Antigen Presentation Machinery Research Articles

Cancer immune escape: the role of antigen presentation machinery.

The mechanisms of antigen processing and presentation play a crucial role in the recognition and targeting of cancer cells by the immune system. Cancer cells can evade the immune system by downregulating or losing the expression of the proteins recognized by the immune cells as antigens, creating an immunosuppressive microenvironment, and altering their ability to process and present antigens. This review focuses on the mechanisms of cancer immune evasion with a specific emphasis on the role of antigen presentation machinery. The study of the immunopeptidome, or peptidomics, has provided insights into the mechanisms of cancer immune evasion and has potential applications in cancer diagnosis and treatment. Additionally, manipulating the epigenetic landscape of cancer cells plays a critical role in suppressing the immune response against cancer. Targeting these mechanisms through the use of HDACis, DNMTis, and combination therapies has the potential to improve the efficacy of cancer immunotherapy. However, further research is needed to fully understand the mechanisms of action and optimal use of these therapies in the clinical setting.

Abstract 3967: CRISPR/Cas9 screening identified novel membrane targets sensitizing hepatocellular carcinoma cells to natural killer cell-mediated cytotoxicity

Abstract Natural killer cells (NK) play a central role in cancer immune surveillance by directly killing cancer cells and producing pro-inflammatory cytokines to stimulate and recruit other immune cells to elaborate long-lasting anti-tumor responses. However, little is known about the immune escape mechanisms and regulatory proteins, particularly those for NK, involved in hepatocellular carcinoma (HCC), one of the most common and lethal cancers. To identify the determinants of NK killing function, we performed an HCC cell line-based CRISPR/Cas9 loss-of-function genetic screen, using a human whole membrane protein library containing ~7000 genes curated from 3 public membrane protein datasets. The human SNU449 was chosen as a representative HCC cell line for CRISPR/Cas9 editing based on the genetic profile analysis, representing one HCC patient subpopulation (> 30%) in the TCGA cohort. The cell line was incubated with NK92 cells at ratios of 2:1, 1:1, and 0.5:1. Bioinformatics methods for prioritizing genes (e.g., MAGeCK) were applied on 3 tested groups with different ratios. Single guide RNAs targeting genes in the antigen presentation machinery were depleted, including HLA-C, B2M, and TAP1/2, whereas components of the tumor-immune synapse, such as NCR3LG1 and ICAM1, were enriched, confirming the validity of the screen. SLC33A1 (an endosomal acetylCoA transporter) and SLC31A1 (the major copper influx transporter) were also depleted, previously unknown as NK regulators. The effects of SLC33A1 and SLC31A1 were validated in individual gene knockout cells generated respectively by lentiviral sgRNA expression. Furthermore, knockout of either transporter in SNU449 cells increased IFNγ secretion, confirming NK92 activation. Our results revealed that CRISPR/Cas9-based functional genomics screens in HCC cell lines could identify novel targets modulating NK-mediated killing. Further analysis of mechanisms of action, and particularly functional validation in in vivo systems, will potentially provide novel targets for the treatment of HCC. Citation Format: Shuo Li, Lin Han, Siyao Zhang, Guangfeng Geng, Lei Huo, Dawei Huang, Zhaoren He, Shuoran Li, Zhaoshi Jiang, James X. Rong. CRISPR/Cas9 screening identified novel membrane targets sensitizing hepatocellular carcinoma cells to natural killer cell-mediated cytotoxicity. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3967.

Abstract 3289: Melanoma patients with high and low target expression can benefit from TCR-CD3 bispecifics through direct and indirect mechanisms of tumor control

Abstract Background: ImmTAC molecules are TCR-CD3 bispecific fusion proteins that can redirect and activate polyclonal T cells to kill tumors. Tebentafusp, which targets gp100, is the first ImmTAC to demonstrate an overall survival benefit and is approved for the treatment of HLA-A*02:01 adult patients with unresectable or metastatic uveal melanoma1. Clinical benefit was observed in patients with both high and low gp100 expression2, suggesting direct and indirect mechanisms of tumor control, which we explored using in vitro models. Method: Uveal and cutaneous melanoma cell lines were pulsed with 0-1 µM peptide concentrations or were treated with supernatants from ImmTAC-activated PBMC, with or without blocking antibodies against IFNβ, IFNγ, TNFα, and IL6. Peptide-HLA (pHLA) molecules on tumor cell surface were quantified by microscopy. ImmTAC-redirected T cell activation and killing were measured by ELISPOT, MSD, and xCelligence assays. Antigen presentation machinery (APM) gene expression was measured by qPCR. Linear regression models were used to assess the influence of variables on ImmTAC-mediated T cell activation. Results: Peptide-pulsing of tumor cells showed a dose response relationship between surface pHLA number and ImmTAC-mediated T cell killing (R2= 0.85), which was confirmed by ImmTAC redirected killing assays using tumor lines with differential endogenous expression of pHLA targets. Higher effector: target (E: T) and higher pHLA number are the key factors, by regression analyses, influencing ImmTAC mediated T cell activation. These account for 53% and 29% variability in IFNγ production (p<0.01), respectively. To assess the indirect impact of ImmTAC-activated T cells on tumor cells, cell lines were pre-treated with supernatants. ImmTAC-activated PBMC supernatants reduced tumor growth (2 to 25-fold) and increased tumor death (20 - 80%) in 5 of 8 cell lines tested. Blocking IFNγ restored tumor growth by over 40%, which was further enhanced by additional blocking of IFNβ, TNFα, and IL6. All cell lines significantly upregulated APM genes and became more susceptible to direct ImmTAC-mediated T cell killing (1.4 to 2-fold increase). Conclusions: In vitro, ImmTAC bispecifics kill cancer cells by both direct and indirect mechanisms. Redirection of polyclonal T cells to kill melanoma cells is greatly influenced by surface pHLA number and E:T ratio. ImmTAC-induced cytokines inhibited tumor growth, increased tumor apoptosis and resulted in higher antigen presentation demonstrating the indirect effect of ImmTAC. These observations highlight how ImmTAC-treated melanoma patients could benefit through direct and indirect mechanisms of tumor control, and in part help to explain how patients with low gp100 expression can derive benefit from tebentafusp.

