Immunotherapy Resistance Research Articles

Beyond the tumor: the gut microbiome as a key player in immunotherapy efficacy and resistance.

Cancer immunotherapy aims to use the immune system of the body for improved therapeutic effects on tumors. Currently, one of the more encouraging interventions under evaluation involves the use of immune checkpoint blockade, which offers longer benefit periods and greater patient tolerance than previous interventions for solid malignancies. Nevertheless, a majority of patients never respond or gradually acquire resistance; hence, a suboptimal effect of the therapy ensues. Resistance to such treatments may arise from tumor-specific factors, host factors, and environmental influences. There is growing evidence that the gut microbiome is an important modulator not only of the efficacy of these treatments but also of toxicities. Current studies are focused on the identification of key microbial profiles from both preclinical and clinical samples associated with immunotherapeutic response and antitumor activities. Elucidation of this complex interaction may provide ways to modulate gut microbial communities to improve patient outcomes. The current review addresses the components responsible for resistance against immune checkpoint inhibitors and highlights the crucial linkage between gut microbiome-immune interactions. We further summarize some recent clinical findings and explore prospective avenues for research in this evolving area of cancer treatment.

A microfluidic tumor-on-chip platform deciphers hypoxia-driven FOXO3a/PD-L1 signaling in gastric cancer immunotherapy resistance

A microfluidic tumor-on-chip platform deciphers hypoxia-driven FOXO3a/PD-L1 signaling in gastric cancer immunotherapy resistance

Regulation of tumor-associated fibroblasts by TACSTD2 in esophageal squamous cell carcinoma and its potential role in immunotherapy resistance.

e16045 Background: Esophageal squamous cell carcinoma (ESCC) has high global incidence and mortality, with poor prognosis due to late diagnosis and limited chemotherapy efficacy. Immune checkpoint inhibitors targeting PD-1/PD-L1 improve survival in some ESCC patients, but benefits are limited to a subset, lacking biomarkers to identify responsive populations. Identifying biomarkers to predict immunotherapy efficacy is crucial for advancing treatment. Methods: Biopsy tissue samples were collected from four ESCC patients before treatment. All patients received three cycles of neoadjuvant therapy with TC regimen plus sintilimab. Post-treatment pathological responses were classified as pCR (pathological complete response), or tumor regression of grades 1 to 3 (P0, P1, P2, and P3). Surgical resection tissue samples were also collected from patients with pCR (P0-after) and tumor regression grade 3 (P3-after). Spatial transcriptomics was performed on these six tissue samples. Additionally, single-cell RNA sequencing data from two untreated patients and one immune therapy-treated ESCC patient from the GSE221561 dataset were included, and cell types were annotated. Results from spatial transcriptomics and single-cell sequencing were analyzed to identify key genes and pathways influencing immunotherapy efficacy, as well as examine cellular distribution, communication, and type-specific effects. The distribution and characteristics of immune cells before and after immunotherapy were explored. Results: (1) Differential genes between non-pCR (P1-P3) and pCR (P0) groups, as well as between Response-good (P0-P1) and Response-poor (P2-P3) groups, included TACSTD2, KRT family, S100A family, membrane proteins, and DSCs. TACSTD2 expression increased across 4 groups, correlating with immunotherapy resistance. Pathway analysis highlighted extracellular matrix organization. (2) Fibroblasts were the second most abundant cell type, with significant differences across 4 groups. These differences matched extracellular matrix-related gene expression, suggesting that tumor-associated fibroblasts (CAFs) influence immunotherapy efficacy by secreting extracellular matrix. (3) TACSTD2 spatial expression significantly overlapped with α-SMA, a CAF marker, suggesting TACSTD2 may induce CAF differentiation. Cell communication analysis showed interactions between ESCC cells and CAFs via TGF-β receptors and ligands. (4) Comparison of non-responding (P3-after) and complete response (P0-after) patients showed differential gene enrichment in extracellular matrix composition and T-cell infiltration, induced by different CAF types. Conclusions: TACSTD2 may regulate CAF differentiation via the TGF-β pathway, promoting immunotherapy resistance through specific extracellular matrix production and T-cell imbalance.

CtDNA-based clinicogenomic analysis of advanced head and neck cancer patients treated with immune checkpoint inhibitors.

