Current Immune Checkpoint Inhibitors Research Articles

PTEN loss shapes macrophage dynamics in high grade serous ovarian carcinoma.

High-grade serous ovarian carcinoma (HGSC) remains a disease of poor prognosis that is unresponsive to current immune checkpoint inhibitors. Although PI3K pathway alterations, such as PTEN loss, are common in HGSC, attempts to target this pathway have been unsuccessful. We hypothesized that aberrant PI3K pathway activation may alter the HGSC immune microenvironment and present a targeting opportunity. Single-cell RNA sequencing identified populations of resident macrophages specific to Pten-null omental tumors in murine models, which were confirmed by flow cytometry. These macrophages derived from peritoneal fluid macrophages and had a unique gene expression program, marked by high expression of the enzyme heme oxygenase-1 (HMOX1). Targeting resident peritoneal macrophages prevented the appearance of HMOX1hi macrophages and reduced tumor growth. Furthermore, direct inhibition of HMOX1 extended survival in vivo. RNA sequencing identified IL33 in Pten-null tumor cells as a likely candidate driver leading to the appearance of HMOX1hi macrophages. Human HGSC tumors also contained HMOX1hi macrophages with a corresponding gene expression program. Moreover, the presence of these macrophages correlated with activated tumoral PI3K/mTOR signaling and poor overall survival in HGSC patients. In contrast, tumors with low numbers of HMOX1hi macrophages were marked by increased adaptive immune response gene expression. These data suggest targeting HMOX1hi macrophages as a potential therapeutic strategy for treating poor prognosis HGSC.

Abstract 5369: Mapping a T cell-cold phenotype to the colorectal cancer genome to identify POFUT1 as a driver of CD8+ T cell inhibition

Abstract CD8+ T cells are a critical anti-cancer immune subset; however, most colorectal cancer (CRC) patients have inadequate levels of CD8+ T cells in their tumor microenvironment (TME), contributing to poor survival and increased CRC aggression. While current immune checkpoint inhibitors are able to elicit anti-cancer CD8+ T cell responses in some patients, a majority of patients are still nonresponsive, and have minimal levels of tumor-infiltrating CD8+ T cells. This effect is potentially due to still-unrecognized cancer-driven mechanisms of immunosuppression. To identify candidate genes with roles in CD8+ T cell exclusion, we used a T cell gene signature (Tsig) to quantify CD8+ T cell infiltration in tumors profiled by RNA-seq. Partial Spearman correlation adjusted for tumor purity was performed for all annotated genes (N=20501) in the TCGA CRC RNA-seq data set (N=373) to identify genes most inversely correlated with Tsig. A locus at chromosome 20q was identified as the region of the genome most inversely correlated with Tsig - both by copy number analysis and by RNA expression. Interestingly, this region is the most copy number-amplified region in CRC tumors, and the genes at this locus are among the most highly expressed genes in CRC tumors. Within this region, the amplified and overexpressed gene Protein O-Fucosyltransferase 1 (POFUT1) had the strongest inverse correlation to Tsig. POFUT1 function is essential to canonical NOTCH signaling, and CRC-expressed NOTCH is implicated in increased CRC secretion of immunosuppressive factors IL-6, TGF-β and VEGF, and reduced CD8+ T cell secretion of IFN-γ and IL-2. POFUT1 overexpressing (OE) CT26 and MC38 cell lines were generated, and confirmed by Western Blot to have elevated levels of both cleaved NOTCH1 (NICD1), and NOTCH pathway transcriptional target, HES1. Murine CD8+ T cells were stimulated in vitro with CD3/CD28 microbeads and IL-2 in conditioned medium (CM) from vector or POFUT1-OE lines. CD8+ T cells cultured in CM from POFUT1-OE lines had significantly lower levels of activation markers (CD45 (p=0.0022), CD8 (p=0.0095), CD25 (p=0.0069, CD69 (p=0.0099)), reduced proliferation (CFSE, p=0.1281), and generated less ATP (Cell-Titer Glo, p=0.0003) than CD8+ T cells cultured in vector CM. In pan-cancer correlational analyses, POFUT1 is in the top 5% of genes most inversely correlated with Tsig in 12/24 solid tumor types annotated in TCGA (SKCM, THCA, COAD, READ, LUSC, CESC, KIRC, STAD, HNSC, KIRP, LIHC, UCEC p<0.0001). POFUT1 is robustly negatively correlated with T cell infiltration in many cancer types. We observed a positive association in CRC between POFUT1 and NOTCH signaling, and observed that POFUT1 expression reduced CD8+ T cell activity, potentially through upregulation of extracellular factors (in progress). Our findings fit a model where POFUT1 amplification in CRC promotes CD8+ T cell dysfunction as a mechanism of immune escape. Citation Format: Calvin J. Wagner, Eric D. Routh, Raven Rutledge, Lance D. Miller. Mapping a T cell-cold phenotype to the colorectal cancer genome to identify POFUT1 as a driver of CD8+ T cell inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5369.

Advances in new targets for immunotherapy of small cell lung cancer.