Abstract 3000: A data-driven and AI-empowered systems biology model of MHC class I antigen presentation pathway

Abstract Immunotherapy is a new frontline therapeutic strategy for multiple cancer types. However, non-response mechanisms of immune checkpoint inhibitors generally exist in patients of solid cancer such breast and colorectal cancer. Multiple factors that parallelly regulate T cell functions may cause the non-response mechanism, such as regulators of T-cell’s recognition of cancer cells and cytotoxicity activities. Our recent study revealed that the presentation and salvage of MHC class I antigen presentation on cancer cells is a key deterministic factor of T-cell recognition in multiple cancer types. In this study, we established a systems biology model of the antigen processing and presentation pathways to identify cancer cell-expressing genes that are associated with immune response of breast cancer and effector T cell-mediated cytotoxicity. To the best of our knowledge, there is no established systems biology model of MHC I class antigen presentation pathway. We first manually collected and curated the biological processes that are involved in the MHC class I antigen presentation pathway via an extensive literature review. MHC I class antigen presentation involves a chain of biological processes, namely ubiquitination, proteosome, transporter associated with antigen processing, chaperone-based formation of MHC I class one complex, ER to Golgi, and Golgi to cell membrane exocytosis. We also include branches such as de-ubiquitination, MHC class I complex checking, and endocytosis-based salvage. We further estimated sample-wise activity level of the MHC class I antigen presentation pathway using transcriptomics data. By treating antigen as a “mass carrying” signal, we developed a systems biology model over the established pathway. The whole biological process was treated as a network model, in which the flux represents antigen presentation activity level. Based on this computational method, we further build a machine-learning pipeline that utilizes omics datasets (cancer genomics, scRNA-seq, spatial transcriptomics) collected from tumor tissue samples to correlate the MHC class I antigen presentation activities with immune response in cancer tumor microenvironment. We identified genes expressed by cancer cells that have high impacts on antigen presentation and are associated with T cell recognition and cytotoxicity. The identified genes are involved in (a) the antigen presentation machinery, (b) the immunoproteasome in antigen processing, (c) the dynamics of antigen presentation on the cell surface, and (d) cytokine and chemokine secretion. Citation Format: Jia Wang, Xiao Wang, Pengtao Dang, Haiqi Zhu, Xinyu Zhou, Kaman So, Sha Cao, Chi Zhang. A data-driven and AI-empowered systems biology model of MHC class I antigen presentation pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3000.

Abstract 2278: Dissecting immuno-genomic pathways of response and resistance to immunotherapy in metastatic urothelial carcinoma in a real-world cohort

Abstract Background: While Immune Checkpoint Inhibitors (ICI) are widely used for the treatment (tx) of patients (pts) with metastatic urothelial carcinoma (mUC), response rates are only around 20%. There is an unmet need to develop biomarkers of response and resistance to avoid unnecessary treatment in the majority of pts who will not respond to ICI, to spare toxicity from ICI as well as mitigate the financial burden. Here we report tumor immune-genomic characteristics associated with response and resistance to ICI in a real-world cohort of mUC pts at the Cleveland Clinic. Methods: We selected 61 mUC pts who received at least 2 cycles of ICI (atezolizumab or pembrolizumab) between 2015-2020 and had pre-tx archival fresh frozen paraffin-embedded (FFPE) tissues available. Pts were categorized by their response to ICI as complete responders (CR; N=8), partial responders (PR; N=14), stable disease (SD; N=9), and progressive disease (PD; N=30). Tumor was macrodissected from 2-5 FFPE slides guided by an H&E stained section of tumor. RNA extraction was done using Qiagen Rneasy FFPE kit. 100ng of RNA was used for transcriptomic analysis using the 770-gene Nanostring PanCancer IO 360 panel. Data normalization and analysis was carried out using nSolver 4.0. Results: Using unsupervised hierarchical clustering, when comparing molecular signatures of pts with CR vs PD respectively, we saw upregulation of exhausted CD8 (log fold change (FC) = 0.7287, p= 0.03289) and IFN downstream (logFC = 0.5929, p = 0.04986). Notably, the molecular signatures upregulated between SD vs PD respectively were IFN Gamma (logFC = 1.503, p = 0.0044), PD-L1 (logFC = 1.136, p = 0.0123), Antigen-presenting machinery (APM) (logFC = 1.103, p = 0.0006), Immunoproteasome (logFC = 0.984, p = 0.00096) and Tumor inflammatory score (logFC = 0.947, p = 0.032). Furthermore, molecular signatures were not differently upregulated between pts with PR vs PD. In our cohort, the proportion of pts who were alive at 35.9 months (median follow-up) was 62.5% for CR (5 of 8), 28.5% for PR (4 of 14), 22.2% for SD (2 of 9) and 6.2% for PD (2 of 30) even though the median overall survival was not significantly different between the groups. Conclusion: In our cohort of ICI-treated mUC pts, we found an association between existing T-cell effector function, particularly IFN-gamma responses, and response to ICI. Signatures associated with T cell exhaustion were also upregulated among pts with CR indicating ongoing anti-tumor activity. These findings need to be further validated in prospective trials. Ongoing genomic and immunologic correlative studies in our cohort will further help understand comprehensive biomarkers of response and resistance to ICI in pts with mUC are currently underway. Citation Format: Nikhil Pramod, Paul Pavicic, Scott Dawsey, Alan Shen, Roshan Lodha, Moshe Ornstein, Omar Mian, Timothy Gilligan, Christopher Wee, Amanda Nizam, Jane Nguyen, C. Marcela Diaz-Montero, Shilpa Gupta. Dissecting immuno-genomic pathways of response and resistance to immunotherapy in metastatic urothelial carcinoma in a real-world cohort [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2278.

