Targeting Immune Checkpoints Research Articles

Abstract P2-20-10: Prognostic impact of lymphocyte-Activation Gene-3 (LAG-3) expression in triple negative breast cancer

Abstract Introduction Recent advances in breast cancer treatment strategies have improved survival outcomes in metastatic breast cancer (mBC). Targeting immune checkpoint, especially programmed death 1 (PD-1)/programmed death ligand 1 (PD-L1), has made a breakthrough in treating advanced malignancies including breast cancer. Immune checkpoint inhibitors (ICIs), such as pembrolizumab and atezolizumab, in combination with chemotherapy have prolonged survival outcomes in mBC with triple negative subtype. However, the low response rate and resistance of ICIs with anti-PD 1/PDL-1 antibodies is a major limitation and a challenge. Lymphocyte Activation Gene-3 (LAG3; CD223) is a potential cancer immunotherapeutic target due to its negative regulatory role on T cells and cytokines, thereby ensuring immune homeostasis. Several studies suggested that combination immunotherapy of anti-LAG-3 and anti-PD-1 has shown promising efficacy in fighting PD-1 resistance. Therefore, we evaluated the prognostic role of LAG3 in metastatic TNBC (mTNBC) treated with ICIs to figure out resistance mechanism of ICIs. Methods mTNBC patients with available archival tumor tissues, who has received ICIs in Samsung Medical Center, were enrolled in this study. For the evaluation of LAG3 expression in tumor microenvironment, Vectra Polaris Multispectral Imaging and Whole Slide Scanning technique (PerkinElmer, Inc. Hopkinton, MA) was used. Results In total, 64 mTNBC patients were treated with ICI’s with or without cytotoxic chemotherapy between 2019.02- 2021.11. Of 70 mTNBC patients, 41 patients had archival tissues and finally 40 patients were included in this study. Median age was 43.0 years of age (range: 24.5 ~ 64.5). Recurrent mTNBC was 92.5% and only 7.5% was de novo mTNBC. Geremline BRCA1 pathogenic variants were detected in 4 (10.0%) patients. Among 37 recurrent mTNBC patients, 72.5% were treated with neoadjuvant chemotherapy with anthracycline (97.3%) and taxane(97.3%). Among ICI’s, 52.5% were treated with pembrolizumab and 47.5% of atezolizumab. LAG3 expression varies among mTNBC tissues (median cell density: 366, range: 48, 2861 cells/mm2). Among cells expressing LAG3, LAG3 was expressed more in CK+ cells compared with CK- cells (median:150 (20, 2503) cells/mm2 in CK+ cells, median: 88 (2, 806) cells/mm2 in CK- cells, p=0.005). Patients with high LAG3 expression in CK+ cells showed short progression free survival(PFS) compared to those with low LAG3 expression in CK+ cells (median PFS of high vs. low LAG3 expression [months]:1.9 vs. 4.2, p=0.01). On the contrary, patients with high LAG3 expression in CK- cells had 9.1 months of PFS compared to 3.1 months of PFS in patients with low LAG3 expression in CK- cells (p=0.10). In addition, patients with high LAG3 in CK- cells had longer overall survival(OS) compared to those with low LAG3 expression in CK- cells (median OS of high vs. low LAG3 expression [months]: not reached, 15.7, p=0.05). Conclusion LAG3 expression was associated with PFS in patients with mTNBC treated with ICI’s independent of PDL-1 expression. And the prognostic significance of LAG3 expression was different between CK+ cells and CK- cells. These findings need to be proved in large scale clinical trials. Citation Format: ji-Yeon Kim, Jeehyun Kim, Hae Hyun Jung, eun Yoon Cho, Yeon Hee Park, Jin Seok Ahn, Kyoung-Mee Kim, Young-Hyuck Im. Prognostic impact of lymphocyte-Activation Gene-3 (LAG-3) expression in triple negative breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-20-10.