Small cell lung cancer (SCLC) is one of the highly aggressive malignancies characterized by rapid growth and early metastasis, but treatment options are limited. For SCLC, carboplatin or cisplatin in combination with etoposide chemotherapy has been considered the only standard of care, but the standard first-line treatment only results in 10-month survival. The majority of patients relapse within a few weeks to months after treatment, despite the relatively sensitive response to chemotherapy. Over the past decade, immunotherapy has made significant progress in the treatment of SCLC patients. However, there have been limited improvements in survival rates for SCLC patients with the current immune checkpoint inhibitors PD-1/PD-L1 and CTLA-4. In the face of high recurrence rates, small beneficiary populations, and low survival benefits, the exploration of new targets for key molecules and signals in SCLC and the development of drugs with novel mechanisms may provide fresh hope for immunotherapy in SCLC. Therefore, the aim of this review was to explore four new targets, DLL3, TIGIT, LAG-3, and GD2, which may play a role in the immunotherapy of SCLC to find useful clues and strategies to improve the outcome for SCLC patients.

Prognostic clinical phenotypes associated with tumor stemness in the immune microenvironment of T-cell exhaustion for hepatocellular carcinoma

T-cell exhaustion (TEX) and high heterogeneity of cancer stem cells (CSCs) are associated with progression, metastasis, and treatment resistance in hepatocellular carcinoma (HCC). Here, we aim to characterize TEX-stemness-related genes (TEXSRGs) and screen for HCC patients who are more sensitive to immunotherapy. The immune cell abundance identifier (ImmuCellAI) was utilized to precisely evaluate the abundance of TEX and screen TEX-related genes. The stemness index (mRNAsi) of samples was analyzed through the one-class logistic regression (OCLR) algorithm. Application of the non-negative matrix decomposition algorithm (NMF) for subtype identification of HCC samples. The different subtypes were assessed for differences in prognosis, tumor microenvironment (TME) landscape, and immunotherapy treatment response. Then, the TEXSRGS-score, which can accurately forecast the survival outcome of HCC patients, was built by LASSO-Cox and multivariate Cox regression, and experimentally validated for the most important TEXSRGs. We also analyzed the expression of TEXSRGs and the infiltration of CD8+ T cells in clinical samples using qRT-PCR and immunohistochemistry (IHC). Based on 146 TEXSRGs, we found two distinct clinical phenotypes with different TEX infiltration abundance, tumor stemness index, enrichment pathways, mutational landscape, and immune cell infiltration through the non-negative matrix decomposition algorithm (NMF), which were confirmed in the ICGC dataset. Utilizing eight TEXSRGs linked to clinical outcome, we created a TEXSRGs-score model to further improve the clinical applicability. Patients can be divided into two groups with substantial differences in the characteristics of immune cell infiltration, TEX infiltration abundance, and survival outcomes. The results of qRT-PCR and IHC analysis showed that PAFAH1B3, ZIC2, and ESR1 were differentially expressed in HCC and normal tissues and that patients with high TEXSRGs-scores had higher TEX infiltration abundance and tumor stemness gene expression. Regarding immunotherapy reaction and immune cell infiltration, patients with various TEXSRGs-score levels had various clinical traits. The outcome and immunotherapy efficacy of patients with low TEXSRGs-score was favorable. In conclusion, we identified two clinical subtypes with different prognoses, TEX infiltration abundance, tumor cell stemness index, and immunotherapy response based on TEXSRGs, and developed and validated a TEXSRGs-score capable of accurately predicting survival outcomes in HCC patients by comprehensive bioinformatics analysis. We believe that the TEXSRGs-score has prospective clinical relevance for prognostic assessment and may help physicians select prospective responders in preference to current immune checkpoint inhibitors (ICIs).

Novel Benzimidazoles as Potent Small-Molecule Inhibitors and Degraders of V-Domain Ig Suppressor of T-Cell Activation (VISTA).

The V-domain Ig suppressor of T-cell activation (VISTA) is a promising negative immune checkpoint and plays a critical role in the regulation of the quiescence of naïve T lymphocytes. Most patients however do not experience durable disease control from current immune checkpoint inhibitors and discovery of inhibitors targeting novel immune checkpoints is necessary. Herein, we report our discovery and optimization of benzimidazoles as the bifunctional inhibitors of VISTA. Compound 1 is identified as a bifunctional inhibitor targeting VISTA, which shows good binding affinity to VISTA and induces VISTA degradation in HepG2 cells through an autophagic mechanism. Compound 1 rescues VISTA-mediated immunosuppression effectively and enhances antitumor activity of immune cells. 1 activates the antitumor immunity in vivo and suppresses tumor growth in a CT26 mouse model significantly. Our results show that compound 1 is a promising VISTA inhibitor and degrader and offers novel approach for cancer immunotherapy through VISTA degradation.

Tumor immune microenvironment in pituitary neuroendocrine tumors (PitNETs): increased M2 macrophage infiltration and PD-L1 expression in PIT1-lineage subset.