Abstract 954: PIK3CA mutation induces immunogenicity and increases the immune checkpoint inhibitor response in urothelial carcinoma

Abstract Background: Immune checkpoint inhibitor (ICI) has been widely used for advanced urothelial carcinoma (mUC) globally. However, little is known about the potential predictive factors of the ICI response. The study aims to identify a potential biomarker to predict ICI response in UC. Material and methods: We performed targeted next generation sequencing (NGS) of pre-treatment tumor Formalin-fixed paraffin-embedded (FFPE) samples by using ACTOnco® comprehensive genomic panel (ACT Genomics, Co. Ltd., Taiwan) to target all coding exons of 440 cancer-associated genes. We retrospective analyzed a total of 70 patients with mUC underwent at least one cycle of ICI with or without chemotherapy at Kaohsiung Chang Gung Memorial Hospital between April 2016 and November 2019. Patients were defined as responders (CR/PR) and non-responders (SD/PD) according to the RECIST evaluation. TMB was estimated by calculating all coding exons variants, including somatic non-synonymous, frameshift and splice site variants. Results: A total of 55 UC tumor samples passed QC process were analyzed. The median age was 67, and 74% of all patients were in ICI monotherapy cohort. The most common genetic alterations were TP53 (66%), KMT2D (42%) and MUC16 (32%). In ICI monotherapy group, responders had a higher TMB than non-responders (median 19.5 vs 6.5 mt/Mb; p = 0.004). Patients with high TMB on ICI monotherapy had a significantly longer PFS (14.6 vs 2.9 months; p = 0.003) and OS (NR vs 11.0 months; p = 0.01) than TMB low patients. We found that PIK3CA (32% vs 3%), UBR5 (23% vs 0%), PRKDC (32% vs 10%) were the top 3 enriched mutations between responders and non-responders. All PIK3CA mutation were hot-spot mutation, and tumor harbored PIK3CA mutation had a median high TMB than PIK3CA wild type in UC, NSCLC, breast and colorectal cancer. By analyzing tumor and immune microenvironment by TISIDB platform, UC with PIK3CA mutation was highly associated with increasing MHC molecules expression (HLA-A, HLA-B, HLA-C, B2M) in UC and gastric cancer. In vitro study, we confirmed anti-PDL1 with PBMC had a synergistic cytotoxicity in PIK3CA mutated cell lines (BFTC-909, AGS and DLD-1), but not in PIK3CA wild type cell lines (T24, RT4, UMUC-14, N87 and SW837). Knocking-down PIK3CA expression diminished the cytotoxic activity of anti-PDL1 with PBMC in BFTC-909 and AGS cells. PIK3CA down-regulation inhibits antigen presentation machinery process. We confirmed that knockout PIK3CA gene function by CRISPR/Cas9 decreased MHC1 and B2M molecules expression, and showing modest resistance to ICI than mice with preserved PIK3CA function. Conclusion: UC with PIK3CA missense mutation exhibits a higher immunogenicity through enhancing MHC class I and B2M expression. PIK3CA missense mutation is a potential predictive marker for ICI response in UC. Citation Format: Harvey Yu-Li Su, Ling-Yi Xiao, Shih-Yu Huang, Li-Chung Chang, Yi-Hua Chen, Hao-Lun Luo, Chia-Ling Wu. PIK3CA mutation induces immunogenicity and increases the immune checkpoint inhibitor response in urothelial carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 954.