6036 Background: Head and Neck cancer (HNC) is among the diverse group of malignancies affecting the head and neck region including the oral cavity. Being the most prevalent malignancy in Southeast Asia, it has a high mortality rate. Despite the advancement in treatment, 5 years survival rate for HNC remains below 50%, and the majority of Pts receiving frontline therapy experience locoregional or incurable metastatic relapse. Immune checkpoint inhibitors (ICI) are recommended for relapsed patients, but only 20% of patients show measurable response. Currently, no predictive biomarkers are available to predict ICI response and there is an urgent need for genomic markers to predict ICI outcomes. Here we report comprehensive genomic profiling (CGP) of advanced HNC patients receiving ICI. Methods: ctDNA from 69 advanced HNC patients receiving combinational immune-chemotherapy were serially profiled at the baseline (BL) and post-treatment (Tx) by targeted, hybridization-based CGP using OncoIndx comprehensive gene panel (CGP) comprising 1080 genes. The ctDNA differential features at BL and post-Tx as well as among responders (R) and non-responders (NR) were correlated with Progression-free survival (PFS) and Overall survival (OS) using Kaplan-Meier statistics and multivariate analysis. Results: Among total patients, 58% (40/69) were responders (R) while the remaining were non-responders (NR). At the population level, HRR pathway tumor suppressors and epigenetic modifiers were the most frequent pathogenic variants. At BL, the NR population was enriched with oncogenic gene mutations compared to the R population. TP53 and BRCA pathway mutations (mTP53 + BRCA) showed a strong association with progression-free survival (PFS) and overall survival (OS). Pts with cooccurring mTP53 + BRCA had significantly lower PFS (median PFS: 2.77 months for mTP53 + BRCA pathway vs 9.1 months for wt TP53 + BRCA pathway. P=<0.0001, HR=3.2-11.6) and OS (median OS: 4.67 months for mTP53 + BRCA pathway vs 12.63 months for wtTP53 + BRCA pathway. P=<0.0001, HR = 11.18-55.27). NOTCH 1 or 2 variants were enriched in R population, with a beneficial effect on survival outcomes. Elevated ctDNA alterations and Tumor fraction (TF) concentrated in the NR population disproportionately contained subclonal potential drivers of immunotherapy resistance including NF1, STAT5 B, and STK11 mutations, and were associated with short survival. Univariate and multivariate analysis suggested that ctDNA mutations, TF, and high mutational heterogeneity emerged as risk factors for shorter PFS and OS. In contrast, total Indel burden and NOTCH mutations had beneficial effects on PFS and OS. Conclusions: Minimally invasive plasma ctDNA CGP showed heterogenous actionable mutations at BL and post Tx and identified immunotherapy resistance conferring genomic markers for stratifying potential responders for immunotherapy guidance.

Application of a novel multiplex imaging-based immunotherapy panel and AI-powered analysis solution for predictive spatial biomarker identification on immunotherapy-treated melanoma patients.

9524 Background: There is an urgent need for more robust methods to differentiate immunotherapy responders from non-responders. In this study, we present a novel multiplex imaging (MI)-based immunotherapy panel and a comprehensive analysis pipeline to characterize the spatial distribution and function of immune cells and its application for spatial biomarker detection in a cohort of immunotherapy-treated melanoma patients. Methods: We designed a 28-plex panel to perform sequential immunofluorescence (seqIF) on the COMET platform to target key biomarkers associated with tumor microenvironment (TME), immune cell infiltration, and immune checkpoint pathways. Pre-treatment biopsies were obtained from 12 patients with known long-term response or rapid progression to immunotherapy combination treatment from the SECOMBIT Trial (NCT02631447) and profiled utilizing Nucleai’s deep-learning-based MI analysis pipeline, aiming to identify spatial biomarkers that can differentiate between long-term responders and non-responders. We identified 15 cell types, including 10 immune cell populations, in addition to 10 cell state markers. Cells were assigned to the tumor area or TME, and spatial features were calculated based on cell type, marker positivity, and gross area assignment. Results: Our novel MI panel and analysis pipeline demonstrated highly balanced accuracy (> 0.8) and F1 scores (> 0.8) in cell typing and protein quantification for most cell types and markers. This analysis pipeline enabled the quantification of known biomarkers such as T cell activation states, T cell infiltration patterns, and tertiary-lymphoid structure maturation. A comparison of calculated spatial features between long-term responders and rapid progressors revealed distinct immune cell interactions and differences in activation status across the tumor areas associated with response. Within the tumor area, the reciprocal interactions of tumor cells, cytotoxic CD8 T-cells and antigen-presenting cells (APC) were associated with a better outcome. In contrast, a high percentage of proliferating regulatory T cells within the tumor invasive margin was associated with a worse outcome. In the adjacent TME, endothelial cell interactions with T-cells and macrophage proliferation were associated with immunotherapy resistance. In contrast, the interaction between HLA-DR-expressing macrophages and APC cells was associated with an improved clinical outcome. Conclusions: Integrating MI with AI analysis has the potential to enhance our understanding of treatment efficacy and resistance mechanisms. Our preliminary data demonstrate that area-specific immune niches contribute to the success or failure of immunotherapy response and highlight the importance of spatial biology in predicting immunotherapy outcomes. Clinical trial information: NCT02631447 .

CCR8 positive Tregs and their correlation with immunotherapy response in advanced non-small cell lung cancer (NSCLC).

