Efficacy Of Immune Checkpoint Blockade Research Articles

Anti-4-1BB×PD-L1 Bispecific Antibody Reinvigorates Tumor-Specific Exhausted CD8+ T Cells and Enhances the Efficacy of Anti-PD-1 Blockade.

To overcome the limited efficacy of immune checkpoint blockade, there is a need to find novel cancer immunotherapeutic strategies for the optimal treatment of cancer. The novel anti-4-1BB×PD-L1 bispecific antibody-ABL503 (also known as TJ-L14B)-was designed to simultaneously target PD-L1 and 4-1BB, and demonstrated strong antitumor T-cell responses without considerable toxicity. Here, we investigated how the combination of ABL503 and anti-PD-1 blockade affected the reinvigoration of exhausted tumor-infiltrating CD8+ T cells (CD8+ TILs) and anti-tumor efficacy. Single cell suspensions of hepatocellular carcinoma and ovarian cancer from treatment-naive patients were used for immunophenotyping of CD8+ TILs and in vitro functional assays. Humanized hPD-1/hPD-L1/h4-1BB triple knock-in mice were used to evaluate the effects of ABL503 and anti-PD-1 blockade in vivo. We observed that ABL503 successfully restored the functions of 4-1BB+ exhausted CD8+ TILs, which were enriched for tumor-specific T cells but unresponsive to anti-PD-1 blockade. Importantly, compared to anti-PD-1 blockade alone, the combination of ABL503 and anti-PD-1 blockade further enhanced the functional restoration of human CD8+ TILs in vitro. Consistently, the combination of ABL503 with anti-PD-1 in vivo significantly alleviated tumor growth, and induced enhanced infiltration and activation of CD8+ TILs. ABL503-a PD-L1 and 4-1BB dual-targeting bispecific antibody-elicits pronounced additive tumor growth inhibition, with increased infiltration and functionality of exhausted CD8+ T cells, which in turn enhances the anti-cancer effects of anti-PD-1 blockade. These promising findings suggest that ABL503 (TJ-L14B) in combination with PD-1 inhibitors will likely further enhance therapeutic benefit in clinical trials.

Abstract PO3-06-09: A phase II study of nivolumab plus ipilimumab and androgen receptor antagonist bicalutamide to stimulate thymic T cell generation in HER2-negative metastatic breast cancer

Abstract Background: Systemic chemotherapy is used commonly in breast cancer and is associated with lymphopenia, potentially limiting efficacy of immune checkpoint blockade. The androgen receptor (AR) serves as a negative regulator of thymic function. AR antagonists, such as bicalutamide, may stimulate thymic production of naïve T-cells, which may help to restore lymphocyte diversity and augment immunotherapy response. The AR is expressed in 50% of hormone receptor negative (HR-) metastatic breast cancer (MBC) and >75% of HR+ MBC, and bicalutamide is clinically active with stable disease as best response. We hypothesize that bicalutamide could be combined with dual immune checkpoint blockade (anti-programmed death 1, nivolumab; plus anti-cytotoxic T-lymphocyte antigen 4, ipilimumab), stimulating T-cell production and resulting in durable clinical response. Methods: This is a phase II Simon 2-stage trial of nivolumab (240 mg IV q2w), ipilimumab (1 mg/kg IV q6w), and bicalutamide (150mg oral daily) for first- or second-line treatment of HER2-negative MBC (NCT03650894). The primary endpoint is iRECIST 24-week clinical benefit rate (CBR), evaluated separately for HR+ MBC (n=15) and AR+/HR- MBC (n=15). An optimum Simon 2-stage design (80% power, 5% one-sided alpha) is employed to evaluate an alternative hypothesis of >50% CBR compared to historical control of < 30% CBR for conventional chemotherapy, with expansion of each cohort planned if ≥6/15 responses are observed. Secondary outcomes include best overall objective response rate (ORR), progression-free and overall survival (PFS,OS), and safety. Exploratory endpoints include assessment of peripheral T-cell counts and T cell receptor excision circles (TREC PCR, Mayo Laboratories, a surrogate of thymic T-cell production) over time. Results: Durable responses were observed, with an ongoing complete response at 41+ months in a patient who discontinued due to toxicity (AR+/HR- cohort, PD-L1 CPS score 3%). Therapy was tolerated with a toxicity profile consistent with prior studies of ipilimumab & nivolumab, with five subjects (19%) requiring treatment discontinuation related to toxicity. Outcomes by cohort and PDL1 are summarized in Table 1. Using mixed effects linear models, therapy was associated with a significant expansion of CD8+ T cells (23/mcL/month, p< .01) but not CD3+ T cells (7/mcL/month, p=.59) or CD4+ T cells (4/mcL/month, p=.65). Baseline TREC counts were higher in younger participants (mean: < 50y 2156/mcL; 50-65y 961/mcL; >65y 311/mcL). TRECs did not increase globally, however TREC expansions were observed in several younger participants. Conclusions: The regimen of ipilimumab, nivolumab, plus bicalutamide is safe and clinically active in 1st/2nd-line HER2-negative MBC. Neither cohort crossed the Simon futility barrier for cohort expansion, however durable responses were observed including in PD-L1-negative patients, highlighting the unmet need for biomarkers to identify candidates for chemotherapy-sparing checkpoint blockade. Further research is indicated to evaluate the impact of AR antagonists on T-cell function and thymic stimulation in MBC. Acknowledgements: Drug and study support was provided by Bristol-Myers Squibb and The BMS International Immuno-Oncology Network (II-ON). Clinical outcomes XX XX HR: hormone receptor; AR: androgen receptor; CPS: PD-L1 combined positive score Citation Format: David Page, Alexander Hall, Krystle Collins, Nicole Moxon, Staci Mellinger, Tracy Kelly, Aysha Kelley, Nicole Fredrich, Amanda Seino, Gabriella Liberatore, Alison Conlin, Zheng Topp, Kelly Perlewitz, Sasha Stanton, Walter Urba, Yaping Wu, Maritza Martel, Zhaoyu Sun, Yoshinobu Kogushi, William Redmond, Tiffany Traina, Ayca Guculp. A phase II study of nivolumab plus ipilimumab and androgen receptor antagonist bicalutamide to stimulate thymic T cell generation in HER2-negative metastatic breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-06-09.