Abstract 6438: Next-generation pan-cancer immunotherapy with patient-derived APC

Abstract Harnessing phagocytic antigen presenting cells (APC) and tumor neoantigen-specific T cells represent the ultimate power of immunotherapy against cancer. The promises of phagocytic APCs lie in their abilities of not only initiating phagocytosis towards cancer cells (to “eat off” tumor) but also, through immunogenic antigen presentation, activating neoantigen-specific Tcells that systemically and durably eliminate cancer cells, as well as the establishment of a long-lasting cellular and hormonal immunity that prevents tumor recurrence. However, the efforts of developing APCs -based therapy have been hindered by the current lack of technology that robustly produce APCs from cancer patients, as none of the traditional methods for differentiating DC or macrophage APCs are effective for cancer patients. Through a proprietary process, we have created a unique reagent combo that enables robust differentiation of cancer patients’ peripheral monocytes (cMo) to be potent APC, termed κAPC. κAPC display a unique gene expression profile, separating from classical DC or macrophage, and profound proinflammatory phenotype with enhanced phagocytic capability and elevated immunogenic antigen presentation machinery. In both in vitro and in vivo settings, κAPC demonstrate direct uptake of tumor antigens and presentation to T cells, the latter leading to activation and large expansion of multi-targeting, polyclonal tumor-specific CD4 and CD8 T populations - termed NeoT- from circulating and tumor-infiltrating lymphocytes. Theexpanded CD8 NeoT are highly tumoricidal and once adoptively transferred in vivo, potently eliminated late-stage metastatic solid tumors, whereas CD4 NeoT contribute to immune memory and repolarize a proinflammatory TME favorable for tumor elimination. Key words: κAPC, NeoT, NeoT adoptive cell therapy, κAPC-based cancer vaccine, combination therapies Citation Format: Zhen Bian, Lei Shi, Yuan Liu, harry Stylli. Next-generation pan-cancer immunotherapy with patient-derived APC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6438.

Abstract 5712: Anti-TIGIT antibody tiragolumab leverages myeloid cells and regulatory T cells to improve PD-L1 checkpoint blockade

Abstract Background: TIGIT is a co-inhibitory receptor and immune checkpoint associated with T cell and natural killer (NK) cell dysfunction in cancer. Tiragolumab is an anti-TIGIT antibody with an active IgG1/kappa Fc. In a randomized double-blind phase 2 clinical trial in non-small cell lung cancer (NSCLC), tiragolumab + atezolizumab (anti-PD-L1) combination treatment demonstrated significant improvement relative to atezolizumab alone. However, the mechanisms underlying the efficacy of this combination are not well understood. Methods: In CITYSCAPE (phase 2, NCT03563716), chemotherapy-naïve patients with locally advanced/metastatic NSCLC received either placebo + 1200 mg atezolizumab or 600 mg tiragolumab + 1200 mg atezolizumab q3w IV. We collected tumor pretreatment samples and serum samples (baseline and on-treatment) from patients enrolled in the trial, which were subject to bulk RNA-seq and proteomics, respectively. In the mouse tumor model, BALB/c mice were implanted with syngeneic CT26 tumors. After tumor establishment, mice were randomized by tumor volume and then treated with control IgG2a, anti-PD-L1, anti-TIGIT IgG2a-LALAPG (Fc-inactive) ± anti-PD-L1, anti-TIGIT IgG2b ± anti-PD-L1, or anti-TIGIT IgG2a ± anti-PD-L1. Three days after treatment, CD45+ immune cells were collected from the peripheral blood and tumor from selected treatment groups and underwent single cell RNA sequencing or flow cytometry profiling. Tumor growth was also monitored to determine efficacy. Results: Here, we show that tiragolumab functions as both a conventional checkpoint inhibitor and, via Fc gamma receptor engagement, as a modulator of immunosuppressive myeloid cells and T regulatory (Treg) cells. Gene expression analysis of patient tumor samples revealed high levels of these cell subsets, were instead associated with treatment benefit in the tiragolumab + atezolizumab arm but not atezolizumab arm. Analysis of patient serum proteins suggested an association of myeloid cell activation with clinical benefit mediated by the combination therapy. In mouse tumor models, treatment with anti-PD-L1 + anti-TIGIT IgG2a (but not anti-TIGIT IgG2b or Fc-silent anti-TIGIT) led to effective tumor control in mice, suggesting a pivotal role for activating Fc receptors. Phenotypic profiling by single cell RNAseq and flow cytometry revealed that Fc-active anti-TIGIT remodels the tumor microenvironment, most prominently by inducing antigen presentation machinery in myeloid cells, counteracting of anti-PDL1-enforced CD8+ T cell exhaustion, and reducing Treg suppressive capacity. Conclusions: These findings reveal that FcR engagement is one of several distinct mechanisms by which tiragolumab unleashes antitumor immune responses, and inform further clinical development of anti-TIGIT therapies. Citation Format: Namrata S. Patil, Shyam Srivats, Yoonha Choi, Xiangnan Guan, Barzin Nabet, Lisa McGinnis, Eugene Chiang, Alexis Dunkle, Bill O’Gorman, Patrick S. Chang, Ruozhen Hu, John Silva, Joy Han, Amelia Au-Yeung, Chikara Takahashi, Nandini Molden, Pallavi Daggumati, Wendy Connolly, Melissa Johnson, Delvys Rodriguez Abreu, Byoung Chul Cho, Antoine Italiano, Ignacio Gil-Bazo, Enriqueta Felip, Ira Mellman, Raymond Meng, Sanjeev Mariathasan, Robert Johnston, David S. Shames. Anti-TIGIT antibody tiragolumab leverages myeloid cells and regulatory T cells to improve PD-L1 checkpoint blockade. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5712.