Combinatorial targeting of immune checkpoints and epigenetic regulators for hepatocellular carcinoma therapy.

Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related mortality worldwide. The five-year survival rate of patients with unresectable HCC is about 12%. The liver tumor microenvironment (TME) is immune tolerant and heavily infiltrated with immunosuppressive cells. Immune checkpoint inhibitors (ICIs), in some cases, can reverse tumor cell immune evasion and enhance antitumor immunity. Rapidly evolving ICIs have expanded systemic treatment options in advanced HCC; however, single-agent ICIs achieve a limited 15-20% objective response rate in advanced HCC. Therefore, other combinatorial approaches that amplify the efficacy of ICIs or suppress other tumor-promoting pathways may enhance clinical outcomes. Epigenetic alterations (e.g., changes in chromatin states and non-genetic DNA modifications) have been shown to drive HCC tumor growth and progression as well as their response to ICIs. Recent studies have combined ICIs and epigenetic inhibitors in preclinical and clinical settings to contain several cancers, including HCC. In this review, we outline current ICI treatments for HCC, the mechanism behind their successes and failures, and how ICIs can be combined with distinct epigenetic inhibitors to increase the durability of ICIs and potentially treat "immune-cold" HCC.

Targeting Immune Checkpoint Pathways in Melanoma: Triumphs and Challenges

The immune checkpoint inhibitors (ICIs) have revolutionized the treatment of advanced melanoma by significantly increasing survival rates, with the promise of durable disease remission in some patients. Herein we review the role of immune checkpoints in melanoma; the history of melanoma immunotherapy; pivotal clinical trial data for ipilimumab, pembrolizumab, nivolumab and relatlimab; and the current clinical role of each ICI. We discuss the challenges that accompany these triumphs in the treatment of melanoma, including: how to distinguish between responders and nonresponders; how to optimize ICI dosing and combinatorial approaches; and the best practices for monitoring response and managing immune-related toxicities. We offer our perspective on the financial toxicity of ICIs and new developments that could deliver answers to current challenges.

A PRRX1 Signature Identifies TIM-3 and VISTA as Potential Immune Checkpoint Targets in a Subgroup of Microsatellite Stable Colorectal Cancer Liver Metastases.

CLM is an important cause of colorectal cancer mortality where the majority of patients have yet to benefit from immunotherapies. In this study of gene expression profiling analyses, we uncovered novel immune checkpoint targets in a subgroup of patients with MSS CLMs harboring a mesenchymal phenotype.

Digital expression profile of immune checkpoint genes in medulloblastomas identifies CD24 and CD276 as putative immunotherapy targets.

Medulloblastoma is the most common and lethal pediatric malignant brain tumor. It comprises four main molecular subgroups: WNT-activated, SHH-activated, Group 3, and Group 4. Medulloblastoma treatment is surgical resection, craniospinal radiation, and chemotherapy. However, many patients do not respond to therapy, and most suffer severe side effects. Cancer immunotherapy targeting immune checkpoints (IC) (PD-1, PD-L1, and CTLA4) has been getting disappointing outcomes in brain tumors. Nevertheless, other less explored immune checkpoints may be promising candidates for medulloblastoma therapy. In the present study, we aimed to characterize the expression profile of 19 immune checkpoints in medulloblastoma. We analyzed 88 formalin-fixed paraffin-embedded medulloblastomas previously classified for each molecular subgroup and three non-tumoral brain tissue. mRNA levels of 19 immune checkpoint-related genes were quantified using the nCounter (PanCancer Immune Profiling Panel) assay. Further in silico analysis was performed in two larger public microarray datasets, one of which enabled comparisons between tumoral and non-tumoral tissues. Immunohistochemistry of PD-L1 was performed in a subset of cases. Microsatellite instability was also molecularly analyzed. We observed an absence of expression of the canonic ICs, namely PDCD1 (PD-1), CD274 (PD-L1), and CTLA4, as well as CD80, CD86, BTLA, IDO1, CD48, TNFSF14, CD160, CEACAM1, and CD244. PD-L1 protein expression was also practically absent. We found higher mRNA levels of CD24, CD47, CD276 (B7-H3), and PVR, and lower mRNA levels of HAVCR2, LAG3, and TIGIT genes, with significant differences across the four molecular subgroups. Compared to the non-tumor tissues, the expression levels of CD276 in all subgroups and CD24 in SHH, Group 3, and Group 4 subgroups are significantly higher. The in silico analysis confirmed the expression profile found in the Brazilian cohort, including the lower/absent expression of the canonic ICs. Moreover, it confirmed the overexpression of CD24 and CD276 in medulloblastomas compared with the non-tumor tissue. Additionally, CD276 and CD24 high levels were associated with worse survival. These results highlight the low or absence of mRNA levels of the canonic targetable ICs in medulloblastomas. Importantly, the analysis revealed overexpression of CD24 and CD276, which can constitute prognostic biomarkers and attractive immunotherapy targets for medulloblastomas.

