Rewiring the tumor microenvironment in hepatocellular carcinoma: mechanism-driven integration of immunotherapy and locoregional strategies

Immune checkpoint inhibitors (ICIs) are immunomodulatory antibodies that intensify the host immune response, thereby leading to cytotoxicity. The primary targets for checkpoint inhibition have included cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), programmed cell death receptor-1 (PD-1) or programmed cell death ligand-1 (PD-L1). ICIs have resulted in a change in treatment landscape of various neoplasms. Among hematologic malignancies, ICIs have been most successful in certain subtypes of lymphomas such as classic Hodgkin lymphoma (cHL) and primary mediastinal B-cell lymphoma (PMBCL). However, there have been several challenges in harnessing the host immune system through ICI use in other lymphomas. The underlying reasons for the low efficacy of ICI monotherapy in most lymphomas may include defects in antigen presentation, non-inflamed tumor microenvironment (TME), immunosuppressive metabolites, genetic factors, and an overall lack of predictive biomarkers of response. In this review, we outline the existing and ongoing studies utilizing ICI therapy in various lymphomas. We also describe the challenges leading to the lack of efficacy with ICI use and discuss potential strategies to overcome those challenges including: chimeric antigen receptor T-cell therapy (CAR-T therapy), bispecific T-cell therapy (BiTE), lymphocyte activation gene-3 (LAG-3) inhibitors, T-cell immunoglobulin and mucin-domain containing-3 (TIM-3) inhibitors, vaccines, promotion of inflammatory macrophages, indoleamine 2,3-dioxygenase 1 (IDO1) inhibitors, DNA methyltransferase inhibitors (DNMTi) and histone deacetylase inhibitors (HDACi). Tumor mutational burden and interferon-gamma release assays are potential biomarkers of ICI treatment response beyond PD-L1 expression. Further collaborations between clinicians and scientists are vital to understand the immunopathology in ICI therapy in order to improve clinical outcomes.

CD33-TIM3 Dual CAR T Cells: Enhancing Specificity While Maintaining Efficacy Against AML

The immune system of people living with HIV (PLWH) is persistently exposed to antigens leading to systemic inflammation despite combination antiretroviral treatment (cART). This inflammatory milieu promotes T-cell activation and exhaustion. Furthermore, it produces diminished effector functions including loss of cytokine production, cytotoxicity, and proliferation, leading to disease progression. Exhausted T cells show overexpression of immune checkpoint molecules (ICs) on the cell surface, including programmed cell death protein 1 (PD-1), cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4), T-cell immunoglobulin and mucin-domain containing-3 (TIM-3), T-cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain (TIGIT), and lymphocyte activation gene-3 (LAG-3). The ICs also play a crucial role in T-cell exhaustion by reducing the immune response to cancer antigens. Immunotherapy based on immune checkpoint inhibitors (ICIs) has changed the management of a diversity of cancers. Additionally, the interest in exploring this approach in the setting of HIV infection has increased, including AIDS-defining cancers and non-AIDS-defining cancers in PLWH. To date, research on this topic suggests that ICI-based therapies in PLWH could be a safe and effective approach. In this review, we provide an overview of the current literature on the potential role of ICI-based immunotherapy not only in cancer remission in PLWH but also as a therapeutic intervention to restore immune response against HIV, revert HIV latency, and attain a functional cure for HIV infection.

Differential expression of TIM-3 in circulation and tumor microenvironment of colorectal cancer patients