Abstract 6433: A novel bacterial strain, CJRB-101, induces anti-cancer effects by repolarization of M2 to CXCL9 and CXCL10 dual expressing M1 macrophages in humanized non-small cell lung cancer mice models

Abstract Backgrounds: Live biotherapeutic products (LBPs) emerged as potential therapeutics to overcome the limitation of ICIs. This research shows that CJRB-101, a novel bacterial strain, can improve anti-tumor effects in synergy with pembrolizumab in non-small cell lung cancer (NSCLC). Objectives and Methods: Tumors from NSCLC patients (anti-PD-1 refractory and resistant) were transplanted into Hu-CD34-NSG to establish humanized patient-derived xenograft (PDX) mice models. Five models (YHIM-2003, 2004, 2009, 2010 and 2014) were treated with CJRB-101 at low (5 × 107 CFU) or high (109 CFU) doses, or with pembrolizumab (10 mg/kg, i.p., Q5D) or in combination. Tumor growth inhibition (TGI) rate was measured. Tumor microenvironment (TME) was analyzed using multiplex IHC, flow cytometry and single cell RNA sequencing. Ex-vivo assays were performed to validate in silico findings. Results: Tumor in PDX models was unresponsive to pembrolizumab alone, however, in combination with CJRB-101 effectively suppressed tumor growth. The synergy was highlighted in YHIM-2009 where TGI was 10-fold higher (56%) than pembrolizumab group (5%). Immune profiling revealed that macrophages may be responsible for the anti-tumor effects of CJRB-101. IHC showed significantly increased antigen presenting specialized DCs (CD16+CD68−CD11c+) and granzyme B+ CD8+ T cells in the tumor by CJRB-101 compared to pembrolizumab (p<0.01). This suggested that CJRB-101 induced infiltration of cytotoxic CD8 T cells into the tumor nest by enhancing antigen presenting machinery. Trajectory analysis showed that CJRB-101 induced repolarization of M2 to M1 macrophages, characterized by high expression of CXCL9/10. CXCL9+/10+ M1 macrophages were comparatively more abundant in the combination group (23.11%) than the pembrolizumab group (0.91%). CXCL9/CXCL10 expression in macrophages was higher in the CJRB-101 group compared to the pembrolizumab group (p<0.0001). The combination group (10.84%) had a higher relative abundance of CD8+ T cells compared to the pembrolizumab group (1.58%) and higher IFNγ expression in CD8+ T cells compared to the pembrolizumab group (p=0.0152), suggesting that CJRB-101 repolarized macrophages and recruited active CD8+ T cells. Co-culture assays using bone marrow-derived macrophages validated that CJRB-101 drove differentiation towards F4/80+ or MHC II+ expressing M1 macrophage (p<0.0001) and repolarized existing M2 (CD206+) to M1 (p=0.0002). Conclusion: Combination treatment of CJRB-101 with anti-PD-1 showed synergistic anti-tumor effects via repolarization of M2 to M1 macrophages, leading to activation of CD8+ T cells in TME. Especially, CXCL9+/10+ M1 macrophage playing a key role in TGI induced by CJRB-101 in NSCLC models. Findings from this study provided rationale for clinical investigation of CJRB-101. Citation Format: Arim Min, Chun-bong Synn, Seong-san Kang, Bo-eun Kwon, Junwon Yang, Hyunkyung Park, Jieun Im, Hyunjeong Kim, Sujeong Beak, Dong Kwon Kim, Jii Bum Lee, Hyeonseok Oh, Seung Min Yang, Yu Jin Han, Mi hyun Kim, Heekyung Han, Kwangmin Na, Young Taek Kim, Sungwoo Lee, Mi Ran Yun, Jae Hwan Kim, Youngseon Byeon, Young Seob Kim, Ji Yun Lee, Chang Gon Kim, Min Hee Hong, Sun Min Lim, Kyoung-Ho Pyo, Byoung Chul Cho. A novel bacterial strain, CJRB-101, induces anti-cancer effects by repolarization of M2 to CXCL9 and CXCL10 dual expressing M1 macrophages in humanized non-small cell lung cancer mice models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6433.

Expression of thimet oligopeptidase (THOP) modulated by oxidative stress in human multidrug resistant (MDR) leukemia cells

Thimet oligopeptidase (THOP) is a cytosolic metallopeptidase known to regulate the fate of post-proteasomal peptides, protein turnover and peptide selection in the antigen presentation machinery (APM) system. Oxidative stress influences THOP expression and regulates its proteolytic activity, generating variable cytosolic peptide levels, possibly affecting the immune evasion of tumor cells. In the present work, we examined the association between THOP expression/activity and stress oxidative resistance in human leukemia cells using the K562 cell line, a chronic myeloid leukemia (CML), and the multidrug-resistant (MDR) Lucena 1 (K562-derived MDR cell line) as model. The Lucena 1 phenotype was validated under vincristine treatment and the relative THOP1 mRNA levels and protein expression compared to K562 cell line. Our data demonstrated increased THOP1 gene and protein levels in K562 cells in contrast to the oxidative-resistant Lucena 1, even after H2O2 treatment, suggesting an oxidative stress dependence in THOP regulation. Further, it was observed higher basal levels of reactive oxygen species (ROS) in K562 compared to Lucena 1 cell line using DHE fluorescent probe. Since THOP activity is dependent on its oligomeric state, we also compared its proteolytic activity under reducing agent treatment, which demonstrated that its function modulation with respect to changes in redox state. Finally, the mRNA expression and FACS analyses demonstrated a reduced expression of MHC I only in K562 cell line. In conclusion, our results highlight THOP redox modulation, which could influence antigen presentation in multidrug resistant leukemia cells.