Tumor immune microenvironment in pituitary neuroendocrine tumors (PitNETs) and application of current immunotherapy for refractory PitNETs remains debated. We aim to evaluate the immune landscape in different lineages of PitNETs and determine the potential role of pituitary transcription factors in reshaping the tumor immune microenvironment (TIME), thus promoting the application of current immunotherapy for aggressive and metastatic PitNETs. Immunocyte infiltration and expression patterns of immune checkpoint molecules in different lineages of PitNETs were estimated via in silico analysis and validated using an IHC validation cohort. The correlation between varying immune components with clinicopathological features was assessed in PIT1-lineage PitNETs. Transcriptome profiles from 210 PitNETs/ 8 normal pituitaries (NPs) and immunohistochemical validations of 77 PitNETs/6 NPs revealed a significant increase in M2-macrophage infiltration in PIT1-lineage PitNETs, compared with the TPIT-lineage, SF1-lineage subsets and NPs. While CD68 + macrophage, CD4 + T cells, and CD8 + T cells were not different among them. Increased M2-macrophage infiltration was associated with tumor volume (p < 0.0001, r = 0.57) in PIT1-lineage PitNETs. Meanwhile, differentially expressed immune checkpoint molecules (PD-L1, PD1, and CTLA-4) were screened and validated in IHC cohorts. The results showed that PD-L1 was highly expressed in PIT1-lineage subsets, and PD-L1 overexpression showed a positive correlation with tumor volume (p = 0.04, r = 0.29) and cavernous sinus invasion (p < 0.0001) in PIT1-lineage PitNETs. PIT1-lineage PitNETs exhibit a distinct immune profile with enrichment of M2 macrophage infiltration and PD-L1 expression, which may contribute to its clinical aggressiveness. Application of current immune checkpoint inhibitors and M2-targeted immunotherapy might be more beneficial to treat aggressive and metastatic PIT-lineage PitNETs.

Effects of treatment and patterns of metastases in upper tract and bladder urothelial cancer and non-urothelial cancer based on the national cancer database (NCDB) analysis.

e16571 Background: Upper tract Urothelial cancer (UTUC) and bladder urothelial and non-urothelial cancers can have varying patterns of metastases and response to treatment. Given worse response of visceral metastases to current immune checkpoint inhibitors (ICI) and worse outcomes of non-urothelial cancers, understanding the incidence, characteristics and survival for different metastatic presentation would be informative. Methods: The primary objective is to describe characteristics, treatment patterns, evaluate trends and differences in metastatic presentation and survival according to NCDB between upper tract urothelial cancers (UTUC) versus non-urothelial (nUTUC) histologies (squamous, sarcomatoid, small cell or neuroendocrine, adenocarcinoma) and bladder urothelial cancer (UC) and bladder non-urothelial cancer (nUC). Results: Extraction of data from diagnosis between year 2004 – 2017 from the NCDB was performed. A total of 649939 cases of whom UC = 630423 and nUC = 19516 for both bladder and upper tract cancers, respectively, were identified. Median age was 71.4 years (UC) and 70.1 years (nUC), with majority made up of men (62% of UTUC and 75% of bladder UC). Stage distribution at diagnosis was higher in nUC with Stage III at 1711 (8.8%, nUC) compared to 15725 (2.5%, UC) and Stage IV the nUC group (21.9%) compared to UC at 4.6%. Overall survival (OS) was worse with nUC for stage IV cancers with median OS (mOS) of 6.14 mos (95% CI 5.78-6.47) for nUC vs 7.92 mos (95% CI 7.75-8.08) for UC; logrank p &lt; 0.001. Metastatic sites evaluated included bone, brain, liver, lung, and distant lymph nodes. mOS of Patients with visceral metastatic sites of disease are as follows: distant lymph nodes: UC: 7.59 mos (95% CI 6.6- 8.8); nUC: 5.62 mos (95% CI 4.1-7); log-rank p = 0.0372; liver: UC: 3.5 mos (95% CI 2.89-4.1); nUC: 4.1 mos (95% CI 2.6-6.83); p = 0.1874; lung: UC: 5.7 mos (95% CI 5.09-6.37); nUC: 4.27 mos (95% CI 2.99-6.74); p = 0.1567; Brain: UC: 3.06 mos (95% CI 2.1-4.24); nUC: 2.34 (95% CI 1.1-5.3), p = 0.5464. Use of chemotherapy resulted in improved OS in the UC group with mOS of 73.53 mos (95% CI 72.61-74.41) compared to nUC at 17.28 mos (95% CI 16.59-17.87), p &lt; 0.0001 and use of ICI similarly yielded improvement in the UC group with mOS of 115.98 mos (95% CI 114.33-117.65) compared to nUC at 64.49 mos (95% CI 45.67-88.51); p &lt; 0.0001 although radiation therapy resulted in less profound treatment effect with UC at 16.39 mos (95% CI 16.07-16.69) and nUC: 13.01 (95% CI 12.32-13.63); p &lt; 0.0001. Conclusions: While treatment using chemotherapy and ICI has led to improvement in OS in higher stages of disease, patterns of metastases reveals visceral presentation portends a poor prognosis in both urothelial and non-urothelial cancers with slightly better mOS in those with distant lymph nodes as main presentation of metastases.