Programmed cell death protein-1 (PD-1), cytotoxic T-lymphocyte antigen 4 (CTLA-4), T-cell immunoglobulin and mucin-domain containing-3 (TIM-3), lymphocyte activation gene-3 (LAG-3), and T-cell immunoglobulin and ITIM domain (TIGIT) are considered major immune co-inhibitory receptors (CIRs) and the most promising immunotherapeutic targets in cancer treatment, but they are largely unexplored in medullary thyroid carcinoma (MTC). We aimed to provide the first evidence regarding the expression profiles and clinical significance of CIRs in a large cohort of MTC patients. In total, 200 MTC patients who received initial surgery in our hospital were included. Immunohistochemistry was performed to evaluate CIR expressions in tissue microarrays (TMAs). Combined with the results of our previous programmed cell death ligand-1 (PD-L1) study, clinicopathologic and prognostic correlations of these proteins were retrospectively analyzed. TIM-3, PD-1, CTLA-4, LAG-3, and TIGIT positivity was detected in 96 (48.0%), 27 (13.5%), 25 (12.5%), 6 (3.0%), and 6 (3.0%) patients, respectively, in whom TIM-3, PD-1, and CTLA-4 expressions were positively correlated. Log-rank tests and multivariate Cox analyses both indicated that TIM-3, CTLA-4 expression, and PD-1/PD-L1 coexpression were associated with worse structural recurrence-free survival. In addition, among 20 patients who developed advanced disease during follow-up, 12 (60%) showed TIM-3 positivity, among whom 6 cases also had concurrent moderate to strong PD-1, PD-L1, or CTLA-4 expression. Using the currently largest TMA cohort of this rare cancer, we delineated the CIR expression profiles in MTC, and identified TIM-3, CTLA-4 expression, and PD-1/PD-L1 coexpression as promising biomarkers for tumor recurrence. Furthermore, a subset of advanced MTCs are probably immunogenic, for which single or combined immunotherapy including TIM-3, PD-1, PD-L1, or CTLA-4 blockade may be potential therapeutic approaches in the future.

BackgroundMetastatic melanoma to the brain carries a particularly poor prognosis that may be associated with an attenuated antitumor response in the presence of central nervous system malignancies. Thus, the development of brain metastases could theoretically accelerate cancer progression both locally and systemically. Although dysregulation of checkpoint markers, such as programmed death-ligand 1 (PD-L1), programmed cell death receptor 1 (PD-1), lymphocyte activation gene 3 (LAG-3), and T-cell immunoglobulin and mucin-domain containing-3 (TIM-3), have been implicated in immune dysfunction, the exact relationship between these markers and brain tumor-mediated immune suppression remains unclear. Thus, the objective of this study was to explore whether there exists a differential expression of the above checkpoint markers in the intracranial milieu as compared to tumors in the periphery, which may shed light on the mechanism behind the diminished antitumor response.MethodsWe identified nine patients with extracranial melanomas and matched intracranial metastases. Formalin-fixed, paraffin-embedded slides were stained for PD-L1, PD-1, LAG-3, and TIM-3 via immunohistochemistry. Qualitative analysis was performed to assess the staining of the markers in the neoplastic and lymphocytic cells, which were the two cell lineages in each biopsy. ResultsExpression of PD-1 and TIM-3 between extracranial and intracranial tumoral sites was conserved. Specifically, in lymphocytes, PD-1 expression was observed in 100% of extracranial and 100% of intracranial slides, whereas TIM-3 expression was seen in 33.33% of extracranial and 33.33% of intracranial slides. Neither marker stained tumor cells, as expected. PD-L1 showed a slight variation in staining between sites, with lymphocyte staining in 100% of extracranial and 88.89% of intracranial slides, and the same percentages per site for tumor cells. The greatest variability was observed in LAG-3 lymphocyte staining, with staining in 77.78% of extracranial and 33.33% of intracranial slides. No LAG-3 staining of tumor cells was noted, as expected.ConclusionPreliminary analysis revealed the conservation of PD-L1, PD-1, LAG-3, and TIM-3 expression intra- and extracranially. This could suggest that these markers are important in maintaining an immunosuppressive phenotype at both sites. Another possibility is that this pattern of expression is associated with patients who develop brain metastasis, as this was the only subset of patients included in this study. Interestingly, LAG-3 staining of lymphocytes appeared more prominent in extracranial over intracranial tumors. Future studies should include more samples to draw out potential patterns masked by the small sample size, as well as to compare checkpoint expression in other patient groups, such as those with non-brain metastasis or those with no metastasis at all.