Oral immune-related adverse events caused by immune checkpoint inhibitors: a retrospective study.

Immune Checkpoint inhibitors (ICI) are linked to a series of adverse systemic and/or oral side effects such as "stomatitis," "oral inflammation" and "mucositis." These oral lesions induced by target therapies and immune checkpoint inhibitors are different from traditional lesions associated with chemo/radiotherapy and they have not yet been correctly characterized. This paper aims to report retrospectively the oral immune-related adverse events caused by immune checkpoint inhibitors. A table in electronic format was prepared and sent by e-mail to several clinical structures in order to collect, for each patient, anamnestic data, discretionary habits, systemic risk factors, the presence and number of comorbidities, and the characteristics of the oral lesions in the course of oncological therapy with anti-PD1 (nivolumab, pembrolizumab). Following the collection of anamnestic and clinical data relating to patients treated with anti-PD1 (nivolumab, pembrolizumab) and the detection of oral lesions, data analysis was carried out. A number of 364 patients treated with nivolumab (209) and pembrolizumab (155), administered intravenously at a therapeutic dose were selected. There have been cases of oral adverse effects in treated patients. The oral adverse effects found fell into the categories of stomatitis, xerostomia, candidiasis and taste disturbances. Analyzing the incidence of oral lesions in patients undergoing treatment with immune checkpoint inhibitors, there was no significant difference between the two drugs examined. Further studies are certainly needed to catalog, focus and identify in advance the adverse effects, including oral ones, in patients treated with ICI type PD1/PDL-1. It is necessary, for the benefit of patients, to pay particular attention to the adverse effects in order to recognize, treat and possibly modulate the therapy with an adequate assessment of the cost/benefit ratio and quality of life.

Biomarkers Associated with Immune Checkpoint, N6-Methyladenosine, and Ferroptosis in Patients with Restenosis.

This study aimed to identify potential diagnostic markers of restenosis after stent implantation and to determine their association with immune checkpoint, ferroptosis, and N6-methyladenosine (m6A). Microarray data were downloaded from the National Center for Biotechnology Information (NCBI: GSE46560 and GSE48060 datasets) to identify differentially expressed genes (DEGs) between in-stent restenosis and no-restenosis samples. We then conducted systematic functional enrichment analyses of the DEGs based on Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG), and further predicted the interactions of different proteins using the Search Tool for the Retrieval of Interacting Genes (STRING). We used the MCC and MCODE algorithms in the cytoHubba plug-in to screen three key genes in the network, and employed receiver operating characteristic (ROC) curves to determine their diagnostic significance using a multiscale curvature classification algorithm. Next, we investigated the relationships between these target genes, immune checkpoint, ferroptosis, and m6A. Finally, quantitative real-time polymerase chain reaction (qRT-PCR) was used to verify the above results. We identified 62 upregulated genes and 243 downregulated genes. Based on GO, KEGG, and screening results, EEF1D, RPL36, and RPSA are promising genes for predicting restenosis. In addition, the methylation of YTHDF2, the ferroptosis-related gene GLS2, and the immune checkpoint-related gene CTLA4 were observed to be associated with restenosis. The qRT-PCR test confirmed that RPSA and RPL36 are useful diagnostic markers of the restenosis that can provide new insights for future studies on its occurrence and molecular mechanisms. We found that RPSA and RPL36, as useful diagnostic markers of restenosis, can provide new insights for future studies on its occurrence and molecular mechanisms.