Chimera and Tandem-Repeat Type Galectins: The New Targets for Cancer Immunotherapy.

In humans, a total of 12 galectins have been identified. Their intracellular and extracellular biological functions are explored and discussed in this review. These galectins play important roles in controlling immune responses within the tumour microenvironment (TME) and the infiltration of immune cells, including different subsets of T cells, macrophages, and neutrophils, to fight against cancer cells. However, these infiltrating cells also have repair roles and are hijacked by cancer cells for pro-tumorigenic activities. Upon a better understanding of the immunomodulating functions of galectin-3 and -9, their inhibitors, namely, GB1211 and LYT-200, have been selected as candidates for clinical trials. The use of these galectin inhibitors as combined treatments with current immune checkpoint inhibitors (ICIs) is also undergoing clinical trial investigations. Through their network of binding partners, inhibition of galectin have broad downstream effects acting on CD8+ cytotoxic T cells, regulatory T cells (Tregs), Natural Killer (NK) cells, and macrophages as well as playing pro-inflammatory roles, inhibiting T-cell exhaustion to support the fight against cancer cells. Other galectin members are also included in this review to provide insight into potential candidates for future treatment(s). The pitfalls and limitations of using galectins and their inhibitors are also discussed to cognise their clinical application.

Abstract 972: VISTA expression in patients with advanced solid tumors: A potential biomarker in VISTA-101 clinical trial

Abstract V-domain Immunoglobulin Suppressor of T cell Activation (VISTA/PD-1H) is a B7 family member highly expressed on circulating and intra-tumoral myeloid cells. It is a negative checkpoint inhibitor that inhibits anti-tumor T cell response. In patients, VISTA is associated with poor overall survival in multiple tumor indications and is also a potential mediator of resistance to anti-CTLA-4 and anti-PD1 therapies. Therefore, VISTA is a unique target for cancer immunotherapy. Kineta has developed a fully human monoclonal antibody targeting VISTA, KVA12123, that is currently being evaluated in a Phase 1/2 clinical trial in cancer patients with advanced solid tumors. This trial also includes a combination arm with pembrolizumab. In order to inform which patients may be susceptible to respond to our anti-VISTA antibody, we hypothesized that the best responders should be associated with a high expression of the target in the tumor microenvironment (TME). Therefore, after assay validation of VISTA labelling by immuno-histochemistry, we analyzed a large set of tumor samples and showed that VISTA was highly expressed on tumor infiltrating immune cells. This was particularly true for patients with non-small cell lung cancers, colorectal cancers, head and neck squamous cell carcinomas, hepatocellular carcinomas, melanomas and squamous cell carcinoma of the skin, and cervical cancers as well as ovarian cancers. VISTA expression was detected mostly on CD163 positive macrophages infiltrating the tumor. These macrophages potentially promote immunosuppression present in the TME and contribute to treatment failure with current immune checkpoint inhibitors like anti-PD1/PD-L1 and CTLA-4. While previous studies reported VISTA expression on cancer cells, we were not able to confirm these results. In all tumor tissues tested, only infiltrating immune cells were labelled for VISTA. We have investigated in parallel the expression level of soluble VISTA in the serum collected from cancer patients independent of this clinical trial. Sera were screened for patients with multiple tumor types. Patients of diverse sex and age were compared to healthy donors. After validation of the ELISA assay, we showed that sera derived from cancer patients exhibit high levels of soluble VISTA, and these levels tend to correlate with age. More data are needed to confirm that high levels of soluble VISTA are associated with advanced disease. In the ongoing Phase 1/2 clinical trial, tumor tissues and serum samples will be collected from cancer patients prior to treatment with KVA12123 to inform the possible significance of these biomarkers. This work could help to better understand the response to KVA12123 in relation to the expression level of VISTA in cancer tissues as well as in the blood. Citation Format: Neda Kabi, Chen Katz, Remington Lance, Jessica Cross, Nathan Eyde, Emily Frazier, Kurt Lustig, Yulia Ovechkina, David Peckham, Shaarwari Sridhar, Carla Talbaux, Isabelle Tihista, Mei Xu, Shawn Iadonato, Thierry Guillaudeux. VISTA expression in patients with advanced solid tumors: A potential biomarker in VISTA-101 clinical trial [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 972.

Abstract 4263: Anti-Siglec 7 antibody displays potent anti-tumor immunity and demonstrates improved tumor control in combination with anti-PD1 in ovarian cancer