Multiple suppressive mechanisms within the tumor microenvironment (TME) are capable of blunting anti-tumor T cell responses. These include engagement of inhibitory receptors expressed in tumor-associated, exhausted CD8 T cells, such as programmed cell death protein 1 (PD-1), T-cell immunoglobulin and mucin-domain containing-3 (TIM-3), lymphocyte-activation gene 3 (LAG-3), 2B4 (also known as CD244), and T cell immunoreceptor with Ig and ITIM domains (TIGIT). While immune checkpoint blockade therapies aimed at reversing the dysfunctional state of tumor-associated T cells have demonstrated clinical effectiveness, not all cancer patients achieve long-term disease control. This is due, at least in part, to the refractory nature of what are categorized as terminally exhausted CD8 T cells to be reinvigorated by, for example, PD-1/PD-L1 blockade. As CD8 T cell exhaustion (or dysfunction) is a major therapeutic challenge, gaps in our understanding of cellular and molecular mechanisms underlying the T cell exhaustion (or dysfunction) program in cancer warrant further study of pathways that program T cells toward exhaustion (or dysfunction). Through comprehensive immune profiling of tumor-infiltrating T lymphocytes (TILs), we found that CD47 expression in CD8 TILs isolated from melanoma patients significantly correlates with expression of several checkpoint inhibitory molecules (e.g., TIM-3, PD-1 and LAG-3). Additionally, our re-analysis of single cell data from melanoma patients revealed that terminally exhausted T cells (Tex) and TCF7hi Tex precursor cells exhibit high levels of CD47 transcripts, suggesting phenotypic association of CD47 with T cell exhaustion. We confirmed our observations in murine B16-F10 melanoma where CD47 expression is significantly upregulated in exhausted CD8 TILs. We also show that CD47 functions as a negative regulator for T cell proliferation and function during T cell priming. To address the role of CD47 during the development of CD8 T cell exhaustion/dysfunction in cancer, we performed adoptive T cell transfer of the naïve-sorted Cd47+/+ (WT) and Cd47+/- (Het) antigen specific Pmel-1 CD8 T cells (but not Cd47-deficient Pmel-1 CD8 T cells as they would be subject to innate immune clearance) into B16 tumor-bearing mice and found that Cd47-Het Pmel-1 CD8 TILs, as compared to the Cd47-WT Pmel-1 CD8 TILs, exhibit less expression of exhaustion-related genes (e.g. Pdcd1, Lag3 and Tox), and increased expression of genes associated with T cell activation and proliferation (e.g. Mki67, Lck, Cd69, Gzma, Gzmk). We further confirmed that thrombospondin-1 (TSP-1), as an extracellular matrix protein and a ligand of CD47, contributes to driving the differentiation of CD8 T cells toward exhaustion. Our data highlight for the first time the potential of extracellular matrix protein TSP-1 in programming CD8 T cell exhaustion in cancer through its interaction with CD47 expressed on CD8 T cells. Citation Format: Chien-Huan Weng, Fadi Samaan, Sadna Budhu, Levi Mangarin, Sébastien Monette, Cailian Liu, Stephane Pourpe, Linda Hamadene, Hong Zhong, Xia Yang, David Schroder, Roberta Zappasodi, Pamela Holland, Jedd D. Wolchok, Taha Merghoub. Potential role of CD47 in T cell exhaustion program [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 6150.

Tumor growth and survival requires a particularly effective immunosuppressant tumor microenvironment (TME) to escape destruction by the immune system. While immunosuppressive checkpoint markers like programmed cell death 1 ligand (PD-L1) are already being targeted in clinical practice, lymphocyte-activation-protein 3 (LAG-3), T-cell immunoglobulin and mucin-domain containing-3 (TIM-3) and V-domain Ig suppressor of T cell activation (VISTA) inhibitors are currently under investigation in clinical trials. Reliable findings on the expression status of those immune checkpoint inhibitors on tumor-infiltrating lymphocytes (TILs) in the TME of oropharyngeal squamous cell carcinoma (OPSCC) are lacking. This work aims to describe the expression of LAG-3, TIM-3, and VISTA expression in the TME of OPSCC. We created a tissue microarray of paraffin-embedded tumor tissue of 241 OPSCC. Expression of the immune checkpoint protein LAG-3, TIM-3, and VISTA in OPSCC was evaluated using immunohistochemistry and results were correlated with CD8+ T-cell inflammation and human papillomavirus (HPV)-status. 73 OPSCC stained positive for LAG-3 (31%; HPV+:44%; HPV-:26%, p = 0.006), 122 OPSCC stained positive for TIM-3 (51%; HPV+:70%; HPV-:44%, p < 0.001) and 168 OPSCC (70%; HPV+:75%; HPV-:68%, p = 0.313) for VISTA. CD8+ T-cells were significantly associated with LAG-3, TIM-3 and VISTA expression (p < 0.001, p < 0.001, p = 0.007). Immune checkpoint therapy targeting LAG-3, TIM-3, and/or VISTA could be a promising treatment strategy especially in HPV-related OPSCC. Future clinical trials investigating the efficacy of a checkpoint blockade in consideration of LAG-3, TIM-3, and VISTA expression are required.