The beginning of a new era in induction treatment for operable non-small cell lung cancer: a narrative review.

The survival benefit of induction therapy for non-small cell lung cancer (NSCLC) remains controversial. Recently, the outcomes of systemic therapy for NSCLC have dramatically changed with the advent of molecular target drugs and immune checkpoint inhibitors (ICIs). The present review was conducted to investigate the outcomes of induction therapy with reference to randomized control trials (RCTs). We reviewed RCTs and ongoing clinical trials between 1990 and 2022 using relevant databases: PubMed, Web of Science, and EMBASE database. We investigated the outcomes of induction therapy. Induction therapy was associated with longer overall survival in comparison to surgery alone in several RCTs for stage III disease. However, its benefit in early-stage (I-II) disease was unclear. Regarding induction chemotherapy and chemoradiotherapy, the safety and survival outcomes did not differ between the two arms. Epidermoid growth factor receptor (EGFR) tyrosine kinase inhibitors as induction therapy in patients with proven EGFR mutations may be a sufficient choice for the improvement of overall survival. In ongoing single arm clinical trials and a randomized control study, the administration of ICIs as induction therapy was associated with a good pathological response and satisfactory safety, which will lead to a better survival outcome. Long-term observation is needed to evaluate the toxicity and survival impact of induction therapy with ICIs. Induction chemotherapy and EGFR-TKIs for stage IIIA NSCLC may contribute to the improvement of survival outcomes although the effect of systemic therapy on stage I-II remains controversial. ICIs may be considered as a valuable treatment option because of their feasibility and safety for induction therapy.

TIM-3 Is a Potential Immune Checkpoint Target in Cats with Mammary Carcinoma.

Recent findings in human breast cancer (HBC) indicate that T-cell immunoglobulin and mucin-domain-containing molecule-3 (TIM-3)-targeted therapies may effectively activate anticancer immune responses. Although feline mammary carcinoma (FMC) is a valuable cancer model, no studies on TIM-3 have been developed in this species. Thus, we evaluated the expression of TIM-3 by immunohistochemistry in total (t), stromal (s), and intra-tumoral (i) tumor-infiltrating lymphocytes (TILs) and in cancer cells, of 48 cats with mammary carcinoma. In parallel, serum TIM-3 levels were quantified using ELISA and the presence of somatic mutations in the TIM-3 gene was evaluated in 19 tumor samples. sTILs-TIM3+ were more frequent than iTILs-TIM-3+, with the TIM-3 ex-pression in sTILs and cancer cells being associated with more aggressive clinicopathological features. In contrast, the TIM-3 expression in iTILs and tTILs was associated with a more benign clinical course. Moreover, the serum TIM-3 levels were lower in animals with FMC when compared to healthy animals (p < 0.001). Only one somatic mutation was found in the TIM-3 gene, at intron 2, in one tumor sample. Altogether, our results suggest that the expression of TIM-3 among TILs subpopulations and cancer cells may influence the clinical outcome of cats with FMC, in line with the previous reports in HBC.

Construction and Periplasmic Expression of a Bispecific Tandem scFv for Dual Targeting of Immune Checkpoints.