Abstract RF01-06: Efficacy and safety of toripalimab plus metronomic chemotherapy in HER2 negative metastatic breast cancer: a multicenter phase II trial based on a Bayesian adaptive randomized design

Abstract Background: Combining Immune checkpoint blockade (ICB) with chemotherapy may significantly improve efficacy in patients with breast cancer. Metronomic chemotherapy is based on more frequent and low-dose drug administrations compared with conventional chemotherapy. Some clinical studies with small samples have tried the combination of metronomic chemotherapy and ICB in patients with advanced tumors. However, whether metronomic chemotherapy is more suitable for ICB than conventional chemotherapy is still unclear. Thus, we performed the first clinical trial, to our knowledge, of metronomic chemotherapy combined with PD-1 blockade comparing with the efficacy of combined conventional chemotherapy and ICB. Methods: This study is a multicenter randomized phase 2 trial using a multi-arm design with Bayesian adaptive randomization and efficacy monitoring. Eligible patients have advanced HER2 negative breast cancer, with no more than one prior line of standard chemotherapy. Patients were randomized to 5 groups: ① metronomic vinorelbine (NVB, 40 mg/day, TIW) monotherapy (the control cohort); ② NVB + Toripalimab (anti-PD1 antibody, 240 mg Q3W); ③Bevacizumab (5 mg/kg Q3W) + NVB+ Toripalimab (the BEV cohort); ④ Cisplatin (50mg/m Q3W) + NVB + Toripalimab (the DDP cohort), ⑤Cyclophosphamide (50mg/day, QD) + Capecitabine (500 mg, TID) + NVB+ Toripalimab (the VEX cohort). The primary endpoint is disease control rate (DCR), and secondary endpoints are objective response rate (ORR), progression free survival (PFS), and overall survival (OS). The safety profile has also been assessed. Mass cytometry time-of-flight (CyTOF) analyses of paired blood samples were performed to demonstrate dynamic changes in systemic immune profile. Results: A total of 103 patients were randomized. Characteristics were as expected for advanced breast cancer and balanced between cohorts. Adverse events of any grade occurred in 64 patients, with 5.2% grade >3. The rate of nausea was significantly higher in the cisplatin cohort than in the others (P< 0.001). Toripalimab was not associated with any previously unreported toxicity. Among the five treatment cohorts, the VEX cohort and the cisplatin cohort had the highest DCR, 69.7% (95% CI 51.7–85.9%) and 73.7% (95% CI 56.1–88.7%), respectively. Followed by the bevacizumab cohort, DCR is 55.7% (95% CI 37.4%–73.6%). Most objective responses were observed in the VEX cohort (ORR: 18.5%; 95% CI 5.6–34.1%), followed by cisplatin (ORR 14.5%; 95% CI 3.3–29.0%). It is worth noting that the PFS of patients in the VEX cohort was the longest, reaching 6.6 months (95% CI 4.0-5.9), followed by 4.0 months (95% CI 2.4-10.4) in patients of the bevacizumab cohort. The PFS of patients receiving the cisplatin regimen was relatively short, only 3.5 months (95% CI 2.2-5.3). Similarly, patients in the cisplatin cohort had shorter OS than those in the VEX and bevacizumab cohorts. In the TNBC subgroup, again, patients in the VEX cohort had the highest DCR (74.1%, 95%CI 47.9%-95.4%) and longest PFS (9.8 months, 95%CI 3.8-21.9). We obtained over 63 million cells in total and clustered CD45+ immune cells into 32 clusters. The systemic immune profile of patients changed dramatically over 2 cycles of PD-1 blockade. However, only the change of cluster 30 differed between responders and non-responders. This is a group of intermediate monocytes with a high expression of CD38, which decreased in responders but significantly increased in non-responders. CD38-NAD+ axis regulates the PD1+ exhausted T cell function. Meanwhile, the overall expression of CD38 in monocytes was significantly increased by DDP and BEV treatment compared with baseline, but not in the VEX groups. DDP significantly increased the expression level of CD38 on NK cells, while BEV or VEX treatment did not affect it. Conclusions: These data suggest promising clinical efficacy and evidence of cooperativity between metronomic VEX chemotherapy and PD-1 blockade. Citation Format: Hongnan Mo, Xiaoying Sun, Jingtong Zhai, Jiashu Han, Hewei Ge, Yuhan Wei, Xiuwen Guan, Haili Qian, Fei Ma. Efficacy and safety of toripalimab plus metronomic chemotherapy in HER2 negative metastatic breast cancer: a multicenter phase II trial based on a Bayesian adaptive randomized design [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr RF01-06.

