed using multiplexed intracellular cytokine staining. Cytolytic activity was observed by staining for surface mobilization of CD107a. Healthy donor responses were quantified using interferon-γ ELISpot, and functionality assessed using a europium release killing assay. Results: We have identified 125 tumor-associated MHC class-I phosphopeptides from CRC, with different HLA-restrictions. There were, on average, 3.5 times more different phosphopeptides identified on cancer than healthy tissues, at 6.7-fold higher levels. Many of these novel epitopes are attributable to signaling events in well-defined cancer pathways and are therefore markers of malignancy. Through analysis of TIL's cytokine responses to these phosphopeptides, we have established that they are playing a key role in tumor-resident immunity. There were multifunctional TILs present in primary and metastatic tumors that recognized and killed in response to these phosphopeptides. Up to 0.7% of expanded TILs targeted each phosphopeptide, comparable with responses seen to viral epitopes. Thus, immunity to tumor-associated phosphopeptides represents a biological strategy for distinguishing tumor from healthy tissue. Furthermore, we have shown that healthy donors have pre-existing, memory T cell responses to many (58%) of these CRC-associated phosphopeptides. These phosphopeptide-specific T cells are readily expanded ex vivo and can kill CRC cell lines. Therefore, MHC class-I associated phosphopeptides are ideal immunotherapeutic targets, as immunity must spare healthy tissue. Conclusion: The identification of this novel class of MHC class-I epitopes in CRC offers new hope for the future of immunotherapy in this malignancy. Citation Format: Sarah A. Penny, Jennifer G. Abelin, Abu Z. Saeed, Stacy A. Malaker, Paisley D. Trantham, Jeffrey Shabanowitz, Stephen T. Ward, Donald F. Hunt, Mark Cobbold. Phosphopeptides as novel T cell epitopes in colorectal cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3627. doi:10.1158/1538-7445.AM2014-3627

The major antigenic targets of endogenous tumor immunity remain unknown although many minor targets have been identified. At a proteomic level, cancer is considered a disease of deregulated cell signal pathway activation; therefore we investigated whether endogenous anti-tumor immunity targeted phosphorylated protein antigens generated through the transformation process. In previous work we identified phosphorylated peptides differentially expressed on primary leukemia samples: 58 for acute myeloid leukemia (AML) samples and 31 for chronic lymphocytic leukemia (CLL). Many of the identified phosphopeptides were derived from established leukemogenic oncoproteins such as MLL, RUNX1, c-Myc, EP300, CXCR4, SKI, GRK, CD19, CD20, NCOA1, and TLK1 among others and differentially expressed between malignant and healthy counterpart tissue (splenic B cells, bone marrow). Using ELISpot and HLA-phosphopeptide multimers, we screened 10 healthy donors, 17 patients in complete remission with underlying AML and 14 patients with CLL. Healthy donors (HD) exhibited a highly heterogeneous reactivity against this panel of phosphoantigens but all responded to at least 10/57 phosphoantigens tested. Further investigation revealed this immunity resided within the central memory CD8 T cell compartment at the same magnitude of responses as those against influenza and adenovirus. By contrast only 2/12 AML and 5/14 CLL patients demonstrated anti-phosphopeptide immunity compared to 10/10 HDs (≥2< 0.01 for both). In contrast immunity against viral and mitogenic stimuli was similar between patients and HDs. Following allogeneic stem cell transplantation the majority (10/12) AML patients reconstituted anti-phosphopeptide immunity. In some cases dramatic expansions, of similar magnitude to cytomegalovirus responses, were observed approaching 1% of CD8 T cells for a single phosphopeptide epitope. In CLL, none of whom were transplanted, we found that over the study period of 12 months that among the patients with anti-phosphopeptide immunity only 1 of 5 patients required treatment compared to 4 of 9 patients requiring treatment where no anti phosphopeptide responses were seen (≥2=NS). Importantly, ex vivo expanded phosphopeptide specific T cells isolated from both healthy individuals and patients post-transplant were able to kill both HLA-matched primary leukemia samples and AML cell lines in a phosphopeptide-specific manner. These data suggest a prominent role for tumor-associated phosphopeptide antigens in the prevention and control of haematolymphoid disease in man. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 512. doi:1538-7445.AM2012-512