Immune checkpoints are molecules that act as regulators of immune system pathways. However, some tumor cells can express the ligands of immune checkpoints to escape from antitumor immune responses. Some agents, such as antibodies, can inhibit these checkpoints that prevent the immune system from targeting and killing cancer cells. The aim of this study was to express a novel bispecific tandem scFv in periplasmic space of Escherichia coli for simultaneous targeting of two immune checkpoints, cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death protein 1 (PD-1). The bispecific tandem scFv was constructed based on the variable regions gene of anti-PD1 and anti-CTLA-4 antibodies. The optimum codon for expression in E. coli was chemically synthesized and subcloned in periplasmic expression plasmid. After transformation, the effect of cultivation conditions on periplasmic expression of the protein in E. coli BL21(DE3) was evaluated. Then, the bispecific tandem scFv was purified and its binding ability to cells expressing PD-1 and CTLA-4 was evaluated. Expression of tandem scFv with a molecular weight of 55 kDa was verified by Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blotting analysis. The best condition for soluble periplasmic expression was obtained to be incubation with 0.5 mM isopropyl β-D-1-thiogalactopyranoside at 23°C. The protein was successfully purified using affinity chromatography with a final yield of 4.5 mg/L. Binding analysis confirmed the bioactivity of purified the tandem scFv. This bispecific tandem scFv could be a potential candidate to cancer immunotherapy, although more biological activity assessments are still required to be carried out.

Targeting immune checkpoints: how to use natural killer cells for fighting against solid tumors.

Natural killer (NK) cells are unique innate immune cells that mediate anti-viral and anti-tumor responses. Thus, they might hold great potential for cancer immunotherapy. NK cell adoptive immunotherapy in humans has shown modest efficacy. In particular, it has failed to demonstrate therapeutic efficiency in the treatment of solid tumors, possibly due in part to the immunosuppressive tumor microenvironment (TME), which reduces NK cell immunotherapy's efficiencies. It is known that immune checkpoints play a prominent role in creating an immunosuppressive TME, leading to NK cell exhaustion and tumor immune escape. Therefore, NK cells must be reversed from their dysfunctional status and increased in their effector roles in order to improve the efficiency of cancer immunotherapy. Blockade of immune checkpoints can not only rescue NK cells from exhaustion but also augment their robust anti-tumor activity. In this review, we discussed immune checkpoint blockade strategies with a focus on chimeric antigen receptor (CAR)-NK cells to redirect NK cells to cancer cells in the treatment of solid tumors.

Intrinsic and Extrinsic Transcriptional Profiles That Affect the Clinical Response to PD-1 Inhibitors in Patients with Non-Small Cell Lung Cancer.

Using a machine learning method, we investigated the intrinsic and extrinsic transcriptional profiles that affect the clinical response to PD-1 inhibitors in 57 patients with non-small cell lung cancer (NSCLC). Among the top 100 genes associated with the responsiveness to PD-1 inhibitors, the proportion of intrinsic genes in lung adenocarcinoma (LUAD) (69%) was higher than in NSCLC overall (36%) and lung squamous cell carcinoma (LUSC) (33%). The intrinsic gene signature of LUAD (mean area under the ROC curve (AUC) = 0.957 and mean accuracy = 0.9) had higher predictive power than either the intrinsic gene signature of NSCLC or LUSC or the extrinsic gene signature of NSCLC, LUAD, or LUSC. The high intrinsic gene signature group had a high overall survival rate in LUAD (p = 0.034). When we performed a pathway enrichment analysis, the cell cycle and cellular senescence pathways were related to the upregulation of intrinsic genes in LUAD. The intrinsic signature of LUAD also showed a positive correlation with other immune checkpoint targets, including CD274, LAG3, and PDCD1LG2 (Spearman correlation coefficient > 0.25). PD-1 inhibitor-related intrinsic gene patterns differed significantly between LUAD and LUSC and may be a particularly useful biomarker in LUAD.