High-Grade Pleomorphic Sarcomas Treated with Immune Checkpoint Blockade: The MD Anderson Cancer Center Experience.

Undifferentiated pleomorphic sarcomas (UPSs) are amongst the most common subtypes of soft-tissue sarcomas. Few real-world data on the use of immune checkpoint blockade (ICB) in UPS patients and other high-grade pleomorphic STS patients are available. The purpose of our study is to describe the efficacy and toxicity of ICB in patients with advanced UPSs and other high-grade pleomorphic sarcomas treated at our institution. This is a retrospective, observational study of all patients with metastatic high-grade pleomorphic sarcomas treated with FDA-approved ICB at MD Anderson Cancer Center between 1 January 2015 and 1 January 2023. Patients included in trials for which results are not yet published were excluded. Thirty-six patients with advanced/metastatic pleomorphic sarcomas were included. The median age was 52 years. A total of 26 patients (72%) had UPSs and 10 patients (28%) had other high-grade pleomorphic sarcomas. The median follow-up time was 8.8 months. The median PFS was 2.9 months. The 3-month PFS and 6-month PFS were 46% and 32%, respectively. The median OS was 12.9 months. The 12-month OS and 24-month OS were 53% and 29%, respectively. The best response, previous RT, and type of ICB treatment were significantly and independently associated with shorter PFS (p = 0.0012, p = 0.0019 and p = 0.036, respectively). No new safety signal was identified, and the toxicity was overall manageable with no toxic deaths and only four patients (11%) stopping treatment due to toxicity. Real-world retrospective data are consistent with the published literature, with a promising 6-month PFS of 32%. Partial or stable responders to ICB treatment have significantly improved PFS compared to progressors.

Circadian tumor infiltration and function of CD8+ Tcells dictate immunotherapy efficacy.

The quality and quantity of tumor-infiltrating lymphocytes, particularly CD8+ Tcells, are important parameters for the control of tumor growth and response to immunotherapy. Here, we show in murine and human cancers that these parameters exhibit circadian oscillations, driven by both the endogenous circadian clock of leukocytes and rhythmic leukocyte infiltration, which depends on the circadian clock of endothelial cells in the tumor microenvironment. To harness these rhythms therapeutically, we demonstrate that efficacy of chimeric antigen receptor Tcell therapy and immune checkpoint blockade can be improved by adjusting the time of treatment during the day. Furthermore, time-of-day-dependent Tcell signatures in murine tumor models predict overall survival in patients with melanoma and correlate with response to anti-PD-1 therapy. Our data demonstrate the functional significance of circadian dynamics in the tumor microenvironment and suggest the importance of leveraging these features for improving future clinical trial design and patient care.

IL1R2 blockade alleviates immunosuppression and potentiates anti-PD-1 efficacy in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with limited therapeutic options. Interleukin-1 receptor type 2 (IL1R2) promotes breast tumor-initiating cell (BTIC) self-renewal and tumor growth in TNBC, indicating that targeting it could improve patient treatment. Here, we observed that IL1R2 blockade strongly attenuated macrophage recruitment and the polarization of tumor-associated macrophages (TAMs) to inhibit BTIC self-renewal and CD8+ T cell exhaustion, which resulted in reduced tumor burden and prolonged survival in TNBC mouse models. IL1R2 activation by TAM-derived IL1β increased PD-L1 expression by interacting with the transcription factor yin yang 1 (YY1) and inducing YY1 ubiquitination and proteasomal degradation in both TAMs and TNBC cells. Loss of YY1 alleviated the transcriptional repression of c-Fos, which is a transcriptional activator of PD-L1. Combined treatment with an IL1R2-neutralizing antibody and anti-PD-1 led to enhanced anti-tumor efficacy and reduced TAMs, BTICs, and exhausted CD8+ T cells. These results suggest that IL1R2 blockade might be a strategy to potentiate immune checkpoint blockade efficacy in TNBC to improve patient outcomes.

CRISPR-Cas9 screening identifies KRAS-induced COX-2 as a driver of immunotherapy resistance in lung cancer.

