Identification of Glycopeptides as Posttranslationally Modified Neoantigens in Leukemia

Abstract 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.</jats:p

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Establishing the prevalence of common tissue-specific autoantibodies following SARS CoV-2 infection.

COVID-19 has been associated with both transient and persistent systemic symptoms that do not appear to be a direct consequence of viral infection. The generation of autoantibodies has been proposed as a mechanism to explain these symptoms. To understand the prevalence of autoantibodies associated with SARS-CoV-2 infection, we investigated the frequency and specificity of clinically relevant autoantibodies in 84 individuals previously infected with SARS-CoV-2, suffering from COVID-19 of varying severity in both the acute and convalescent setting. These were compared with results from 32 individuals who were on ITU for non-COVID reasons. We demonstrate a higher frequency of autoantibodies in the COVID-19 ITU group compared with non-COVID-19 ITU disease control patients and that autoantibodies were also found in the serum 3-5 months post COVID-19 infection. Non-COVID patients displayed a diverse pattern of autoantibodies; in contrast, the COVID-19 groups had a more restricted panel of autoantibodies including skin, skeletal muscle and cardiac antibodies. Our results demonstrate that respiratory viral infection with SARS-CoV-2 is associated with the detection of a limited profile of tissue-specific autoantibodies, detectable using routine clinical immunology assays. Further studies are required to determine whether these autoantibodies are specific to SARS-CoV-2 or a phenomenon arising from severe viral infections and to determine the clinical significance of these autoantibodies

Establishing the prevalence of common tissue-specific autoantibodies following severe acute respiratory syndrome coronavirus 2 infection.

Coronavirus 19 (COVID-19) has been associated with both transient and persistent systemic symptoms that do not appear to be a direct consequence of viral infection. The generation of autoantibodies has been proposed as a mechanism to explain these symptoms. To understand the prevalence of autoantibodies associated with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, we investigated the frequency and specificity of clinically relevant autoantibodies in 84 individuals previously infected with SARS-CoV-2, suffering from COVID-19 of varying severity in both the acute and convalescent setting. These were compared with results from 32 individuals who were on the intensive therapy unit (ITU) for non-COVID reasons. We demonstrate a higher frequency of autoantibodies in the COVID-19 ITU group compared with non-COVID-19 ITU disease control patients and that autoantibodies were also found in the serum 3-5 months post-COVID-19 infection. Non-COVID patients displayed a diverse pattern of autoantibodies; in contrast, the COVID-19 groups had a more restricted panel of autoantibodies including skin, skeletal muscle and cardiac antibodies. Our results demonstrate that respiratory viral infection with SARS-CoV-2 is associated with the detection of a limited profile of tissue-specific autoantibodies, detectable using routine clinical immunology assays. Further studies are required to determine whether these autoantibodies are specific to SARS-CoV-2 or a phenomenon arising from severe viral infections and to determine the clinical significance of these autoantibodies

Tumor Infiltrating Lymphocytes Target HLA-I Phosphopeptides Derived From Cancer Signaling in Colorectal Cancer

There is a pressing need for novel immunotherapeutic targets in colorectal cancer (CRC). Cytotoxic T cell infiltration is well established as a key prognostic indicator in CRC, and it is known that these tumor infiltrating lymphocytes (TILs) target and kill tumor cells. However, the specific antigens that drive these CD8+ T cell responses have not been well characterized. Recently, phosphopeptides have emerged as strong candidates for tumor-specific antigens, as dysregulated signaling in cancer leads to increased and aberrant protein phosphorylation. Here, we identify 120 HLA-I phosphopeptides from primary CRC tumors, CRC liver metastases and CRC cell lines using mass spectrometry and assess the tumor-resident immunity against these posttranslationally modified tumor antigens. Several CRC tumor-specific phosphopeptides were presented by multiple patients’ tumors in our cohort (21% to 40%), and many have previously been identified on other malignancies (58% of HLA-A*02 CRC phosphopeptides). These shared antigens derived from mitogenic signaling pathways, including p53, Wnt and MAPK, and are therefore markers of malignancy. The identification of public tumor antigens will allow for the development of broadly applicable targeted therapeutics. Through analysis of TIL cytokine responses to these phosphopeptides, we have established that they are already playing a key role in tumor-resident immunity. Multifunctional CD8+ TILs from primary and metastatic tumors recognized the HLA-I phosphopeptides presented by their originating tumor. Furthermore, TILs taken from other CRC patients’ tumors targeted two of these phosphopeptides. In another cohort of CRC patients, the same HLA-I phosphopeptides induced higher peripheral T cell responses than they did in healthy donors, suggesting that these immune responses are specifically activated in CRC patients. Collectively, these results establish HLA-I phosphopeptides as targets of the tumor-resident immunity in CRC, and highlight their potential as candidates for future immunotherapeutic strategies