2635 Background: Regulatory T cells (Tregs) expressing the chemokine receptor CCR8 are pivotal modulators of the tumor immune microenvironment. CCR8 has recently emerged as a promising therapeutic target due to its selective expression on activated Tregs in the tumor microenvironment and its role in promoting immunosuppression. This study investigates the prognostic and therapeutic implications of CCR8-positive Tregs in non-small cell lung cancer (NSCLC), focusing on their impact in relation to tertiary lymphoid structure (TLS) status. Methods: A validated 6-plex multiplex immunofluorescence (mIF) panel was used to analyze tumor samples from NSCLC patients treated with immune checkpoint blockers (ICB) in the BIP precision medicine study (ClinicalTrials.gov: NCT04389143). Markers included CD4, CD8, CD20, FoxP3, PanCK, and CCR8, alongside DAPI staining to assess immune contexture (infiltrated, excluded, desert), TLS status, and CCR8/FOXP3 double-positive Tregs. Clinical outcomes, including progression-free survival (PFS) and objective response rate (ORR), were analyzed in 50 responders and 50 non-responders. Findings were validated using transcriptomic data from the POPLAR (NCT01903993) and OAK (NCT02008227) studies, which evaluated atezolizumab versus docetaxel in advanced NSCLC. Kaplan-Meier curves, hazard ratios, and Cox regression models were used for survival analyses. Results: CCR8-expressing Tregs were significantly enriched in infiltrated tumors, showing a 1.5-fold increase compared to excluded tumors (p = 0.057), a 3.3-fold increase compared to desert tumors (p = 0.001), and a 1.8-fold increase in TLS-positive tumors compared to TLS-negative tumors (p = 0.003). These findings highlight that activated Tregs co-infiltrate with CD8 T cells and other immune cell types. This enrichment was confirmed in samples from the POPLAR and OAK studies using transcriptomic analyses (3-fold increase, p = 2e-16). Due to their correlation with overall immune cell infiltration, the presence of CCR8-positive Tregs was significantly associated with better survival (HR 0.45, p < 0.001) across the entire patient cohort. However, when stratified for TLS-positive tumors, the presence of activated Tregs was associated with diminished objective response rate and progression-free survival suggesting a negative impact of these immunosuppressive cells on response to ICB. Conclusions: This study provides the first evidence linking CCR8-positive Tregs with immunotherapy resistance in NSCLC, particularly in TLS-positive tumors. These findings parallel observations in TLS-positive sarcomas, where Treg abundance predicted poor outcomes (Italiano et al., Nature Medicine , 2022). This study supports the exploration of CCR8-targeted therapies to deplete immunosuppressive Tregs and enhance the efficacy of immunotherapy in TLS-positive NSCLC.

Delineation of immunotherapeutic predictive versus prognostic transcriptional programs to identify SLC22A5-centric carnitine metabolism-driven resistance to anti-PD-L1 treatment in advanced non–small-cell lung cancer.

2623 Background: Prognostic factors indicate the natural course of a disease regardless of treatment, whereas predictive factors determine the likelihood of response to specific therapies. Distinguishing between predictive and prognostic factors is essential for separating treatment-specific outcomes from the inherent progression of cancer, thereby guiding clinical decision-making. We aim to dissect the predictive and prognostic transcriptional programs underlying the efficacy of anti-PD-L1 versus chemotherapy in advanced non-small cell lung cancer (NSCLC) to uncover mechanisms specific to immunotherapy resistance. Methods: Clinical and baseline tumor transcriptomic data were collected from two randomized controlled trials comparing atezolizumab with docetaxel: OAK (n=697, discovery cohort) and POPLAR (n=192, validation cohort). Transcriptional program scores for each biological process and metabolic pathway from the Reactome database were calculated using gene set variation analysis for each patient. Cox regression and P-value for interaction tests were conducted to differentiate predictive versus prognostic effects of transcriptional programs. Tumor microenvironment and cell-cell communication underlying immunotherapy resistance were explored using bulk and single-cell transcriptomic data. Results: Transcriptional programs in the OAK discovery cohort were divided into four categories associated with different predictive effects specific to atezolizumab or docetaxel. Carnitine metabolism was the most prominent process contributing to atezolizumab-specific resistance, while porphyrin metabolism drove docetaxel-specific resistance. SLC22A5, the only high-affinity carnitine transporter, was upregulated in atezolizumab-resistant patients. The predictive effect of SLC22A5-centric carnitine metabolism for resistance to atezolizumab rather than docetaxel was confirmed in the POPLAR validation cohort. Integrative analyses of bulk and single-cell transcriptomes revealed that cancer cell-specific SLC22A5 expression induced M2 macrophage polarization and decreased CD8+ T cell infiltration via carnitine uptake, thus forming an immunosuppressive microenvironment. Conclusions: Our study elucidates the distinction between predictive and prognostic factors in advanced NSCLC from a metabolic perspective. Cancer cells uptake of carnitine via SLC22A5 mediates resistance to anti-PD-L1 treatment. Combining inhibition of SLC22A5-centric carnitine metabolism with anti-PD-L1 agents might be a promising strategy to reverse immune escape in advanced NSCLC. Keywords: Predictive, Prognostic, Non-small cell lung cancer, Carnitine metabolism, Resistance.