Oncogenic KRAS impairs anti-tumor immune responses. As effective strategies to combine KRAS inhibitors and immunotherapies have so far proven elusive, a better understanding of how oncogenic KRAS drives immune evasion is needed to identify approaches that could sensitize KRAS-mutant lung cancer to immunotherapy. In vivo CRISPR-Cas9 screening in an immunogenic murine lung cancer model identified mechanisms by which oncogenic KRAS promotes immune evasion, most notably via upregulation of immunosuppressive cyclooxygenase-2 (COX-2) in cancer cells. Oncogenic KRAS potently induced COX-2 in both mouse and human lung cancer, which was suppressed using KRAS inhibitors. COX-2 acted via prostaglandin E2 (PGE2) to promote resistance to immune checkpoint blockade (ICB) in lung adenocarcinoma. Targeting COX-2/PGE2 remodeled the tumor microenvironment by inducing pro-inflammatory polarization of myeloid cells and influx of activated cytotoxic CD8+ T cells, which increased the efficacy of ICB. Restoration of COX-2 expression contributed to tumor relapse after prolonged KRAS inhibition. These results provide the rationale for testing COX-2/PGE2 pathway inhibitors in combination with KRASG12C inhibition or ICB in patients with KRAS-mutant lung cancer.

Inhibition of autophagy-related protein 7 enhances anti-tumor immune response and improves efficacy of immune checkpoint blockade in microsatellite instability colorectal cancer

BackgroundThe efficacy of anti-PD-1 therapy is primarily hindered by the limited T-cell immune response rate and immune evasion capacity of tumor cells. Autophagy-related protein 7 (ATG7) plays an important role in autophagy and it has been linked to cancer. However, the role of ATG7 in the effect of immune checkpoint blockade (ICB) treatment on high microsatellite instability (MSI-H)/mismatch repair deficiency (dMMR) CRC is still poorly understood.MethodsIn this study, patients from the cancer genome altas (TCGA) COAD/READ cohorts were used to investigate the biological mechanism driving ATG7 development. Several assays were conducted including the colony formation, cell viability, qRT-PCR, western blot, immunofluorescence, flow cytometry, ELISA, immunohistochemistry staining and in vivo tumorigenicity tests.ResultsWe found that ATG7 plays a crucial role in MSI-H CRC. Its knockdown decreased tumor growth and caused an infiltration of CD8+ T effector cells in vivo. ATG7 inhibition restored surface major histocompatibility complex I (MHC-I) levels, causing improved antigen presentation and anti-tumor T cell response by activating reactive oxygen species (ROS)/NF-κB pathway. Meanwhile, ATG7 inhibition also suppressed cholesterol accumulation and augmentation of anti-tumor immune responses. Combining ATG7 inhibition and statins improved the therapeutic benefit of anti-PD-1 in MSI-H CRC. Importantly, CRC patients with high expression of both ATG7 and recombinant 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) experienced worse prognosis compared to those with low ATG7 and HMGCR expression.ConclusionsInhibition of ATG7 leads to upregulation of MHC-I expression, augments immune response and suppresses cholesterol accumulation. These findings demonstrate that ATG7 inhibition has therapeutic potential and application of statins can increase the sensitivity to immune checkpoint inhibitors.

D-mannose targets PD-1 to lysosomal degradation and enhances T cell-mediated anti-tumor immunity

High expression of programmed cell death protein 1 (PD-1), a typical immune checkpoint, results in dysfunction of T cells in tumor microenvironment. Antibodies and inhibitors against PD-1 or its ligand (PD-L1) have been widely used in various malignant tumors. However, the mechanisms by which PD-1 is regulated are not fully understood. Here, we report a mechanism of PD-1 degradation triggered by d-mannose and the universality of this mechanism in anti-tumor immunity. We show that d-mannose inactivates GSK3β via promoting phosphorylation of GSK3β at Ser9, thereby leading to TFE3 translocation to nucleus and subsequent PD-1 proteolysis induced by enhanced lysosome biogenesis. Notably, combination of d-mannose and PD-1 blockade exhibits remarkable tumor growth suppression attributed to elevated cytotoxicity activity of T cells in vivo. Furthermore, d-mannose treatment dramatically improves the therapeutic efficacy of MEK inhibitor (MEKi) trametinib in vivo. Our findings unveil a universally unrecognized anti-tumor mechanism of d-mannose by destabilizing PD-1 and provide strategies to enhance the efficacy of both immune checkpoint blockade (ICB) and MEKi -based therapies.

Efficient CAR T cell targeting of the CA125 extracellular repeat domain of MUC16

BackgroundOvarian cancer (OC) is the leading cause of death from gynecologic malignancies in the Western world. Contributing factors include a high frequency of late-stage diagnosis, the development of chemoresistance, and...

Ketogenic Diet Alters the Epigenetic and Immune Landscape of Prostate Cancer to Overcome Resistance to Immune Checkpoint Blockade Therapy.

Optimized cyclic ketogenic diet and 1,3-butanediol supplementation regimens enhance the efficacy of immune checkpoint blockade in prostate cancer through epigenetic and immune modulations, providing dietary interventions to sensitize tumors to immunotherapy.