Specific cannabinoids revive adaptive immunity by reversing immune evasion mechanisms in metastatic tumours.

Emerging cancers are sculpted by neo-Darwinian selection for superior growth and survival but minimal immunogenicity; consequently, metastatic cancers often evolve common genetic and epigenetic signatures to elude immune surveillance. Immune subversion by metastatic tumours can be achieved through several mechanisms; one of the most frequently observed involves the loss of expression or mutation of genes composing the MHC-I antigen presentation machinery (APM) that yields tumours invisible to Cytotoxic T lymphocytes, the key component of the adaptive cellular immune response. Fascinating ethnographic and experimental findings indicate that cannabinoids inhibit the growth and progression of several categories of cancer; however, the mechanisms underlying these observations remain clouded in uncertainty. Here, we screened a library of cannabinoid compounds and found molecular selectivity amongst specific cannabinoids, where related molecules such as Δ9-tetrahydrocannabinol, cannabidiol, and cannabigerol can reverse the metastatic immune escape phenotype in vitro by inducing MHC-I cell surface expression in a wide variety of metastatic tumours that subsequently sensitizing tumours to T lymphocyte recognition. Remarkably, H3K27Ac ChIPseq analysis established that cannabigerol and gamma interferon induce overlapping epigenetic signatures and key gene pathways in metastatic tumours related to cellular senescence, as well as APM genes involved in revealing metastatic tumours to the adaptive immune response. Overall, the data suggest that specific cannabinoids may have utility in cancer immunotherapy regimens by overcoming immune escape and augmenting cancer immune surveillance in metastatic disease. Finally, the fundamental discovery of the ability of cannabinoids to alter epigenetic programs may help elucidate many of the pleiotropic medicinal effects of cannabinoids on human physiology.

Next-generation antigen-presenting cell immune therapeutics for gliomas.

Antigen presentation machinery and professional antigen-presenting cells (APCs) are fundamental for an efficacious immune response against cancers, especially in the context of T cell-centric immunotherapy. Dendritic cells (DCs), the gold standard APCs, play a crucial role in initiating and maintaining a productive antigen-specific adaptive immunity. In recent decades, ex vivo-differentiated DCs from circulating CD14+ monocytes have become the reference for APC-based immunotherapy. DCs loaded with tumor-associated antigens, synthetic peptides, or RNA activate T cells with antitumor properties. This strategy has paved the way for the development of alternative antigen-presenting vaccination strategies, such as monocytes, B cells, and artificial APCs, that have shown effective therapeutic outcomes in preclinical cancer models. The search for alternative APC platforms was initiated by the overall limited clinical impact of DC vaccines, especially in indications such as gliomas, a primary brain tumor known for resistance to any immune intervention. In this Review, we navigate the APC immune therapeutics' past, present, and future in the context of primary brain tumors.

Clustering by antigen-presenting genes reveals immune landscapes and predicts response to checkpoint immunotherapy

Immune checkpoint blockade (ICB) has demonstrated efficacy by reinvigorating immune cytotoxicity against tumors. However, the mechanisms underlying how ICB induces responses in a subset of patients remain unclear. Using bulk and single-cell transcriptomic cohorts of melanoma patients receiving ICB, we proposed a clustering model based on the expression of an antigen-presenting machinery (APM) signature consisting of 23 genes in a forward-selection manner. We characterized four APM clusters associated with distinct immune characteristics, cancer hallmarks, and patient prognosis in melanoma. The model predicts differential regulation of APM genes during ICB, which shaped ICB responsiveness. Surprisingly, while immunogenically hot tumors with high baseline APM expression prior to treatment are correlated with a better response to ICB than cold tumors with low APM expression, a subset of hot tumors with the highest pre-ICB APM expression fail to upregulate APM expression during treatment. In addition, they undergo immunoediting and display infiltration of exhausted T cells. In comparison, tumors associated with the best patient prognosis demonstrate significant APM upregulation and immune infiltration following ICB. They also show infiltration of tissue-resident memory T cells, shaping prolonged antitumor immunity. Using only pre-treatment transcriptomic data, our model predicts the dynamic APM-mediated tumor-immune interactions in response to ICB and provides insights into the immune escape mechanisms in hot tumors that compromise the ICB efficacy. We highlight the prognostic value of APM expression in predicting immune response in chronic diseases.

Yes-Associated Protein 1 Inhibition Induces Immunogenic Cell Death and Synergizes With Radiation and PD-1 Blockade