Mechanism study of reversing tumor immune desertification and immunotherapy resistance by low dose radiotherapy.

e15181 Background: The immune classification of tumor can be divided into immune infiltrating type, immune rejection type and immune desert type. There is little infiltration of CD8 + T cells in immune desert tumors, which are less responsive to immune checkpoint inhibitor (ICB) therapy. Therefore, it is particularly important to identify effective and feasible combination treatment options for these patients. Methods: To construct an animal model of metastatic non-small cell lung cancer (A549 cells), the experimental group was treated with low dose radiotherapy (LDRT: The expression of local immune cells and their related molecules in the tumor microenvironment was evaluated at different time points after 1Gy) irradiation, and the effect and mechanism of LDRT and reasonable immunotherapy combination in the treatment of immune desert tumors were discussed. Follow-up clinical trials to evaluate the benefits and evidence of low-dose radiotherapy combined with immunotherapy induced advanced non-small cell lung cancer in humans. Results: 1. Low dose radiotherapy (LDRT) induced local immune cell infiltration in a mouse model of locally advanced in-situ non-small cell lung cancer 1Gy irradiation of tumors induces important transcriptional changes in vivo, particularly significant upregulation of inflammatory pathways, including IFN-α and IFN-γ responses, complement activation, IL6/JAK/STAT3 signaling pathways, and attracts the expression of key chemokines of T and NK cells, as well as cross-presented dendritic cells. Low-dose radiation therapy enhanced immunotherapy responsiveness in a mouse model of locally advanced in-situ non-small cell lung cancer Utilizing the pro-inflammatory effects of LDRT, the combination therapy showed a significant therapeutic effect on the initial low-T-cell inflammatory tumors. Low-dose radiation therapy combined with immunotherapy increases the number of local CD4 + and CD8 + T cells in the tumor. All components of the combination therapy help mobilize an effective anti-tumor immune response. Low dose radiotherapy combined with immune checkpoint blocking induced responses to advanced immune desert human tumors In patients with locally advanced non-small cell lung cancer treated with immunotherapy (IT) and low-dose radiotherapy (LDRT: 1Gy), low dose radiotherapy combined with immune checkpoint blocking has been clinically observed to induce responses to advanced immune desert human tumors. Conclusions: This paper demonstrates a novel and important synergy between LDRT and rationally developed combination immunotherapies for the treatment of tumors with poor immunoinfiltration that relies on simultaneous activation of multiple innate and adaptive immune pathways, revealing the interdependence between LDRT and immune regulation.

The dual role of chaperone-mediated autophagy in the response and resistance to cancer immunotherapy.

The dual role of chaperone-mediated autophagy in the response and resistance to cancer immunotherapy.

Efficacy and safety of cadonilimab in recurrent/metastatic solid tumors including ICI-treated tumors in clinical practice: Cohorts of head and neck squamous cell carcinoma and cervical cancer.

e17502 Background: Checkpoint inhibitors (ICIs) has been approved for treating multiple solid tumors, the real-world clinical benefits are under widely discussion. In addition, as more patients failed previous ICIs, the following treatment strategies and effectiveness warrant investigation. Cadonilimab (AK104) is a PD-1/CTLA-4 bispecific antibody, which is expected to exert enhanced anti-tumor activity or reverse immunotherapy resistance after single target ICI. This study focused on the real-world efficacy and safety of cadonilimab in R/M solid tumors, first reported performance of cadonilimab in R/M head and neck squamous cell carcinoma (HNSCC), enriched performance of cadonilimab in R/M cervical cancer (CC), and also summarized the effectiveness of re-treatment with cadonilimab for patients failed previous ICIs. Methods: We reviewed histologically confirmed CC and HNSCC in our center. Those with R/M disease and received ≥ one cycle of cadonilimab were enrolled. Patients received cadonilimab (10mg/kg, Q3W) mono- or combination therapy. The primary endpoints were ORR and PFS (RECIST1.1). Secondary endpoints included DCR, OS, and AE (CTCAE 5.0). Results: As of December 15, 2024, a total of 36 patients (median age: 57 years) were enrolled, including 29 CC and 7 HNSCC patients. The median follow-up time was 12.1 months (9.9-14.3 months). Patients with CC were treated with cadonilimab monotherapy or combined with chemotherapy ± radiotherapy or ± anti-angiogenic therapies. The ORR were 100% (7/7), 60% (9/15), 28.6%(2/7) for 1 st , 2 nd and ≥ 3 rd line therapy, the mPFS were not reached, 6.2 months (95% CI[3.3 9.0] ), and 4.8 months (95% CI[0.3 9.4]), respectively. The mOS was not mature. Patients with HNSCC received cadonilimab plus anti-EGFR therapy (one 1 st line, five 2 nd line, one ≥3 rd line). The overall ORR, DCR and mPFS are 71.4% (5/7), 85.7% (6/7) and 8.0 months (95%CI[3.2-12.9]). The ORR and DCR of 2 nd line treatment were 80% (4/5), mPFS was 8.0 months (95CI[3.954-12.113]). mOS was not mature. Of the 11 patients who received cadonilimab for immunotherapy rechallenging, three achieved PR and six maintained SD, with ORR of 27.3% (3/11), DCR of 81.8%(9/11), and mPFS was 4.7 months (95%CI[0-10.3]). Of all patients, 19.4%(7/36) developed grade 3 AE(4 leukopenia, 1 anemia, 2 rash, and 1 pancreatitis). Conclusions: This real-world study first reported performance of cadonilimab (combined with anti-EGFR therapy) in R/M HNSCC, showing excellent ORR and PFS. In R/M CC, the combination regimen of cadonilimab based on clinical practice improved ORR (COMPASSION-16, ORR 82.9%; COMPASSION-03, ORR 32.3%). The overall safety is manageable. Furthermore, cadonilimab showed promising anti-tumor efficacy and potential to reverse resistance of single target ICI. Analysis with more samples and cancer types will continue.