Comparative efficacy and tolerability of targeted and immunotherapy combined with chemotherapy as first-line treatment for advanced gastric cancer: a Bayesian network meta-analysis

The use of target agents and immune checkpoint inhibitors have changed the treatment landscape for AGC in the first-line setting. However, the crosswise comparison between each regimen is rare. Therefore, we estimated the efficacy and safety of targeted therapy or immunotherapy with chemotherapy in AGC patients as the first-line treatment. Included studies were divided into “average” or “specific positivity” group according to whether the patients were selected by a certain pathological expression. We conducted a Bayesian network meta-analysis for all regimens in both groups. In average group, no regimen showed significant improvements in overall survival (OS) and progression free survival (PFS), while pembrolizumab and nivolumab combined with chemotherapy were ranked first and second respectively without an obvious safety difference. In specific positivity group, zolbetuximab plus chemotherapy significantly prolonged OS (HR 0.53, 95% CI 0.36–0.79) and PFS (HR 0.45, 95% CI 0.25–0.81). The top three regimens were zolbetuximab-chemotherapy, trastuzumab plus pertuzuma-chemotherapy and nivolumab-chemotherapy respectively, with no significant safety risk. For average patients, immune checkpoint inhibitor PD-1 plus chemotherapy will be the promising regimen. For patients with overexpression of CLDN18.2, zolbetuximab combined with chemotherapy comes with greater survival benefits, while for patients who have PD-L1 expression with no HER-2 or CLDN18.2 positivity, additional immune checkpoint inhibitor of PD-1 will be a good considered option.

Combination TIGIT/PD-1 blockade enhances the efficacy of neoantigen vaccines in a model of pancreatic cancer.

Cancer neoantigens are important targets of cancer immunotherapy and neoantigen vaccines are currently in development in pancreatic ductal adenocarcinoma (PDAC) and other cancer types. Immune regulatory mechanisms in pancreatic cancer may limit the efficacy of neoantigen vaccines. Targeting immune checkpoint signaling pathways in PDAC may improve the efficacy of neoantigen vaccines. We used KPC4580P, an established model of PDAC, to test whether neoantigen vaccines can generate therapeutic efficacy against PDAC. We focused on two immunogenic neoantigens associated with genetic alterations in the CAR12 and CDK12 genes. We tested a neoantigen vaccine comprised of two 20-mer synthetic long peptides and poly IC, a Toll-like receptor (TLR) agonist. We investigated the ability of neoantigen vaccine alone, or in combination with PD-1 and TIGIT signaling blockade to impact tumor growth. We also assessed the impact of TIGIT signaling on T cell responses in human PDAC. Neoantigen vaccines induce neoantigen-specific T cell responses in tumor-bearing mice and slow KPC4580P tumor growth. However, KPC4580P tumors express high levels of PD-L1 and the TIGIT ligand, CD155. A subset of neoantigen-specific T cells in KPC4580P tumors are dysfunctional, and express high levels of TIGIT. PD-1 and TIGIT signaling blockade in vivo reverses T cell dysfunction and enhances neoantigen vaccine-induced T cell responses and tumor regression. In human translational studies, TIGIT signaling blockade in vitro enhances neoantigen-specific T cell function following vaccination. Taken together, preclinical and human translational studies support testing neoantigen vaccines in combination with therapies targeting the PD-1 and TIGIT signaling pathways in patients with PDAC.

A Totipotent "All-In-One" Peptide Sequentially Blocks Immune Checkpoint and Reverses the Immunosuppressive Tumor Microenvironment.

Immune checkpoint blockade combined with reversal of the immunosuppressive tumor microenvironment (TME) can dramatically enhance anti-tumor immunity, which can be achieved by using multiple-agent therapy. However, the optimal dose and order of administration of different agents remain elusive. To address this dilemma, multiple agents are often grafted together to construct "all-in-one" totipotent drugs, but this usually comes at the cost of a lack of synergy between the agents. Herein, by comprehensively analyzing the conserved sites of the immune checkpoint and TME drug targets, peptide secondary structures, assembly properties, and other physicochemical properties, a high-content peptide library is designed. By using the "3D-molecular-evolution" screening strategy, an efficient and totipotent "all-in-one" peptide (TAP) is obtained, which possesses the abilities of self-assembling, blocking the PD-1/PD-L1axis, inhibiting Rbm38-eIF4E complex formation, and activating p53. It is shown that in mice treated with TAP, with either subcutaneous tumors or patient-derived xenografts, PD-L1is blocked, with increased activation of both T and NK cells whilst reversing the immunosuppressive TME. Moreover, TAP can mitigate tumor activity and suppress tumor growth, showing superior therapeutic effect over antibody-based drugs.