Abstract High grade serous ovarian cancer (OC) and ovarian carcinosarcoma, referred as “Cold” tumors have restricted treatment choices and being associated with high mortality. Current immune checkpoint inhibitors (CPIs) have had modest impact for OC treatment; with anti PD-1/PD-L1 single therapies reported response rates of 4-15%. Accordingly, additional approaches are important for these patients. Natural killer (NK) cells represent an important subset of effector lymphocytes with strategic approaches for maximizing NK reactivity to target OC are likely important. Of interest, an inhibitory receptor and glyco immune checkpoint- Siglec 7 is expressed on 100% of peripheral blood and umbilical cord NK cells. We have generated antibodies against Siglec 7 through an optimized human Siglec 7 DNA/protein immunization approach in humanized next generation transgenic mice. By high throughput flow screening, we identified several Siglec 7 antibodies and down selected relevant clones. A unique clone DB7.2 was highly specific for Siglec 7. We demonstrated the ability of DB7.2 (human IgG1) to activate NK cells and induce OC cell killing using xCELLigence RTCA for in vivo OC challenge studies. DB7.2 (1μg/ml) bound to &amp;gt;90% of NK cells including both CD56dim and CD56bright subsets, evaluated in the PBMCs of multiple donors. It showed strong binding to recombinant Siglec 7 and Siglec 7 transduced HEK293T but not to wild type 293T cells supporting specificity. DB7.2 induced specific killing of multiple OC lines (OVCAR3&amp;10,TOV21G, CaOV3, OVISE, PEO4) carrying different mutations; BRCA1&amp;2, AKT, TP53, PIK3CA, BRAF etc. and resistant against a wide array of cancer drug targets; HSP90, HDAC, MTORC, DNA alkylating agents, EGFR, PARP, PI3K, and WEE1. Tumor killing was indicated to be mediated via enhanced secretion of soluble Fas, perforin, granulysin as well as granzyme A. Of note, OVISE (BRCA1mutated) and PEO4 (BRCA2 mutated, PARPi resistant) were susceptible to DB7.2 killing with EC50- 82.67 and 68.67 nM respectively. A single dose of in vivo expressed DB7.2 significantly reduced the tumor burden in an OVISE challenged humanized mice model enhancing median survival by 57 days. As OCs are highly diverse in nature and likely to require combinatorial approaches for simultaneous targeting of immune pathways; we combined DB7.2 with anti-PD1 for further investigation. In xCELLigence assay, anti-PD1 demonstrated killing of PEO4 cells with EC50 680 nM. The combination of anti PD-1 with DB7.2 showed further enhancement of OC killing in the presence of human PBMCs. This is the first demonstration of the impact of Siglec 7 targeting mAb alone as well as in combination with anti-PD1 (NK and T cell CPIs) studied for targeting OC or any human tumor. These studies have important implications for tumor therapy and provide a novel non T cell CPI potential approach to augment current immune therapy strategies. Citation Format: Devivasha Bordoloi, Abhijeet J. Kulkarni, Opeyemi S. Adeniji, M Betina Pampena, Pratik S. Bhojnagarwala, Shushu Zhao, Elizabeth M. Parzych, Rugang Zhang, Michael R. Betts, Mohamed Abdel-Mohsen, David B. Weiner. Anti-Siglec 7 antibody displays potent anti-tumor immunity and demonstrates improved tumor control in combination with anti-PD1 in ovarian cancer. [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 4263.

Abstract 2361: Prevalence of mature tertiary lymphoid structures and association with tumor mutational burden in patients with solid tumors

Abstract Background: Mature Tertiary Lymphoid Structures (mTLS) are organized lymphoid structures containing B-lymphocytes admixed to CD23+ follicular dendritic cells, which play a key role in antitumor immune response. We and others have demonstrated that presence of mature TLS is higly predictive of benefot of anti-PD1/PDL1 antibodies in patients with solid tumors (Vanhersecke et al Nature Cancer 2021, Italiano et al Nature Medicine 2022). However, the relative prevalence of mature TLS in the different tumor types has not been investigated with an uniform methodological approach. Methods: We screened the presence of mature TLS (mTLS) on tumor samples from patients included in an institutional molecular profiling program (BIP, NCT02534649). mTLS were screened using HES, CD20 (clone L26, Ventana) and CD23 (clone 1B12, Novocastra) stainings, performed on serial sections. TLS were defined as lymphoid aggregates of CD20+ B-lymphocytes of more than 50 immune cells within 1mm of the tumor front. mTLS were defined as TLS containing either a visible germinal center on HES or a meshwork of CD23+ follicular dendritic cells or isolated CD23+ dendritic cell if the cell harbored a morphology fitting with dendritic differentiation. If plasma sample of patient was available, bTMB was assessed by Foundation Medicine, with bTMB-high defined as at least 10 mutations/megabase (mut/Mb). Results: We characterized 1,393 samples. mTLS were present in 467 cases (33.5%) across all tumor histotypes: 36.2% of carcinoma (N = 392/1080 cases), 24.8% of sarcoma (N =69/209 cases) and 25% of neuroendocrine tumor (N= 4/12 cases). Among 569 cases with evaluable bTMB, the proportion of patients with bTMB-high was significantly higher in the mTLS group (16.7%, N=31/185) compared to absence of mTLS group (8.5%, N=30/353) (p=0.036). Conclusion: mTLS are present in variable proportions in almost all tumor types even in those considered as resistant to current immune checkpoint inhibitors. Prostate Bladder Kidney Head and neck Gynecologic (except ovary) Ovary mTLS+% of patients (N) 34% (28) 33% (19) 25% (6) 53% (17) 33% (19) 44% (26) mTLS-% of patients (N) 66% (55) 67% (38) 75% (18) 47% (15) 67% (39) 55% (32) Upper gastro intestinal tract Pancreatic Biliary tract Breast Thyroid Sarcoma mTLS+% of patients (N) 37% (19) 50% (13) 40% (21) 30% (38) 49% (24) 25% (69) mTLS-% of patients (N) 63% (33) 50% (13) 60% (32) 70% (90) 51% (25) 75% (209) Citation Format: Maxime Brunet, Lucile Vanhersecke, Francois Le Loarer, Isabelle Soubeyran, Antoine Italiano. Prevalence of mature tertiary lymphoid structures and association with tumor mutational burden in patients with solid tumors [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 2361.