Gut microbiome and serum cytokines as biomarkers for pathological complete response (pCR) and recurrence free survival in early locally advanced triple negative breast cancer (LA TNBC) receiving neoadjuvant chemotherapy (NA) and immune checkpoint blocker (ICI): Updated analysis from a pilot study.

e12630 Background: Microbial metabolic pathways are known to influence immunotherapy resistance and these may similarly contribute to resistance in LA TNBC treated with NA ICIs. We previously reported differential microbial response predictors of treatment in this population. In this analysis, we are presenting updated data demonstrating differences in the expanded profile of microbiome, metabolites and cytokines. Methods: 35 patients who received NA pembrolizumab and chemotherapy for LA TNBC were identified at Moffitt Cancer Center and Tampa General Hospital between February 2022 to October 2023. Blood and stool samples were collected at initiation and completion of NA therapy and/or when the patients developed grade 3 or higher toxicity to therapy. The goal was to study the gut microbiome, composition and metabolites associated with pCR We conducted shotgun metagenomic sequencing on 59 stool samples, GC-MS sequencing in 59 stool and 56 plasma for metabolites and 25 serum cytokines. Patient characteristics were as follows: 26 Caucasian, 4 African-American, 2 Hispanic, 3 unknown, median age 52 years. 23 patients had stage II, while 12 patients had stage III, 17 patients were node negative while 18 were node positive disease. Results: Response was assessed in 34 patients, of which 12 had pCR (ypT0/is ypN0) while 22 had residual disease (RD), 5 (14%) patients had recurrence. At median follow up of 20.38 months, the percentage survival was 87.3% (95% CI: 69.3-95.1). One patient experienced disease progression while on NA therapy, and 6 developed grade 3-4 immune-related adverse events, resulting in treatment discontinuation, with one patient succumbing to these complications. Butyric acid is a known microbial metabolite associated with immunosuppressive effects. However, its role in TNBC role has not been explored. We report that lower butyric acid is associated with a significantly higher likelihood of pCR in LA TNBC,HR 0.9, p < 0.05. Meanwhile amino acid metabolites tyrosine and phenylalanine are associated with better pCR with HR 3.99 and HR 1.2, p < 0.05 respectively. No difference in alpha diversity at baseline or follow-up comparing patient pCR vs RD. Beta diversity at follow-up was statistically different between patients with pCR vs RD while no difference noted at baseline. Higher levels of CXCL5, CCL2 were noted in patients with RD. Conclusions: To our knowledge this is the first analysis in patients with LA TNBC treated with NA ICB showing differential immunosuppressive microbial metabolites, particularly Short Chain Fatty Acids which are associated with pCR. Biomarkers play a vital role in identifying patients who are most likely to respond to immune checkpoint blockade (ICB). The gut microbiome may act as a predictive biomarker for neoadjuvant ICB response in TNBC, necessitating validation in larger studies.

A novel adenosine 2A receptor antagonist HZ-086 enhances the efficiency of immunotherapy and alleviates the acquired resistance to PD-L1 by restoration of T cell functions.

A novel adenosine 2A receptor antagonist HZ-086 enhances the efficiency of immunotherapy and alleviates the acquired resistance to PD-L1 by restoration of T cell functions.

Ultrasound-triggered drug-loaded nanobubbles for enhanced T cell recruitment in cancer chemoimmunotherapy.

Ultrasound-triggered drug-loaded nanobubbles for enhanced T cell recruitment in cancer chemoimmunotherapy.

Validation and refinement of Society of Immunotherapy of Cancer (SITC) definitions for PD-(L)1 resistance: An analysis of more than 1,300 participants from SWOG.

2656 Background: New immuno-oncology (IO) agents are commonly used in patients who previously received PD-(L)1 inhibitors. In order to facilitate clinical trial interpretation and better delineate therapeutic contributions of novel IO agents, consensus definitions of PD-(L)1 single-agent and combination immunotherapy resistance were published in 2020 (PMC7174063) and 2022 (PMC10016305) by the Society of Immunotherapy of Cancer (SITC); definitions outlined in Table. Validation of these expert-derived definitions is currently lacking. Herein we analyze two SWOG trials to evaluate the proposed definitions for the advanced and adjuvant settings. Methods: S1609/DART (NCT02834013) was a basket trial for patients with rare cancers treated with ipilimumab (1mg/kg intravenously [IV] every 6 weeks) plus nivolumab (240mg IV every 2 weeks). S1404 (NCT02506153) included an arm where patients with high-risk resectable Stage III melanoma received adjuvant pembrolizumab (200mg IV every 3 weeks for 1 year). In both trials, overall survival (OS) was measured from study registration to death from any cause with those last known to be alive censored. OS was evaluated with Kaplan-Meier and martingale residual plots and Cox regression models. Results: In S1609 (advanced setting), 733 participants were analyzed: 127 (17%) were not evaluable for primary resistance due to death or off treatment before 6 weeks. Among the 570 evaluable, 366 met the SITC primary resistance definition and 204 did not. Martingale residuals plots indicated a positive association between time to progression and OS with no evidence of a threshold. With a 6-month landmark, participants with primary resistance had significantly shorter OS compared to those who did not: hazard ratio (HR)=2.84, 95% confidence interval (CI) 2.28-3.55, p<0.001. In S1404 (adjuvant setting), 626 participants were analyzed with 12 (2%) meeting the definition of early recurrence/primary resistance and 138 (22%) meeting the definition of late recurrence/secondary resistance. Using a 12-month landmark, there was no significant difference in OS between early and late recurrences (HR=0.98, 95% CI:0.35-2.70, p=0.25), however late recurrences were associated with significantly shorter OS than no recurrence (HR=7.69, 95% CI:2.71-20.1,p<0.001). Conclusions: In the advanced cancer cohort, the SITC definitions were validated. In the adjuvant cohort, early recurrences were uncommon and there was no significant difference in OS between early and late recurrences suggesting a 12-month cutoff may be more appropriate than 12 weeks. Additional analyses in other patient cohorts are needed to further understand if the SITC definitions for advanced cancers validate more broadly and if data-drive refinements are needed for the adjuvant setting. Advanced Primary Treatment > 6 weeks; no response or < 6 months Secondary Treatment > 6 months; response/stable > 6 months Adjuvant Early/primary < 12 weeks last dose Late/secondary 12 weeks