Danger signals released by ionizing radiation (IR) can theoretically stimulate immune activation in the tumor environment (TME), but IR alone is not sufficient to induce an effective immune response in clinical practice. In this study, we investigated whether inhibition of yes-associated protein 1 (YAP1) could induce immunogenic cell death (ICD) and whether the combination of YAP1 inhibition with IR could increase in vivo immune infiltration and thereby boost a tumor response to immunotherapy. First, the expression of ICD markers, markers of T-cell activation, and key proteins involved in innate immune signaling were measured after YAP1 inhibition. Next, the expression level of YAP1 protein was measured after different doses of IR. Then, the antitumor effect of YAP1 inhibition combined with IR was investigated in vivo, and the immune status of the TME was evaluated. Finally, the efficacy of a triple therapy including YAP1 inhibition combined with IR and programmed cell death protein 1 blockade in the treatment of resistant tumors was determined. We found that YAP1 inhibition induced ICD and increased the levels of antigen presentation machinery, effectively causing the activation of T cells. Mechanistically, YAP1 inhibition induced cell DNA damage and activated the cyclic GMP-AMP synthase (cGAS)/stimulator of interferon genes (STING) pathway. Surprisingly, IR upregulated YAP1 expression. IR combined with YAP1 inhibition significantly inhibited cancer growth and prolonged survival, which was related to the augmented infiltration, activation, and function of CD8+ T cells in the TME. Moreover, the addition of YAP1 inhibition significantly improved the efficacy of pancreatic cancer treatment when neither radiation nor programmed cell death protein 1 inhibitors were ideal. YAP1 inhibition could trigger ICD and is a potential approach to potentiating the therapeutic efficacy of radiation therapy and anti-PD1 immunotherapy.

Conventional and non-conventional antigen presentation by mast cells.

Mast cells (MCs) are multifunctional immune cells that express a diverse repertoire of surface receptors and pre-stored bioactive mediators. They are traditionally recognized for their involvement in allergic and inflammatory responses, yet there is a growing body of literature highlighting their contributions to mounting adaptive immune responses. In particular, there is growing evidence that MCs can serve as antigen-presenting cells, owing to their often close proximity to T cells in both lymphoid organs and peripheral tissues. Recent studies have provided compelling support for this concept, by demonstrating the presence of antigen processing and presentation machinery in MCs and their ability to engage in classical and non-classical pathways of antigen presentation. However, there remain discrepancies and unresolved questions regarding the extent of the MC's capabilities with respect to antigen presentation. In this review, we discuss our current understanding of the antigen presentation by MCs and its influence on adaptive immunity.

Response to Immune Checkpoint Inhibitors Is Affected by Deregulations in the Antigen Presentation Machinery: A Systematic Review and Meta-Analysis.

Immune checkpoint inhibitors (ICI) targeting programmed death 1 (PD-1), its ligand (PD-L1), or cytotoxic T-lymphocyte antigen 4 (CTLA-4) have shown promising results against multiple cancers, where they reactivate exhausted T cells primed to eliminate tumor cells. ICI therapies have been particularly successful in hypermutated cancers infiltrated with lymphocytes. However, resistance may appear in tumors evading the immune system through alternative mechanisms than the PD-1/PD-L1 or CTLA-4 pathways. A systematic pan-cancer literature search was conducted to examine the association between alternative immune evasion mechanisms via the antigen presentation machinery (APM) and resistance towards ICI treatments targeting PD-1 (pembrolizumab and nivolumab), PD-L1 (durvalumab, avelumab, and atezolizumab), and CTLA-4 (ipilimumab). The APM proteins included the human leucocyte antigen (HLA) class I, its subunit beta-2 microglobulin (B2M), the transporter associated with antigen processing (TAP) 1, TAP2, and the NOD-like receptor family CARD domain containing 5 (NLRC5). In total, 18 cohort studies (including 21 original study cohorts) containing 966 eligible patients and 9 case studies including 12 patients were reviewed. Defects in the APM significantly predicted poor clinical benefit with an odds ratio (OR) of 0.39 (95% CI 0.24−0.63, p < 0.001). The effect was non-significant, when considering complete and partial responses only (OR = 0.52, 95% CI 0.18−1.47, p = 0.216). In summary, the APM contains important targets for tumorigenic alterations which may explain insensitivity towards ICI therapy.

Mutant and non-mutant neoantigen-based cancer vaccines: recent advances and future promises.

Major advances in cancer treatment have emerged with the introduction of immunotherapies using blocking antibodies that target T-cell inhibitory receptors, such as programmed death-1 (PD-1) and cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4), known as immune checkpoints. However, most cancer patients do not respond to immune checkpoint blockade (ICB) therapies, suggesting the development of resistance mechanisms associated with either an insufficient number of preexisting tumor-specific T-cell precursors and/or inappropriate T-cell reactivation. To broaden clinical benefit, anti-PD-1/PD-1 ligand (PD-L1) neutralizing antibodies have been combined with therapeutic cancer vaccines based on non-mutant and/or mutant tumor antigens, to stimulate and expand tumor-specific T lymphocytes. Although these combination treatments achieve the expected goal in some patients, relapse linked to alterations in antigen presentation machinery (APM) of cancer cells often occurs leading to tumor escape from CD8 T-cell immunity. Remarkably, an alternative antigenic peptide repertoire, referred to as T-cell epitopes associated with impaired peptide processing (TEIPP), arises on these malignant cells with altered APM. TEIPP are derived from ubiquitous non-mutant self-proteins and represent a unique resource to target immune-edited tumors that have acquired resistance to cytotoxic T lymphocytes (CTLs) related to defects in transporter associated with antigen processing (TAP) and possibly also to ICB. The present review discusses tumor-associated antigens (TAAs) and mutant neoantigens and their use as targets in peptide- and RNA-based therapeutic cancer vaccines. Finally, this paper highlights TEIPP as a promising immunogenic non-mutant neoantigen candidates for active cancer immunotherapy and combination with TAA and mutant neoantigens. Combining these polyepitope cancer vaccines with ICB would broaden T-cell specificity and reinvigorate exhausted antitumor CTL, resulting in the eradication of all types of neoplastic cells, including immune-escaped subtypes.