Simultaneous inhibition of PD-1 and LAG-3: the future of immunotherapy?

Immunotherapy has improved the prognosis of many cancers, yet a large number of patients have demonstrated resistance to current immune checkpoint inhibitors. LAG-3 is an immune checkpoint expressed on tumor-infiltrating lymphocytes CD4+ and CD8+, Tregs and other immune cells. Coexpression of PD-1 and LAG-3 in solid or hematological cancers is generally associated with a poor prognosis and may be responsible for immunotherapy resistance. Dual inhibition therapy in the RELATIVITY-047 trial significantly improved progression-free survival in metastatic melanoma. This article discusses the presence of a possible synergistic interaction between LAG-3 and PD-1 in the tumor microenvironment and the utility of targeting both immune checkpoint inhibitors as an effective way to bypass resistance and increase treatment efficacy.

Abstract 1371: Rho-GEF hampers CD8+T cells-mediated immunosurveillance by reducing antigen presentation

Abstract Immune response is a critical anti-tumor response to overcome tumorigenesis. In line with this, immune checkpoint blockade shows tremendous anti-tumor efficacy by enhancing cytotoxicity of CD8+ cytotoxic T lymphocytes (CTLs). However, cancer cells develop different mechanisms of immune evasion, among which suppression of immune recognition of somatic mutations via tumor antigen is prevalent. In fact, 65% to 90% of the cancers show reduced MHC-I antigen presentation. There is, nonetheless, little achievement toward the therapeutic approaches to restore antigen presentation on tumor cells. Colorectal cancer (CRC), the second deadliest cancer in U.S., shows increasing incidence and mortality rates over the past years. Standard conventional CRC treatments, surgery, chemotherapy and radiotherapy, often lead to many side effects due to their non-specificity and cytotoxicity. While immunotherapy is very promising, only a small population of CRC patients respond to the current immune checkpoint inhibitors. In this study, our objective was to harness a novel immunological approach to target CRC tumors. We developed a bioinformatics tool to create a pool of genes whose expression negatively correlates with cytotoxicity of CD8+ CTLs in the tumor microenvironment of CRC patients. Given the central role of antigen presentation in activating CD8+ CTLs, we further screened these genes based on the scope of their effects on antigen presentation. To this end, we developed stable clones of OVA expressing MC38 cells (MC38OVA) where OVA-derived peptide, SIINFEKL, will be bound to MHC-I complex for cell surface presentation. 4 genes with highest capacity to alter MHC-I cell surface levels were identified. Our consequent in vivo experiments and in vitro cytotoxicity assays identified that “Rho-GEF” gene was the top actionable target with striking suppressive effects on tumor growth. TCGA database analysis showed Rho-GEF is upregulated in 22% of CRC patients accompanied by a poor prognosis. Rho-GEF knockdown (KD) significantly inhibited tumor growth in immunocompetent mice whereas it only showed minor effects on immunocompromised mice, suggesting that the Rho-GEF may modulate tumor growth by regulating the interaction of tumor cells with non-tumor cells such as immune cells. Consistent with the major contribution of antigen presentation in promoting CD8+ CTLs activity, TNFα, IFNγ, and GZMB levels in CD8+ T cells isolated from Rho-GEF KD MC38-derived tumors showed a significant increase compared to the wild type tumors. Taken together, we introduced Rho-GEF as a potential clinical target involved in CRC immune evasion by modulating antigen presentation and CD8+ CTLs anti-tumor responses. Citation Format: Haniyeh Eyvani, Xinna Zhang. Rho-GEF hampers CD8+T cells-mediated immunosurveillance by reducing antigen presentation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1371.

Characterization of stem cell landscape and identification of stemness-relevant prognostic gene signature to aid immunotherapy in colorectal cancer