Molecular Docking, Molecular Dynamics Simulation, and Pharmacophore-Based Virtual Screening Unveil Natural Compounds with TIM-3 Inhibitory Activity

ABSTRACTIntroduction:The aberrant expression of T cell immunoglobulin mucin-3 (TIM-3) has been linked to adaptive immunotherapy resistance and poor prognosis. Targeting TIM-3 has gained popularity due to positive outcomes in preclinical settings.Materials and method:This research explored the Supernatural 3.0 database for potential TIM-3 inhibitors. A pharmacophore-based virtual screening, molecular docking, and molecular dynamics simulations were conducted for 449,008 compounds. Compounds SN0085417 (-7.542 kcal/mol), SN0261906 (-7.036 kcal/mol), and SN0276180 (-6.871 kcal/mol) showed better docking scores. These compounds revealed remarkable hydrogen bonds and π–π stackings interactions with TIM-3 IgV domain.Results:The analysis of root-mean-square deviation (RMSD) and root-meansquare-fluctuation (RMSF) trajectories displayed stable interaction patterns with no substantial conformational changes in the TIM-3 backbone.Conclusion:These findings proposed SN0085417, SN0276180, and SN0261906 as TIM-3 inhibitors, which, with more extensive analysis, could undoubtably add to the realm of feasible TIM-3 based cancer immunotherapies.

Preclinical evaluation of the efficacy of anti-PVR antibody-drug conjugates combined with anti-PD-1 therapy for bladder cancer.

e16547 Background: Poliovirus receptor (PVR) is a ligand for TIGIT and plays a crucial role in tumor immune evasion. We have identified that PVR is highly expressed in bladder cancer (BC), particularly in cases that are resistant to immunotherapy. This study aims to investigate whether antibody-drug conjugate (ADC) targeting PVR can provide both antitumor activity and inhibition of immune escape in BC. Methods: Forty-one BC patients who received anti-PD1 neoadjuvant therapy were analyzed to identify proteins linked to immunotherapy resistance. Briefly, laser-capture microdissection was employed to isolate 3 urothelial and 3 stromal regions from each FFPE sample, enabling high-sensitivity mass spectrometry to spatially define the proteomic profiles in immunotherapy-responsive and unresponsive samples. Expression levels of PVR and common ADC targets were head-to-head compared by IHC in a cohort includes 347 BC samples. A fully humanized anti-PVR monoclonal antibody was conjugated to the toxin MMAE and the linker MC-VC-PAB to produce the potential therapeutic ADC, PVR-MMAE. The antitumor efficacy of PVR-MMAE was assessed in BC cell lines and organoids. Humanized NGS mice were engrafted with human peripheral blood mononuclear cells following by orthotopic xenografted with 5637 cells. Rats given 0.1% BBN in drinking water for 28 weeks produced bladder tumors. Animals were administered either isotype-MMAE, PVR-MMAE (2 mg/kg), or a combination of PVR-MMAE and anti-PD-1twice weekly for 4 weeks intravenously. Finally, the potential toxicity of PVR-MMAE was evaluated by dose escalation in normal rats. Results: PVR was found to be a significantly upregulated tumor-associated membrane antigen in immunotherapy-unresponsive patients. Objective response rates to immunotherapy were 19% in the PVR high group and 90% in the PVR low group. The positive expression rate of PVR in BC was 76%, comparable to HER-2 (74%), Trop-2 (69%), nectin-4 (82%) and EGFR (80%). High PVR expression correlated with advanced stage and poorer prognosis. Functional assays indicated that PVR is not only a tumor-associated antigen but also conferred strong oncogenic effects. PVR-MMAE showed significant antitumor effect in PVR-positive cells and organoids, with an IC50 of 5-10 µg/mL. Tumor growth was significantly inhibited by PVR-MMAE versus isotype-MMAE in the orthotopic xenograft model and BBN-induced BC model. Combined therapy with PVR-MMAE and anti-PD-1 showed superior efficacy over monotherapy. Additionally, intravenous administration of PVR-MMAE was safe within effective therapeutic dose ranges. Conclusions: A de novo anti-PVR monoclonal antibody conjugated with MMAE was developed. Preclinical data on bladder cancer cell lines, organoids and animal models showed the potent antitumor efficiency of anti-PVR antibody-drug conjugate as a single agent or in combination with ICIs in bladder cancer.