233 Evolutionary Analysis Identifies Early Responsive Macrophage Representing as Anti-tumor Myeloid Cell in Glioblastoma

INTRODUCTION: Glioblastoma (GBM) has no effective treatments. Although immunotherapy has shown promise in certain cancer types, it has not been effective against GBM, largely due to its highly immunosuppressive tumor microenvironment (TMEs), which is rich in tumor-associated macrophages/microglia (TAMs). TAMs in late-stage GBM contribute to T-cell exhaustion and worsen prognosis, but the role of TAMs in earlier stages of tumor development is unclear. METHODS: By employing genetically engineered mouse models and human samples, we used spatiotemporal single-cell transcriptomics to investigate TAM evolution during GBM progression. RESULTS: We identified a previously unknown macrophage state, termed early responsive macrophage (ERM), that exhibited antitumoral activity. ERMs were found to be abundant in the early stages of GBM progression and spatially enriched in the invading edges of GBMs. ERMs are characterized by upregulated immune response signatures, particularly pathways related to antigen processing and presentation, as well as genes associated with phagocytosis and chemotaxis. Mechanistically, we identified QKI, a transcriptional co-activator, as a key player that activates antigen processing and presentation and MHCII expression in ERMs. Loss of QKI in TAMs is associated with the transformation of ERMs into exhausted tumor-infiltrating macrophages (TIMs), which promote an immune-suppressive TME. Therapeutically, enhancement of QKI's transcriptional co-factor activity greatly improved the efficacy of immune checkpoint blockade. CONCLUSIONS: Our study sheds light on the evolutionary trajectory of TAMs in GBM and presents a novel therapeutic approach to enhance their antitumoral activity, thereby constraining tumor growth.

Type III interferon inhibits bladder cancer progression by reprogramming macrophage-mediated phagocytosis and orchestrating effective immune responses

BackgroundInterferons (IFNs) are essential for activating an effective immune response and play a central role in immunotherapy-mediated immune cell reactivation for tumor regression. Type III IFN (λ), related to type...

Having the cake and eating it? Clofazimine boosts immunotherapy while limiting side effects

Combined immune checkpoint blockade (ICB) for cancer exhibits good efficacy in a subset of patients but also associates with immune-related adverse events. Xue etal. use an elegant drug screening strategy to identify the antimicrobial drug clofazimine as an agent that both potentiates ICB efficacy and decreases immune-related adverse events.

Abstract 1423: Harnessing the gut microbiome to modulate immune checkpoint blockade response in triple-negative breast cancer

Abstract Triple-negative breast cancer (TNBC) is one of the most aggressive subtypes of breast cancer. The discovery that this subtype has the highest levels of tumor-infiltrating immune cells and programmed death ligand 1 (PD-L1) expression in the tumor microenvironment (TME) has propelled the use of anti-PD-1 immune checkpoint blockade (ICB) therapy, in combination with chemotherapy, as a treatment for TNBC patients. This has advantageously impacted TNBC patient outcomes such as survival, yet there remains a need to improve patient response to ICB therapies. The gut microbiome, in particular, increased abundance of Akkermansia muciniphila was associated with ICB therapy response in other cancer types. A. muciniphila can degrade mucins and produce short-chain fatty acid (SCFA) metabolites. Therefore, we investigated the interplay between A. muciniphila, SCFAs, and ICB therapy efficacy in TNBC models. As diet is the main modifier of the gut microbiome, we first investigated diet-gut microbiome interactions on ICB efficacy. Using the EO771 syngeneic murine model of TNBC, female C57BL/6 tumor-bearing mice consuming a low-fat control, or high-fat Western, or Mediterranean diet, were treated with 3 doses of 200µg of IgG or anti-PD-L1 antibodies and tumor progression was monitored. To assess changes in tumoral immune cell populations, end-of-study tumor tissue was used for immunohistochemistry (IHC). To evaluate modulation on the gut microbiome, metagenomic sequencing was performed on DNA isolated from fecal samples collected at the end-of-study, and targeted SCFA metabolomic analysis was performed on plasma collected at the study endpoint. Results show mice consuming a Mediterranean diet treated with anti-PD-L1 had significant increases in the abundance of gut A. muciniphila and plasma SCFAs, as well as significantly reduced primary tumor volume and increased immune cell activity within the TME. To then investigate if direct supplementation of A. muciniphila or SCFAs could enhance ICB efficacy, EO771 tumor-bearing mice were treated with 3 doses of 200µg of IgG, anti-PD-L1 or anti-PD-1 antibodies with a subset of mice stratified to also receive A. muciniphila or exogenoussupplementation of SCFAs. Tumor progression was measured and changes in tumoral immune cell populations were assessed with end-of-study tumor tissue prepared for flow cytometry and IHC. Results show mice supplemented with A. muciniphila and treated with anti-PD-L1 therapy demonstrated response to treatment, with a significant reduction in tumor volume as well as increased levels of immune cell populations in the TME. Mice supplemented with SCFAs and treated with a combination of anti-PD-1 and chemotherapy demonstrated enhanced response to treatment, with a significant reduction in tumor volume. In conclusion, data from our lab suggest that increased levels of gut A. muciniphila and SCFAs drive ICB efficacy in TNBC. Citation Format: Kenysha YJ. Clear, Adam S. Wilson, Elizabeth R. Stirling, Yu-Ting Tsai, Valerie Payne, David R. Soto-Pantoja, Katherine L. Cook. Harnessing the gut microbiome to modulate immune checkpoint blockade response in triple-negative breast cancer [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 1423.