BackgroundIt is generally accepted that colorectal cancer (CRC) originates from cancer stem cells (CSCs), which are responsible for CRC progression, metastasis and therapy resistance. The high heterogeneity of CSCs has precluded clinical application of CSC-targeting therapy. Here, we aimed to characterize the stemness landscapes and screen for certain patients more responsive to immunotherapy.MethodsTwenty-six stem cell gene sets were acquired from StemChecker database. Consensus clustering algorithm was applied for stemness subtypes identification on 1,467 CRC samples from TCGA and GEO databases. The differences in prognosis, tumor microenvironment (TME) components, therapy responses were evaluated among subtypes. Then, the stemness-risk model was constructed by weighted gene correlation network analysis (WGCNA), Cox regression and random survival forest analyses, and the most important marker was experimentally verified.ResultsBased on single-sample gene set enrichment analysis (ssGSEA) enrichments scores, CRC patients were classified into three subtypes (C1, C2 and C3). C3 subtype exhibited the worst prognosis, highest macrophages M0 and M2 infiltrations, immune and stromal scores, and minimum sensitivity to immunotherapies, but was more sensitive to drugs like Bosutinib, Docetaxel, Elesclomol, Gefitinib, Lenalidomide, Methotrexate and Sunitinib. The turquoise module was identified by WGCNA that it was most positively correlated with C3 but most negatively with C2, and five hub genes in turquoise module were identified for stemness model construction. CRC patients with higher stemness scores exhibited worse prognosis, more immunosuppressive components in TME and lower immunotherapeutic responses. Additionally, the model’s immunotherapeutic prediction efficacy was further confirmed from two immunotherapy cohorts (anti-PD-L1 in IMvigor210 cohort and anti-PD-1 in GSE78220 cohort). Mechanistically, Gene Set Enrichment Analysis (GSEA) results revealed high stemness score group was enriched in interferon gamma response, interferon alpha response, P53 pathway, coagulation, apoptosis, KRAS signaling upregulation, complement, epithelial–mesenchymal transition (EMT) and IL6-mediated JAK-STAT signaling gene sets.ConclusionsOur study characterized three stemness-related subtypes with distinct prognosis and TME patterns in CRC patients, and a 5-gene stemness-risk model was constructed by comprehensive bioinformatic analyses. We suggest our stemness model has prospective clinical implications for prognosis evaluation and might facilitate physicians selecting prospective responders for preferential use of current immune checkpoint inhibitors.

Novel Immune Checkpoints in Esophageal Cancer: From Biomarkers to Therapeutic Targets

Esophageal cancer ranks as the sixth most common cause of cancer death worldwide. Due to the limited efficacy of conventional therapeutic strategies, including surgery, chemotherapy, and radiotherapy, treatments are still far from satisfactory in terms of survival, prompting the search for novel treatment methods. Immune checkpoints play crucial roles in immune evasion mediated by tumor cells, and successful clinical outcomes have been achieved via blocking these pathways. However, only a small fraction of patients can benefit from current immune checkpoint inhibitors targeting programmed cell death ligand-1 (PD-L1) and cytotoxic T-lymphocyte-associated protein-4. Unfortunately, some patients show primary and/or acquired resistance to immune checkpoint inhibitors. Until now, novel immune checkpoint pathways have rarely been studied in esophageal cancer, and there is a great need for biomarkers to predict who will benefit from existing strategies. Herein, we primarily discuss the roles of new immune checkpoints as predictive biomarkers and therapeutic targets for esophageal cancer. In addition, we summarize the ongoing clinical trials and provide future research directions targeting these pathways.

New Strategies and Combinations to Improve Outcomes in Immunotherapy in Metastatic Non-Small-Cell Lung Cancer.

Immune checkpoint inhibitors have transformed the treatment of metastatic non-small-cell lung cancer, yielding marked improvements in survival and the potential for durable clinical responses. Primary and acquired resistance to current immune checkpoint inhibitors constitute a key challenge despite the remarkable responses observed in a subset of patients. Multiple novel combination immunotherapy and adoptive cell therapy strategies are presently being developed to address treatment resistance. The success of these strategies hinges upon rational clinical trial design as well as careful consideration of the immunologic mechanisms within the variable tumor immune microenvironment (TIME) which underpin resistance to immunotherapy. Further research is needed to facilitate a deeper understanding of these complex mechanisms within the TIME, which may ultimately provide the key to restoring and enhancing an effective anti-tumor immune response. This review aims to provide an introduction to some of the recent and notable combination immunotherapy and cell therapy strategies used in advanced non-small-cell lung cancer (NSCLC), and the rationale for their use based on current understanding of the anti-tumor immune response and mechanisms of resistance within the TIME.

The Emerging Role of T-Cell Immunoglobulin Mucin-3 in Breast Cancer: A Promising Target For Immunotherapy.

Cancer treatment through immune checkpoint receptor blockade has made significant advances in the recent years. However, resistance to the current immune checkpoint inhibitors (ICIs) has been observed in many patients, who consequently do not respond to these treatments. T-cell immunoglobulin mucin-3 (Tim-3) is a novel immune checkpoint molecule emerging as a potential therapeutic target for cancer immunotherapy. Epidemiologic findings reveal that genetic polymorphisms in the Tim-3 gene are associated with increased susceptibility to breast cancer. In patients with breast cancer, Tim-3 is expressed both on immune and tumor cells. Accumulating evidence demonstrates that Tim-3 can notably affect breast cancer treatment outcome and prognosis. Therefore, Tim-3 is being regarded as a high-potential target for improving breast cancer therapy. In this review, we summarize the role of Tim-3 in breast cancer and the regulation mechanisms of Tim-3 to furnish evidences for future research and therapy.