Abstract B020: Biomarkers of IDO1 inhibition in patients with locally advanced rectal cancer treated with radiation and chemotherapy

Abstract Background: To date, immunotherapy has not advanced the initial care of locally advanced rectal cancer (LARC) where a combination of radiation and chemotherapy remains the standard of care. In evaluation of other targets, we identified that the immune-oncology target indoleamine 2,3 dioxygenase-1 (IDO1) is universally overexpressed in rectal cancers after radiation therapy (RT) regardless of MSI status. IDO1 initiates tryptophan metabolism along the kynurenine pathway (KP) and activation of the KP exerts immune-suppressive effects in the tumor microenvironment. Our preclinical data identified that epacadostat, a potent inhibitor of IDO1, reduced CRC growth in vitro and in vivo. Furthermore, we found that epacadostat augmented the anti-tumor effects of RT by directly increasing apoptosis in neoplastic cells as well as by relieving RT induced immune-suppression. Based on these findings, we initiated a Phase I study in patients with LARC where epacadostat was used in combination with short-course RT (5 Gy x 5) and chemotherapy. This study is ongoing with the primary endpoint of determining the recommended phase II dose (RP2D) of epacadostat in this population. The study is a dose escalating design including 300, 400, and 600 mg BID. Aim: To present preliminary results from the corollary studies on sequentially acquired biospecimens. Methods: We collected tumor tissue and blood pre-treatment, post-radiation/pre-chemo, sequentially throughout chemo, and at treatment completion or surgery. Analyses include global metabolomics, focused evaluation of tryptophan pathway metabolites, single cell RNAseq, flow cytometry, ctDNA and tissue analysis for IDO1 and other immune checkpoint targets. We also established tumor-derived organoids/tumoroids and have established a “tumor-on-a-chip” model to conduct ex vivo tumor modeling. Results: Preliminary studies confirm an elevation of kynurenine to tryptophan ratio after SCRT which is reversed by epacadostat. Changes in the global metabolomics profiles and immune profiles shift from baseline through the stages of SCRT and chemotherapy revealing consistent patterns. Changes in ctDNA were assessed using the CAPP-Seq based AVENIO platform providing a personalized oncogenomic analysis and demonstrated of rapid clearance from post-SCRT which persisted throughout chemotherapy. Studies with tumoroids and the analysis of scRNAseq data are ongoing and will be presented. Conclusions: Preliminary analysis of biospecimens from this actively accruing study of epacadostat plus RT and chemotherapy reveals distinct changes in metabolomic, immunologic, and genomic profiling across the stages of treatment. These results have the potential to inform a precision design for the Phase II study and will also inform the most promising disease and treatment related biomarkers. Citation Format: Matthew A. Ciorba, Katrina Pedersen, Lauren Henke, Aadel Chaudhuri, Matthew Mutch, Hyun Kim, Haeseong Park. Biomarkers of IDO1 inhibition in patients with locally advanced rectal cancer treated with radiation and chemotherapy [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 B020.

13P Tumor-infiltrating CD8/CD103/TIM-3-expressing lymphocytes in epithelial ovarian cancer co-express CXCL13 and associate with improved survival

Reactivation of tumor-infiltrating T lymphocytes (TILs) with immune checkpoint inhibitors or co-stimulators has proven to be an effective anti-cancer strategy for a broad range of malignancies. However, epithelial ovarian cancer (EOC) remains largely refractory to current T cell-targeting immunotherapeutics. Therefore, identification of novel immune checkpoint targets and biomarkers with prognostic value for EOC is warranted. We here identified a TIM-3/CXCL13-positive tissue-resident memory (CD8/CD103-positive) T cell (Trm) population in EOC.