Abstract 1360: TIGIT blockade combined with local radiotherapy and PD-1 blockade in a murine triple-negative breast cancer model

Abstract Background: Immune checkpoint blockades (ICB) improve outcomes of patients with some solid tumors such has melanoma and lung cancer, however the efficacy of ICB alone showed limited efficacy in many other tumors such as breast cancer. Radiation therapy is a promising combination partner of ICB as a strong immune stimulator so called in situ tumor vaccine, however it also can increase immune suppressive repertoires. TIGIT (T cell Immunoglobulin and ITIM domain) is an inhibitory receptor expressed on activated T cells and NK cells. We evaluated the effects of TIGIT blockade in addition to local RT and PD-1 blockade as a strategy to overcome therapeutic resistance of ICI in a murine breast cancer model. Materials and Methods: 4T1-Luc tumors were treated with various strategies including control, RT, anti-PD-1, anti-TIGIT, RT+anti-PD-1, RT+anti-TIGIT, anti-PD-1+anti-TIGIT, and RT+anti-PD-1+anti-TIGIT. A total of 24 Gy in 3 fractions at 1 week was delivered to the tumor at hindlimb (primary tumor) while the tumor at flank (secondary tumor) was left unirradiated. 10 mg/kg of anti-PD-1 blocking antibodies and 10 mg/kg of anti-TIGIT blocking antibodies were intraperitoneally injected for 6 times for 2 weeks. Flow cytometry, IHC staining, and ELISA were performed to assess the immunologic status after treatments. Results: The triple combination therapy (TCT) group showed the most superior growth delay of primary and secondary tumor growth among treatments. The number of metastatic lung nodule was significantly reduced by TCT as well. Plasma levels of interferon-β and interferon-γ were the highest after TCT. Moreover, TCT significantly increased the proportion of splenic CD8+ dendritic cells among myeloid cells, and effector-memory (CD44+CD62L−) cells among splenic Foxp3− non-regulatory CD4+ and CD8+ T cells. Furthermore, expression of CD226, which is an activating counter-receptor of TIGIT, on splenic CD8+ T cells was elevated by TIGIT blockade, possibly suggesting the activation of systemic immune response by addition of TIGIT blockade to local RT and PD-1 blockade. Meanwhile, TCT decreased splenic Foxp3+ regulatory T cells, as well as the expression of CD39 on splenic Foxp3+ regulatory T cells. The increase of CD8+ T cell infiltration in primary and secondary tumors was most prominent in TCT group. Conclusion: TIGIT blockade elicits systemic immune responses in a murine triple-negative breast cancer model. when combined with local RT and PD-1 blockade, which were correlated with significant delay in the growth of both irradiated and unirradiated tumors. These results suggest that TIGIT blockade could be a viable approach to increase the efficacy of RT and immune checkpoint inhibitors in breast cancer which is a relatively immunologically cold tumor. (Work supported by a grant from National Research Foundation of Korea #2023R1A2C3003782 to In Ah Kim). Citation Format: Seongmin Kim, Seung Hyuck Jeon, In Ah Kim. TIGIT blockade combined with local radiotherapy and PD-1 blockade in a murine triple-negative breast cancer model [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 1360.

Abstract 7646: Evaluation of tumor immune microenvironment after neoadjuvant immunotherapy in patients with esophageal squamous cell carcinoma (ESCC): Towards a prediction for recurrence risk