Construction of a Promising Tumor-Infiltrating CD8+ T Cells Gene Signature to Improve Prediction of the Prognosis and Immune Response of Uveal Melanoma.

BackgroundCD8+ T cells work as a key effector of adaptive immunity and are closely associated with immune response for killing tumor cells. It is crucial to understand the role of tumor-infiltrating CD8+ T cells in uveal melanoma (UM) to predict the prognosis and response to immunotherapy.Materials and MethodsSingle-cell transcriptomes of UM with immune-related genes were combined to screen the CD8+ T-cell-associated immune-related genes (CDIRGs) for subsequent analysis. Next, a prognostic gene signature referred to tumor-infiltrating CD8+ T cells was constructed and validated in several UM bulk RNA sequencing datasets. The risk score of UM patients was calculated and classified into high- or low-risk subgroup. The prognostic value of risk score was estimated by using multivariate Cox analysis and Kaplan–Meier survival analysis. Moreover, the potential ability of gene signature for predicting immunotherapy response was further explored.ResultsIn total, 202 CDIRGs were screened out from the single-cell RNA sequencing of GSE139829. Next, a gene signature containing three CDIRGs (IFNGR1, ANXA6, and TANK) was identified, which was considered as an independent prognostic indicator to robustly predict overall survival (OS) and metastasis-free survival (MFS) of UM. In addition, the UM patients were classified into high- and low-risk subgroups with different clinical characteristics, distinct CD8+ T-cell immune infiltration, and immunotherapy response. Gene set enrichment analysis (GSEA) showed that immune pathways such as allograft rejection, inflammatory response, interferon alpha and gamma response, and antigen processing and presentation were all positively activated in low-risk phenotype.ConclusionOur work gives an inspiration to explain the limited response for the current immune checkpoint inhibitors to UM. Besides, we constructed a novel gene signature to predict prognosis and immunotherapy responses, which may be regarded as a promising therapeutic target.

Identification of immune subtypes of cervical squamous cell carcinoma predicting prognosis and immunotherapy responses

BackgroundThe main limitation of current immune checkpoint inhibitors (ICIs) in the treatment of cervical cancer comes from the fact that it benefits only a minority of patients. The study aims to develop a classification system to identify immune subtypes of cervical squamous cell carcinoma (SCC), thereby helping to screen candidates who may respond to ICIs.MethodsA real-world cervical SCC cohort of 36 samples were analyzed. We used a nonnegative matrix factorization (NMF) algorithm to separate different expression patterns of immune-related genes (IRGs). The immune characteristics, potential immune biomarkers, and somatic mutations were compared. Two independent data sets containing 555 samples were used for validation.ResultsTwo subtypes with different immunophenotypes were identified. Patients in sub1 showed favorable progression-free survival (PFS) and overall survival (OS) in the training and validation cohorts. The sub1 was remarkably related to increased immune cell abundance, more enriched immune activation pathways, and higher somatic mutation burden. Also, the sub1 group was more sensitive to ICIs, while patients in the sub2 group were more likely to fail to respond to ICIs but exhibited GPCR pathway activity. Finally, an 83-gene classifier was constructed for cervical SCC classification.ConclusionThis study establishes a new classification to further understand the immunological diversity of cervical SCC, to assist in the selection of candidates for immunotherapy.

Generation of triacyl lipopeptide-modified glycoproteins by metabolic glycoengineering as the neoantigen to boost anti-tumor immune response.

The lack of tumor specific antigens (TSA) and the immune tolerance are two major obstacles for the immunotherapy of cancer. Current immune checkpoint inhibitors (ICIs) show clinical responses in only limited subsets of cancer patients, which, to some extent, depends on the mutation load of tumor cells that may generate neoantigens. Here, we aimed to generate a neoantigen MDP to exhibit stronger anti-tumor efficacy.Methods: In this study, we utilized chemically modified sialic acid precursor tetra acetyl-N-azidoacetyl-mannosamine (AC4ManNAZ) to engineer the glycoproteins on the membranes of tumor cells for the covalent ligation of hapten adjuvant Pam3CSK4 in vivo, which eventually generated a neoantigen, i.e., ManNAZ-DBCO-Pam3CSK4 (MDP), on tumor cells. The high labeling efficiency, relatively specific biodistribution in tumor tissues and the anti-tumor efficacy were confirmed in the syngeneic murine models of the breast cancer and the lung cancer.Results: The generation of MDP neoantigen in tumor-bearing mice significantly evoked both the humoral and the T-cell-dependent antitumor immune responses, resulting in a strong inhibition on the growth of the breast cancer and the lung cancer allografts and significantly prolonged survival of tumor-bearing mice. Interestingly, MDP neoantigen was able to dramatically increase the sensitivity of cancer cells to ICIs and greatly enhance the anti-tumor efficacy in the murine models of both breast cancer and the lung cancer, which showed no or low responses to the immunotherapy with anti-PD1 antibody alone.Conclusions: We developed a simple metabolic glycoengineering method to artificially generate neoantigens on tumor cells to enhance tumor cell immunogenicity, which is able to significantly improve the response and the clinical outcome of ICIs.