Novel phthalimides regulating PD-1/PD-L1 interaction as potential immunotherapy agents.

Programmed cell death 1(PD-1)/programmed cell death ligand 1(PD-L1) have emerged as one of the most promising immune checkpoint targets for cancer immunotherapy. Despite the inherent advantages of small-molecule inhibitors over antibodies, the discovery of small-molecule inhibitors has fallen behind that of antibody drugs. Based on docking studies between small molecule inhibitor and PD-L1 protein, changing the chemical linker of inhibitor from a flexible chain to an aromatic ring may improve its binding capacity to PD-L1 protein, which was not reported before. A series of novel phthalimide derivatives from structure-based rational design was synthesized. P39 was identified as the best inhibitor with promising activity, which not only inhibited PD-1/PD-L1 interaction (IC50=8.9nmol/L), but also enhanced killing efficacy of immune cells on cancer cells. Co-crystal data demonstrated that P39 induced the dimerization of PD-L1 proteins, thereby blocking the binding of PD-1/PD-L1. Moreover, P39 exhibited a favorable safety profile with a LD50>5000mg/kg and showed significant invivo antitumor activity through promoting CD8+ T cell activation. All these data suggest that P39 acts as a promising small chemical inhibitor against the PD-1/PD-L1 axis and has the potential to improve the immunotherapy efficacy of T-cells.

Development of Effective Siglec-9 Antibodies Against Cancer.

This study aims to review state-of-the-art advances in Siglec-9-directed antibodies and to highlight specific aspects of Siglec-9 antibodies that are suitable to mount anti-tumor immunity. Controversies surrounding studies on Siglec-9 antibodies can confound future studies. In this review, we have highlighted some controversies, explained the distinction between Siglec-9 agonistic and antagonistic (endocytic) antibodies, and discussed their suitability in sustaining anti-tumor immunity. Siglec-9 is an immune checkpoint target and an immunoinhibitory receptor that can engage either sialic acid ligands or agonistic antibodies. Through Siglec-9 sialic acid interactions, activated immunoreceptor tyrosine-based inhibitory signaling of the immune cells can lead to unfavorable immunosuppression. To overcome tumor-related immunosuppression, different types of Siglec-9 antibody blockade need to be developed. However, whether a Siglec-9-directed antibody is agonistic or antagonistic is probably affinity-dependent and not epitope-dependent. Additionally, unlike immune-modulatory antibodies such as agonistic antibodies (OX40, CD28, ICOS, and 4-1BB) or Fc-inert antibodies (PD1 and PD-L1) directed against cancer cells, the nature of antagonistic Siglec-9 antibodies is more suitable to enhance anti-tumor immunity and will be discussed.

Disruption of the NKG2A:HLA-E Immune Checkpoint Axis to Enhance NK Cell Activation against Cancer.

Ligation of the inhibitory receptor NKG2A by its ligand HLA-E negatively regulates the activation of natural killer (NK) cells, as well as subsets of CD8+ T cells and innate T cell populations. NKG2A has recently become a novel immune checkpoint target for the treatment of cancer and direct antibody mediated blockade of NKG2A function is currently under assessment in two phase 3 clinical trials. In addition to direct targeting, the NKG2A:HLA-E axis can also be disrupted indirectly via multiple different targeted cancer agents that were not previously recognised to possess immunomodulatory properties. Increased understanding of immune cell modulation by targeted cancer therapies will allow for the design of rational and more efficacious drug combination strategies to improve cancer patient outcomes. In this review, we summarise and discuss the various strategies currently in development which either directly or indirectly disrupt the NKG2A:HLA-E interaction to enhance NK cell activation against cancer.