An exploratory study to predict the efficacy and prognosis of immunotherapy for extensive-stage small cell lung cancer based on peripheral blood dynamic immune profiles.

2549 Background: The high-dimensional classification information of peripheral blood mononuclear cells can provide abundant efficacy and prognosis-related data. However, in the field of immunotherapy for extensive-stage small cell lung cancer (ES-SCLC), the biomarkers that can predict the efficacy and prognosis need to be explored and clarified. Methods: Cytometry by Time-Of-Flight (CyTOF) was applied to the dynamic monitoring of immunotherapy using clinical resources such as dynamic peripheral blood from ES-SCLC patients. By labeling the following proteins: CD45, CD3, CD4, CD8, CD25, CD127, CD45RA, CD45RO, CCR7, TCRγδ, CD19, CD66b, CD14, CD56, CD16, CD11c, CD123, HLA-DR, CD38, CD57, CXCR3, CCR6, CCR4, CXCR5, CD95/Fas, LAG-3, Tim-3, CTLA-4, PD-L1, PD-1, CD278/ICOS, and TIGIT, this study performed high-dimensional fine-phenotyping of peripheral blood immune cells from ES-SCLC patients. We further explored the dynamic immune profile of peripheral blood that could predict the efficacy and prognosis of immunotherapy in combination with efficacy assessment and survival indicators. Results: 81 dynamic peripheral blood samples (baseline, after two cycles of treatment[C2], and progressive disease) were collected from ES-SCLC patients who received first-line immunotherapy combined with chemotherapy (n = 20) and chemotherapy alone (n = 7) in this study. In the immunotherapy group, a high percentage of senescent CD4+TEM/CD4+TEM at baseline (P = 0.029) was significantly associated with longer PFS. High TIGIT expression at baseline (P = 0.016) was significantly associated with shorter PFS. In addition, PD-1 (CD4+TCM, P = 0.017; Naive CD4+T, P = 0.031; pDCs, P = 0.031; NK, P = 0.007; Early NK, P = 0.007; Late NK, P = 0.02) and TIGIT (CD8+TEM, P = 0.046; NK, P = 0.038) expression levels at baseline in multiple cell subpopulations were significantly negatively correlated with OS. In contrast, the above peripheral blood immune profile was not a predictor in the chemotherapy group. In the immunotherapy group, peripheral blood dynamic monitoring showed that increased γδT cell percentage after treatment was significantly associated with longer PFS and OS (PFS, P = 0.035; OS, P = 0.032). Increased CD4+ TEM and CD4+ TCM percentage after treatment was significantly associated with shorter PFS and OS (CD4+ TEM: PFS, P = 0.021, OS, P = 0.036; CD4+ TCM: PFS, P = 0.01; OS, P = 0.014). Meanwhile, CTLA-4 and ICOS expression in total cells at progressive disease was significantly higher than C2, suggesting that it might be related to immunotherapy resistance. In the chemotherapy group, the above peripheral blood dynamic immune profile did not predict the efficacy and prognosis of chemotherapy. Conclusions: Dynamic peripheral blood immune profile can predict the efficacy and prognosis of immunotherapy in ES-SCLC.

Clinical Investigation of Tyrosinase Inhibitors: Past, Present, and Future.

Tyrosinase (EC 1.14.18.1) is a pivotal enzyme that catalyzes the conversion of L-tyrosine to dopaquinone through a dual oxidation process, initiating melanin biosynthesis. Melanin plays a critical role in various biological processes, and its overproduction is associated with multiple conditions. Tyrosinase plays a crucial role in immune regulation by regulating the activity of immune cells and enhancing the immune response of the body. It is essential for maintaining skin health and preventing autoimmune diseases. In addition, tyrosinase has shown potential in immunotherapy, especially in the treatment of malignant melanoma and autoimmune diseases such as vitiligo. Inhibiting tyrosinase to reduce melanin synthesis has emerged as a promising therapeutic strategy with applications in skin whitening, melasma treatment, acne management, Parkinson's disease (PD) intervention, melanoma prevention, and overcoming immunotherapy resistance. By leveraging the tyrosinase-related comprehensive data documented in the BRENDA database, we have systematically summarized the effective information, including its classification, structural characteristics, catalytic functions, biosynthesis pathways, substrate specificity profiles, reaction products, and associated disease mechanisms, and so forth. This review comprehensively examines the therapeutic mechanisms, development history, and current clinical status of tyrosinase inhibitors at preclinical and advanced stages. We highlight recent research progress, focusing on evidence from animal models, preclinical studies, and human clinical trials across different indications. Additionally, we critically analyze the challenges and limitations in the field and provide insights into future directions for optimizing tyrosinase inhibitors. By synthesizing current knowledge and advancements, this review aims to underscore the therapeutic potential of tyrosinase inhibition and its role in addressing diverse medical needs.

Resistance to anti-PD-1 immunotherapy for stage III and IV melanoma: Results from a global multi-site chart review.