Abstract Background: Despite the promising potential of neoadjuvant immune checkpoint blockade (ICB) in ESCC, postoperative recurrence remains a critical challenge. Besides pathological response, there remains no biomarkers for recurrence risk prediction after neoadjuvant ICB. Recent studies have highlighted a strong association between ICB efficacy and tumor immune microenvironment (TIME). Thus, we sought to investigate the TIME features in surgically resected tumors following neoadjuvant ICB from patients with ESCC to develop a potential way for recurrence risk prediction. Methods: This is a side study of a phase 2 NICE trial (ChiCTR1900026240) of 51 patients with resectable locally advanced ESCC who received neoadjuvant ICB plus chemotherapy followed by surgery from 2019 to 2020. TIME features of surgically resected tumors after neoadjuvant therapy were assessed by multiplex immunohistochemistry (mIHC) for immune markers (CD8, PD-L1, CD45RO, PD-1, Pan-CK, CD4, FoxP3, CD68, CD163). We first analyzed TIME features in tumors evaluated as pathological complete response (pCR) and major pathological response (MPR). Then, we divided 51 patients into a training and validation set; and machine learning was applied to identify a list of TIME features that predict recurrence-free survival (RFS) and to develop a TIME-based signature (TIS) on neoadjuvant ICB prognosis. Results: Fifty-one patients from NICE trial were used for primary analysis. A higher positive rate of CD8+ cells, CD163−CD68+ macrophages and CD8+CD45RO+ cells was observed in patients with pCR. Similarly, MPR patients exhibited a higher positive rate of CD8+ cells and CD8+CD45RO+ cells. Regarding RFS prediction, machine learning selection of TIS were identified: CD8+PD1−(cytotoxic T cells), CD4+Foxp3+ (Treg), CD8+CD45RO+ (memory T cells), and M1 macrophages (CD163−CD68+) exhibited significant predictive value. Stratifying patients into low- and high-risk groups using TIS for RFS revealed a prolonged prolonged RFS in the low-risk group (p<0.001; HR: 0.08, 95% CI [0.03-0.25]). Notably, when applying TIS to pCR patients, we identified a subset of high-risk pCR patients (median RFS = 8.9 months, 1- year RFS=32%) relapsed earlier than low-risk pCR patients (median RFS = not reached, 1-year RFS=100%, p<0.001). Multivariable Cox regression analysis demonstrated that TIS was an independent predictor for RFS as well as overall survival (both p<0.001). Further independent validation (ICB and non-ICB cohort) of TIS is ongoing. Conclusion: The establishment of a risk prediction model based on TIME demonstrated the potential to enhance postoperative monitoring management for ESCC patients who underwent surgery after neoadjuvant ICB. Our study provides insights into development of optimal post-immunotherapy management strategy. Citation Format: Zhichao Liu, Boyao Yu, Yuxin Yang, Cong Qi, Chang Yuan, Yang Yang, Jinzhi Wei, Sidong Chen, Yang Wang, Zhigang Li. Evaluation of tumor immune microenvironment after neoadjuvant immunotherapy in patients with esophageal squamous cell carcinoma (ESCC): Towards a prediction for recurrence risk [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 7646.

Abstract 3972: Personalized immune-organoid co-culture system for modeling the anti-tumor immune response in breast cancer patients

Abstract Recent studies have reported the effectiveness of immune checkpoint blockade (ICB) therapy in breast cancer patients. However, only a subset of patients benefits from ICB therapy due to various immunosuppressive mechanisms. The development of personalized predictive models for the anti-tumor immune response is essential to broaden the indication of ICB therapy. Here, we suggest a novel model platform using a co-culture system with patient tumor-derived organoids and matched immune cells to predict the immune response of individual patients. We successfully established fifty-nine cases of patient-derived breast cancer organoids with matched peripheral blood mononuclear cells (PBMCs) (subtype: Luminal A, 18.6%; Luminal B, 28.8%; Triple-negative breast cancer, 35.6%; HER2-positive, 17.0%). Three rapidly proliferating organoids (one Luminal B and two TNBC organoids) were selected to develop an immune-organoid co-culture (IOC) system. To mimic the anti-tumor immune response, cytotoxic immune cells were expanded from PBMCs by modified cytokine-induced killer (CIK) cell expansion protocol utilizing IL-2/IL-15. The CIK cells were co-cultured with patient-derived tumor organoids for killing assay to enable the prediction of ICB responsiveness in vitro. The CIK cells were expanded in the IOC system by stimulation of PBMC with IL-2/IL-15 in the presence of matched tumor organoids for 11 to 14 days. The CIK cells exhibited tumor organoid-specific killing (measured by Caspase-3, 15.22% ± 8.58, mean ± SEM) on organoid co-culture and showed induction of CD107a and CD137 expression in CD8+ T cells and NK cells with the PD-1 blocking antibody. The CIK-mediated tumor organoid deaths and immune cell activation were reduced by HLA class I blocking antibody (W6/32) treatment, confirming HLA-TCR mediated immune response and killing of tumor organoids in the IOC system (-14.85% in organoid-specific lysis and -7.46% in CD107a expression). In the live cell imaging of the IOC system, the interaction between T cells and tumor organoid cells and subsequent induction of Caspase 3 was noted. Our IOC system could directly measure the immunogenic tumor organoid killing by immune cells on ICB treatment in individual breast cancer patients. Moreover, the analysis of immune receptor profiles may reveal immune modulating mechanisms in each patient. We expect the IOC system to serve as a useful platform for personalized immunotherapy and exploration of combinatory therapy to enhance the anti-tumor efficacy of ICB in breast cancer patients. Citation Format: Cheol-Hwa Hong, Won-Ji Ryu, Shinyoung Park, Tae Yeong Kim, Yumi Hwang, Hyunju Han, Seongyeon Jo, Taeyong Kweon, Gun Min Kim, Min Hwan Kim, Hyung Seok Park, Joo Hyuk Sohn. Personalized immune-organoid co-culture system for modeling the anti-tumor immune response in breast cancer patients [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 3972.