B-cell maturation antigen chimeric antigen receptor T-cell re-expansion in a patient with myeloma following salvage programmed cell death protein 1 inhibitor-based combination therapy.

Hepatocellular carcinoma (HCC) is one of most common cancers and the fourth leading cause of death worldwide. Commonly, HCC development occurs in a liver that is severely compromised by chronic injury or inflammation. Liver transplantation, hepatic resection, radiofrequency ablation (RFA), transcatheter arterial chemoembolization (TACE), and targeted therapies based on tyrosine protein kinase inhibitors are the most common treatments. The latter group have been used as the primary choice for a decade. However, tumor microenvironment in HCC is strongly immunosuppressive; thus, new treatment approaches for HCC remain necessary. The great expression of immune checkpoint molecules, such as programmed death-1 (PD-1), cytotoxic T-lymphocyte antigen 4 (CTLA-4), lymphocyte activating gene 3 protein (LAG-3), and mucin domain molecule 3 (TIM-3), on tumor and immune cells and the high levels of immunosuppressive cytokines induce T cell inhibition and represent one of the major mechanisms of HCC immune escape. Recently, immunotherapy based on the use of immune checkpoint inhibitors (ICIs), as single agents or in combination with kinase inhibitors, anti-angiogenic drugs, chemotherapeutic agents, and locoregional therapies, offers great promise in the treatment of HCC. This review summarizes the recent clinical studies, as well as ongoing and upcoming trials.

Fetal bovine serum (FBS) or human serum is widely used in the production of chimeric antigen receptor (CAR) T-cells. In order to overcome a lot-to-lot inconsistency, the use of chemically defined medium that is free of animal-components would be highly desirable. The present study compared three serum-free media [Prime-XV™ T Cell CDM, Fujifilm™ (FF), LymphoONE™ T-Cell Expansion Xeno-Free Medium, Takara Bio™ (TB) and TCM GMP-Prototype, CellGenix™ (CG)] to the standard CAR T-cell medium containing FBS (RCF). After 12 days of CD19.CAR T-cell culture, the expansion, viability, transduction efficiency and phenotype were assessed using flow cytometry. The functionality of CAR T-cells was evaluated using intracellular staining, a chromium release assay and a long-term co-culture assay. Expansion and viability did not differ between the CAR T-cells generated in serum-free media compared to the standard FBS-containing medium. The CG CAR T-cells had a statistically significant higher frequency of IFNγ+ and IFNγ+TNF-α+ CAR T-cells than the CAR T-cells cultured with FBS (22.5 vs. 7.6%, P=0.0194; 15.3 vs. 6.2%, P=0.0399, respectively) as detected by intracellular cytokine staining. The CAR T-cells generated with serum-free media exhibited a higher cytotoxicity than the CAR T-cells cultured with FBS in the evaluation by chromium release assay [CG vs. RCF (P=0.0182), FF vs. RCF (P=0.0482) and TB vs. RCF (P=0.0482)]. Phenotyping on day 12 of CAR T-cell production did not reveal a significant difference in the expression of the exhaustion markers, programmed cell death protein 1, lymphocyte-activation gene 3 and T-cell immunoglobulin and mucin-domain containing-3. The CAR T-cells cultured in FF had a higher percentage of central memory CAR T-cells (40.0 vs. 14.3%, P=0.0470) than the CAR T-cells cultured with FBS, whereas the CAR T-cells in FF (6.2 vs. 24.2%, P=0.0029) and CG (11.0% vs. 24.2%, P=0.0468) had a lower frequency of naïve CAR T-cells. On the whole, the present study demonstrates that in general, the functionality and expansion of CAR T cells are maintained in serum-free media. Given the advantages of freedom from bovine material and consistent quality, serum-free media hold promise for the future development of the field of GMP manufacturing of CAR T-cells.