9532 Background: Anti-PD-1 immunotherapy has been approved for the treatment of stage III and IV melanoma. Real-world data on its resistance is needed to facilitate the development of combinatorial approaches to overcome anti-PD-1 resistance. Objectives: To estimate the percentage and understand patient/disease characteristics and survival of those receiving anti-PD1 therapy who experience primary resistance and late relapse in the adjuvant (resectable) setting, and primary, secondary resistance and late progression in the advanced (unresectable/metastatic) setting. Methods: A retrospective chart review was conducted in 22 sites in Australia, France, Germany, South Korea, UK and USA. Adult patients who began anti-PD-1 therapy for stage III or IV melanoma from 1 Jan 2018 until 12 months before the start of data collection were included. SITC Immunotherapy Resistance Taskforce definitions of resistance were used, and late relapse/progression defined if occurring more than 12 weeks after last dose of anti-PD-1. The percentage of patients with primary, secondary resistance or late relapse/progression were calculated. Time to death was analysed using Kaplan-Meier. Univariate tests were used to compare baseline characteristics and survival by type of resistance. Results: Of 981 eligible patients, 738 were included in the full analysis set. In the adjuvant setting (n=240), 53 (22.1%) patients developed primary resistance and 60 (25.0%) experienced late relapse. In the advanced setting (n=498), 222 (44.6%), 50 (10.0%) and 64 (12.9%) patients developed primary, secondary resistance, and late progression, respectively. In the adjuvant setting, a greater proportion of patients with primary resistance (66%) or late relapse (75%) were male than in those with no relapse (52%) (p=0.007). Asian patients experienced less late relapse (0.0%) than White patients (28.2%) but more primary resistance (61.5% vs. 18.4%) (p<0.001). In the advanced setting, 48.6% of Asian patients developed primary resistance, 37.8% secondary resistance, 5.4% late progression vs. 40.8%, 8.8% and 12.7% of White patients (p<0.001). No significant difference was observed in age, BMI, disease severity, Charlson index score and comorbid conditions by type of resistance. In both settings, time to death varied significantly by type of resistance (p<0.001). Patients with primary resistance had the poorest survival: a mean of 42 months in the adjuvant setting and 31 months in the advanced setting (39 months in those with secondary resistance). Conclusions: A large proportion of patients developed resistance or late relapse/progression and require alternative therapy after anti-PD-1, highlighting a substantial unmet medical need. A greater proportion of Asian patients developed resistance compared to White patients, possibly due to differences in melanoma subtype. Patients with primary resistance had the poorest survival.

Safety, efficacy, and tumor immune microenvironment changes with neoadjuvant chemotherapy and cemiplimab with or without alirocumab in stage 1B-3A non-small cell lung cancer.

TPS8118 Background: The addition of immune checkpoint blockade to neoadjuvant and adjuvant therapy is now standard of care in early-stage surgically resectable non-small cell lung cancer (NSCLC). However, resistance to immunotherapy limit their benefit for most patients. The pathological complete response (pCR) rate, a surrogate for long term survival, remains close to 20%, leaving many patients at a high risk of recurrence and death. Thus, there is a need to apply strategies to overcome immunotherapy resistance in earlier stages of NSCLC to improve cure. Proprotein convertase subtilisin/kexin type 9 (PCSK9), a major cholesterol regulator, has emerged as an inhibitory modulator of anti-tumor immunity. Preclinical evidence showed that PCSK9 downregulated MHC class I antigen expression on tumor cells. This effect was reversed by genetic or pharmacologic inhibition of PCSK9. PCSK9 inhibitor synergized with immune checkpoint blockade to increase cytotoxic T-cell mediated tumor death. Retrospective clinical analyses of NSCLC patients treated with immune checkpoint inhibitors also showed a correlation between higher PCSK9 levels and poorer survival. Methods: TOP 2301 is a multi-center, open label, two-arm, randomized, phase 2 trial of chemotherapy and cemiplimab (350mg IV every 3 weeks) with or without the PCSK-9 inhibitor, alirocumab (150 mg SC every 4 weeks), prior to surgery. Eligible patients will have stage IB-3A NSCLC, deemed surgical candidates, and have no EGFR or ALK mutations. One hundred and twenty-six patients will be randomized 1:1 to receive neoadjuvant SOC chemotherapy and cemiplimab versus SOC chemotherapy, cemiplimab and alirocumab. Approximately 64 participants are required in each arm to have 90% power to reject the null hypothesis. The primary objective is to compare the pCR rates for neoadjuvant chemotherapy plus cemiplimab versus chemotherapy, cemiplimab, and alirocumab. Secondary efficacy objectives for the experimental arm include: the objective response rate (ORR), disease free survival (DFS), and overall survival (OS). A secondary safety objective is to determine the safety and tolerability of neoadjuvant chemotherapy and cemiplimab with alirocumab in early-stage NSCLC. The correlative science objective will evaluate the difference in tumor infiltrating lymphocytes and dendritic cells through IHC, FACs analysis, and bulk RNA-seq with CIBERSORT from postsurgical specimens of patients treated with neoadjuvant chemotherapy and cemiplimab with or without alirocumab. The trial was open to enrollment on 12/15/2024. Clinical trial information: NCT06385262 .