Combined ROS, ki67 and BCL2 staining of control and AML stem/progenitor cell subsets.

<p>Plots show representative immunophenotypic blasts stained with DCF, ki67 and BCL2 in control BM (A) CD34⁺AML BM (B) and CD34⁻ AML BM (C). Isotype controls for each sample were performed to establish negative gates (shown as green dotted rectangle). An expanded population with pro-survival phenotype, ki67^lowBCL2^high, is observed in both AML types (B and C) and highlighted by black rectangle.</p

ROS-separated ki67 and BCL2 expression in AML stem/progenitor cells.

<p>AML progenitors stained with mAb for surface markers, DCF, ki67 and BCL2 were separated into ROS^lowest and ROS^highest cells based on the 20% dimmest and 20% brightest DCF staining populations respectively. Charts show ki67 and BCL2 expression respectively in ROS^lowest and ROS^highest subsets in (A-B) CD34⁺ AMLs, (C-D) in CD34⁻ AMLs and (E-F) in CD34⁺ cells in MDS patients with no excess blasts (no EB) and MDS patients with excess blasts (RAEB-1/RAEB-2). Profiles from 5 representative control/normal BMs are shown for CD34⁺ and CD34⁻CD117⁺ cells in the respective charts (grey triangles). AML patients were subdivided into <i>Flt3</i>ITD⁺/<i>NPM1</i>wt (simplified to F+/N-), <i>Flt3</i>ITD⁻/<i>NPM1</i>mut (F-/N+), <i>Flt3</i>ITD⁺/<i>NPM1</i>mut (F+N+), CBF-AMLs, t(9;11)-AMLs and other cases. AML patients with poor early response (refractory/ early-relapse/ delayed remission) indicated by red squares. Patients achieving stable remission after one course of treatment are shown as black squares. Patients for whom early outcome data was unavailable are shown as grey squares. Thresholds for high BCL2 expression are shown as dashed lines on each BCL2 chart. The threshold was set at >mean+2SD of normal CD34⁺ (>6.9) or CD34⁻ blasts (>3.3).</p

Summarised ki67 and BCL2 expression in control, AML and MDS subsets.

<p>CD34⁺CD38^low, CD34⁺CD38^high and CD34⁻ (CD117⁺) blast subsets were compared for ki67-positivity (A), BCL2 expression (using BCL2-specific MFI defined by fold increase over staining with appropriate fluorescent isotype-control mAb) and the aberrant ki67^lowBCL2^high phenotype (C), which is defined using isotype control staining (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163291#pone.0163291.s003" target="_blank">S3E Fig</a>). Data includes 16 control BM, 29 CD34⁺ diagnosis AML samples (mixed BM/PB), 6 MDS with no excess blasts (no EB) and 6 MDS-RAEB samples. The CD34⁻ plots include data from 11 CD34⁻ diagnosis AMLs. Median expression and interquartile range is shown on each plot. Data is shown for all AML patients (grey squares, filled for CD34⁺ and open for CD34⁻ AMLs) and genetic subgroups. F+N- denotes ITD⁺/<i>NPM1</i>wt patients (red filled squares, all CD34⁺), F+N+ denotes ITD⁺/<i>NPM1</i>mut patients (red open squares, all CD34⁻) and F-N+ denotes ITD⁻<i>/NPM1</i>mut (blue filled open/blue open squares for CD34⁺/CD34⁻ respectively). CBF-AMLs are shown as green squares.</p