Abstract 6751: In situ vaccination with Flt3l gene modified CD103+ type 1 conventional dendritic cells synergizes with anti-PD-1 checkpoint blockade in murine models of non-small cell lung cancer

Abstract A major hurdle in treatment of Non-Small Cell Lung Cancer (NSCLC) with anti-PD-1 immune checkpoint blockade (ICB) therapy is a lack of response (primary resistance) and relapse after an initial response (acquired resistance). Recent studies reveal that responses to PD-1/PD-L1 blockade are associated with high tumor mutational burden (TMB), increased CD8+ T cell infiltration and high baseline PD-L1 expression within the tumor microenvironment (TME), while impaired tumor antigen presentation and the immunosuppressive TME have been associated with resistance to ICB. One approach to overcome anti-PD-1 resistance is to intratumorally vaccinate NSCLC tumors with gene modified conventional dendritic cells (cDC), specifically the type I conventional DC (cDC1) lineage. Recent studies have established that generation of an anti-tumor immune response driven by CD8+ T cells requires the cross presentation of tumor associated antigens and that cDC1s are the primary cross presenting APC subtype in vivo, which can license CD8+ T cells to initiate an adaptive anti-tumor immune response. In addition, previous studies have shown that intratumoral administration of the FMS-like tyrosine kinase 3 ligand (FLT3L) protein can expand endogenous CD103+ cDC1s in the TME and augment anti-tumor immune responses to ICB therapy. Here, we engineered murine CD103+ cDC1s to constitutively secrete soluble FLT3L (FLT3L_cDC1) and performed in situ vaccination studies on anti-PD1 resistant murine models of NSCLC with LKB1-deficiency and elevated TMB that better represents human disease. In situ vaccination with FLT3L_cDC1 enhances anti-tumor efficacy compared to non-modified cDC1 vaccination and synergizes with anti-PD-1 ICB to inhibit tumor growth. FLT3L_cDC1 + anti-PD-1 combination therapy induces significant activation and expansion T cells and cDC1s within the TME. Furthermore, combination therapy significantly increases DC progenitor numbers within the tumor draining lymph node, including DC progenitors that are committed to the cDC1 lineage. Our data suggests in situ vaccination with FLT3L_cDC1 may represent a promising strategy to potentiate the efficacy of ICB and improve outcomes for patients with primary resistance PD-1/PD-L1 monotherapy. Citation Format: Jensen W. Abascal, Raymond J. Lim, Ramin Salehi-Rad, Zhe Jing, Michael S. Oh, William P. Crosson, Bitta P. Kahangi, Edgar Perez Reyes, Camelia Dumitras, Diana Reyimjan, Jessie Zhu, Linh M. Tran, Manash Paul, Kostyantyn Krysan, Bin Liu, Steven M. Dubinett. In situ vaccination with Flt3l gene modified CD103+ type 1 conventional dendritic cells synergizes with anti-PD-1 checkpoint blockade in murine models of non-small cell lung cancer [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 6751.

Abstract 6524: Single-cell landscape of cancer-associated fibroblasts in immunotherapy resistance of hepatocellular carcinoma

Abstract Introduction: The advent of immune checkpoint blockade (ICB) therapy has revolutionized the treatment approach for hepatocellular carcinoma (HCC). However, the response rate to ICB therapy remains low (below 20%). This limited response rate can be attributed to the considerable heterogeneity observed in HCC cells and the tumor microenvironment (TME). Recent research has highlighted the pivotal role of cancer-associated fibroblasts (CAFs) in orchestrating various components within the TME of HCC. Although the significance of lipid metabolism-associated CAFs in the efficacy of ICB therapy has been demonstrated in HCC murine models [1], the functions and contributions of other CAF subtypes remain largely unknown. Therefore, identifying CAF heterogeneity could advance our comprehension of HCC TME and potentially augment ICB efficacy. Methods: We performed single-cell RNA sequencing on tumor biopsy specimens obtained from patients with advanced HBV-related HCC who had participated in a pembrolizumab (anti-PD1) phase II clinical trial (NCT03419481). Our objective was to examine the dynamic alterations in the HCC TME before and during anti-PD1 treatment (upon 2-cycle). We utilized the Seurat and Cellchat packages for bioinformatics analysis which enabled us to investigate the associations between changes in the proportions of CAF subtypes and the patients' clinical responses, as well as the potential cell-cell interactions involving CAF and other TME components. Results: We identified four CAF subtypes within the TME: cycling, inflammatory, matrix, and vascular CAFs in our anti-PD1 HCC patient cohort. Among the anti-PD1 non-responders, there was a significant increase in the proportions of cycling CAFs (cCAFs) and matrix CAFs (matCAFs) compared to the responders. Notably, matCAFs exhibited a high enrichment of Gene Ontology (GO) Terms related to the extracellular matrix (ECM), whose higher proportion correlated with poor prognosis of patients. Moreover, a distinct collagen pathway signal was found between matCAFs and two immunosuppressive cell types, namely regulatory T cells and M2 macrophages in the non-responders. Discussion and Conclusion: The presence of matCAFs within the HCC tumor may associate with the development of an immunosuppressive TME and unfavorable prognosis for patients. Targeting this specific CAF subtype holds promise as a potential strategy to overcome resistance to ICB therapy. Acknowledgments: The CUHK Strategic Seed Funding for Collaborative Research Scheme and The Li Ka Shing Foundation.