YH29407 with anti-PD-1 ameliorates anti-tumor effects via increased T cell functionality and antigen presenting machinery in the tumor microenvironment.

Among cancer cells, indoleamine 2, 3-dioxygenase1 (IDO1) activity has been implicated in improving the proliferation and growth of cancer cells and suppressing immune cell activity. IDO1 is also responsible for the catabolism of tryptophan to kynurenine. Depletion of tryptophan and an increase in kynurenine exert important immunosuppressive functions by activating regulatory T cells and suppressing CD8+ T and natural killer (NK) cells. In this study, we compared the anti-tumor effects of YH29407, the best-in-class IDO1 inhibitor with improved pharmacodynamics and pharmacokinetics, with first and second-generation IDO1 inhibitors (epacadostat and BMS-986205, respectively). YH29407 treatment alone and anti-PD-1 (aPD-1) combination treatment induced significant tumor suppression compared with competing drugs. In particular, combination treatment showed the best anti-tumor effects, with most tumors reduced and complete responses. Our observations suggest that improved anti-tumor effects were caused by an increase in T cell infiltration and activity after YH29407 treatment. Notably, an immune depletion assay confirmed that YH29407 is closely related to CD8+ T cells. RNA-seq results showed that treatment with YH29407 increased the expression of genes involved in T cell function and antigen presentation in tumors expressing ZAP70, LCK, NFATC2, B2M, and MYD88 genes. Our results suggest that an IDO1 inhibitor, YH29407, has enhanced PK/PD compared to previous IDO1 inhibitors by causing a change in the population of CD8+ T cells including infiltrating T cells into the tumor. Ultimately, YH29407 overcame the limitations of the competing drugs and displayed potential as an immunotherapy strategy in combination with aPD-1.

Abstract IA025: Stem and metaplastic cell states and their influences on the tumor immune microenvironment in colonic pre-cancerous lesions

Abstract The immune microenvironment is critical to colorectal cancer (CRC) pathogenesis, regarding both prognosis and therapeutic responses. While much work has been done to characterize tumor microenvironments, less is known about those environments are established following transformation of epithelial cells. We hypothesize that mapping routes towards CRCs, from normal human colonic tissues and precursor polyps, will uncover additional mechanisms that define the CRC cellular landscape and identify targets with diagnostic or therapeutic utility. Here, we present a comparative atlas of the two most common colorectal precursor lesions, conventional adenomas and serrated polyps, and their resulting CRC counterparts using single-cell transcriptomics and multiplex imaging across two cohorts of human participants. Integrative analysis reveals adenomas arise from dysregulated WNT-driven expansion of stem cells, while serrated polyps are depleted of stem cell populations and are derived from differentiated cells transitioning through a pyloric metaplasia program. Mucosal damage associated with metaplasia is coupled to a cytotoxic immune microenvironment that precedes hypermutation. Mouse and organoid experiments validate the importance of the differentiation states of colonic tumor cells in driving cytotoxic immunity partly through increased expression of antigen-presentation machinery. These data provide constitute a rich resource into malignant progression of colorectal polyps and their microenvironments, serving as a framework for precision surveillance and prevention of CRC. Citation Format: Ken S. Lau. Stem and metaplastic cell states and their influences on the tumor immune microenvironment in colonic pre-cancerous lesions [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer; 2022 Oct 1-4; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_1):Abstract nr IA025.

Short-course pembrolizumab and continuous afatinib therapy for recurrent or metastatic head and neck squamous cell carcinoma: a real-world data analysis

ObjectivesThe optimal duration of anti-PD-1 for cancer therapy has not been tested, especially when using combination therapy. Epidermal growth factor receptor (EGFR) pathway blocker was the top compound that enhanced T-cell killing of tumor cells in a high-throughput immune-oncology screen, possibly by stimulate the antigen presentation machinery and other mechanisms. We explored the effect of combination of EGFR inhibition with a short course of anti-PD-1 therapy in patients with recurrent or metastatic head and neck squamous cell carcinoma (R/M HNSCC).MethodWe analyzed the effect of a short course of anti-PD-1 with continuous afatinib on the survival of a real-world cohort of R/M HNSCC patients. Patient characteristics, treatments, efficacies, and toxicities were reviewed and recorded for analysis.ResultsFrom November 2016 to May 2018, 51 consecutive patients received pembrolizumab and afatinib. The cutoff date was June 30, 2022. The most common toxicities (all grades) were diarrhea (62.7%), skin rash (43.1%), mucositis (31.4%), and paronychia (23.5%). The objective response rate was 54.9% (95% confidence interval [CI] 40.3–68.9%). Median progression-free survival was 5.9 months (95% CI: 4.4–7.6 months), and the median overall survival was 10.5 months (95% CI: 6.8–16.5 months). The 12-month, 24-month, 36-month, and 48-month survival rate was 47.0%, 22.5%, 17.7%, and 12.6% respectively.ConclusionsThis retrospective study showed that short course pembrolizumab with afatinib therapy has acceptable efficacy in R/M HNSCC patients. The durable response and long-term survival rates were similar to prospective clinical trials. Short course anti-PD-1 therapy, especially in combination with EGFR blocker, is worth for further prospective study.