Sarah Penny

MHC class-I associated phosphopeptides are the targets of memory-like immunity in leukemia

Immunity against phosphopeptide antigens lacking in leukemia patients can be restored with stem cell transplantation. Adding to the Toolkit for Cancer Therapy The immune system is increasingly being used as a tool for cancer therapy. Researchers have harnessed the body’s own defense system to specifically target tumors. However, one limitation of immune-targeting strategies is the relative lack of targets. Because cancer cells are derived from normal human tissue, ideal antigens would be specifically or differentially expressed by tumor cells and healthy tissues. Now, Cobbold et al. find that phosphoproteins may broaden the pool of tumor antigens that can be targeted with immunotherapy. One difference between cancer and normal cells is the way in which they are regulated. Indeed, signal transduction pathways are frequently dysregulated in cancer cells. The authors now use a hallmark of signal transduction—protein phosphorylation—to identify and characterize new phosphoantigens that stimulate immune cells. They identified 95 phosphopeptides presented on the surface of leukemic cells and demonstrated that they could be recognized and killed by phosphopeptide-specific cytotoxic T lymphocytes. Somewhat surprisingly, healthy individuals had high levels of responses to phosphopeptides, but these responses were muted in leukemia patients with poor prognosis. What’s more, allogeneic stem cell transplant could restore phosphoprotein immune response in patients. These data suggest that phosphopeptides could be developed as new targets for cancer immunotherapy. Deregulation of signaling pathways is a hallmark of malignant transformation. Signaling-associated phosphoproteins can be degraded to generate cancer-specific phosphopeptides that are presented by major histocompatibility complex (MHC) class I and II molecules and recognized by T cells; however, the contribution of these phosphoprotein-specific T cells to immune surveillance is unclear. We identified 95 phosphopeptides presented on the surface of primary hematological tumors and normal tissues, including 61 that were tumor-specific. Phosphopeptides were more prevalent on more aggressive and malignant samples. CD8+ T cell lines specific for these phosphopeptides recognized and killed both leukemia cell lines and human leukocyte antigen–matched primary leukemia cells ex vivo. Notably, healthy individuals showed robust CD8+ T cell responses against many of these phosphopeptides within the circulating memory compartment. This immunity was significantly reduced or absent in some leukemia patients. This reduction correlated with clinical outcome; however, immunity was restored after allogeneic stem cell transplantation. These results suggest that phosphopeptides may be targets of cancer immune surveillance in humans, and point to their importance for development of vaccine-based and T cell adoptive transfer immunotherapies.

Identification of Glycopeptides as Posttranslationally Modified Neoantigens in Leukemia

The identification of neoepitopes expressed by tumors will aid the effectiveness of antitumor therapies. Four classes of posttranslationally modified tumor neoantigens were identified on primary tumors. Healthy donors had detectable natural immunity to a subset of these. 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.

Accelerated Loss of TCR Repertoire Diversity in Common Variable Immunodeficiency

Although common variable immunodeficiency (CVID) has long been considered as a group of primary Ab deficiencies, growing experimental data now suggest a global disruption of the entire adaptive immune response in a segment of patients. Oligoclonality of the TCR repertoire was previously demonstrated; however, the manner in which it relates to other B cell and T cell findings reported in CVID remains unclear. Using a combination approach of high-throughput TCRβ sequencing and multiparametric flow cytometry, we compared the TCR repertoire diversity between various subgroups of CVID patients according to their B cell immunophenotypes. Our data suggest that the reduction in repertoire diversity is predominantly restricted to those patients with severely reduced class-switched memory B cells and an elevated level of CD21lo B cells (Freiburg 1a), and may be driven by a reduced number of naive T cells unmasking underlying memory clonality. Moreover, our data indicate that this loss in repertoire diversity progresses with advancing age far exceeding the expected physiological rate. Radiological evidence supports the loss in thymic volume, correlating with the decrease in repertoire diversity. Evidence now suggests that primary thymic failure along with other well-described B cell abnormalities play an important role in the pathophysiology in Freiburg group 1a patients. Clinically, our findings emphasize the integration of combined B and T cell testing to identify those patients at the greatest risk for infection. Future work should focus on investigating the link between thymic failure and the severe reduction in class-switched memory B cells, while gathering longitudinal laboratory data to examine the progressive nature of the disease.

G462(P) Capturing t–cell receptors. a potential new modality for targeting hepatic tumours and post–transplantation lymphoproliferative disease (ptld)

Background The immune system plays an important part in recognising infection and diseases including cancer. Malignant cells express specific proteins on their cell surface. It is widely believed that it is these proteins that the immune system uses to recognise tumours and eventually eradicate them. When this process goes wrong, a tumour forms. The immune response to cancers in general is widely accepted to play an important role in control of tumorigenic processes, by using tumour infiltrating lymphocytes. How this process works is still being established, however there remains a potential target for cancer specific immunomodulatory treatment regimens. Aim (1) To identify tumour specific MHC class I phosphopeptide antigens on lymphoblastoid cell lines LCL’s (an in vitro model for PTLD) as well as hepatic tumour tissues. (2) T-cells are immune cells which are notoriously difficult to maintain in long–term culture and as a result it is difficult to establish an ‘off the shelf’ T-cell product, however the aim of this project was to explore potential modalities for capturing the T-cell receptor (TCR), important in recognising tumour specific antigens and the resultant product could be used to establish a non patient–specific, but tumour specific product. Patients and methods Paediatric and adult patients were identified with hepatic malignancy and consented as per current policy. Cells were isolated and tumour specific phosphopeptide antigens were identified. These provide the targets for T-cells, and more specifically TCR’s. Having identified these antigens, modalities have been explored for expanding these cells. Hybridoma technology is long established in immortalising B-cells, and this study aims to explore its potential with immortalisation of T-cells. Results A number of novel phosphopeptide antigens have been identified both in vitro as well as on patient tissues. This information has been used to identify potential T-cell targets and by formation of hybridomas we have established a method for expanding specific T-cell’s in vitro. Although these hybridomas are currently unstable due to their tetraploid status, we aim to modify this protocol further to allow for stable expansion of hybridoma cells which possess the relevant TCR motif. Conclusions Identifying a modality for expanding cells with a specific TCR repertoire clearly allows us to target tumour specific phosphopeptide antigens and has the potential to be developed as an immunomodulatory therapy in patients with hepatic tumours or PTLD.

Abstract 2893: Engineered release and presentation of antibody-bound viral antigens: A highly specific and novel immunotherapeutic approach to target cancer in vivo

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA 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 Pena, 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

Abstract 3627: Phosphopeptides as novel T cell epitopes in colorectal cancer

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 TIL9s 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

Abstract 512: Leukemia-specific immunity in healthy individuals and patients post-transplant targets phosphorylated tumor antigens

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL 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