Endogenous control mechanisms, including immune checkpoints and immunosuppressive cells, are exploited in the process of tumorigenesis to weaken the anti-tumor immune response. Cancer treatment by chemotherapy or immune checkpoint inhibition can lead to changes of checkpoint expression, which influences therapy success. Peripheral blood lymphocytes (PBL) and tumor-infiltrating lymphocytes (TIL) were isolated from head and neck squamous cell carcinoma (HNSCC) patients (n = 23) and compared to healthy donors (n = 23). Immune checkpoint expression (programmed cell death ligand 1 (PD-1), tumor necrosis factor receptor (TNFR)-related (GITR), CD137, tumor necrosis factor receptor superfamily member 4 (TNFRSF4) (OX40), t-cell immunoglobulin and mucin-domain containing-3 (TIM3), B- and T-lymphocyte attenuator (BTLA), lymphocyte-activation gene 3 (LAG3)) was determined on immune cells by flow cytometry. PD-L1 expression was detected on tumor tissue by immunohistochemistry. Immune cells were treated with immuno- and chemotherapeutics to investigate treatment-specific change in immune checkpoint expression, in vitro. Specific changes of immune checkpoint expression were identified on PBL and TIL of HNSCC patients compared to healthy donors. Various chemotherapeutics acted differently on the expression of immune checkpoints. Changes of checkpoint expression were significantly less pronounced on regulatory T cells compared to other lymphocyte populations. Nivolumab treatment significantly reduced the receptor PD-1 on all analyzed T cell populations, in vitro. The specific immune checkpoint expression patterns in HNSCC patients and the investigated effects of immunomodulatory agents may improve the development and efficacy of targeted immunotherapy.

3133 Background: Natural killer (NK) cells play a crucial role in tumor progression and anti-tumor immunity. However, they often exhibit an exhausted phenotype within the tumor microenvironment (TME), limiting their full cytotoxic potential. T-cell immunoglobulin and mucin-domain containing-3 (TIM-3) has emerged as a novel immune checkpoint that is highly expressed on NK cells and suppresses their cytotoxic function. TIM-3 is closely associated with immune evasion and anti-tumor immune tolerance. This study aims to investigate the effects and mechanisms by which radiation modulates NK cell function, providing a foundation for developing strategies that specifically target TIM-3 on NK cells. Methods: First, mRNA high-throughput sequencing, RT-qPCR, and Western blot experiments were used to analyze changes in the expression of related genes in NK92 cells after radiotherapy. The LDH release assay was employed to evaluate the effect of radiation on the viability of NK92 cells. ELISA was conducted to detect changes in the release levels of tumor necrosis factor TNF-α and other factors after radiotherapy. Dual-luciferase reporter assays and chromatin immunoprecipitation (ChIP) experiments confirmed that the transcription factor MYB mediates radiation-induced regulation of NK cell activation by targeting and binding to the TIM-3 promoter region. A non-contact co-culture system was established, and flow cytometry demonstrated that radiation combined with MYB overexpression enhanced the cytotoxicity of NK cells against tumor cells. A colon cancer mouse model was constructed to evaluate the anti-tumor effect of combining anti-TIM-3 antibodies with radiotherapy. Results: We found that radiation can activate NK92 cells in vitro and enhance TIM-3 expression,promoting the secretion of granzyme B, perforin, TNF-α, IFN-γ, and other cytokines and chemokines that modulate the TME and enhance anti-tumor immune responses. Moreover, the transcription factor MYB inhibits TIM-3 expression by directly binding to the TIM-3 promoter region, mediating the effects of radiation on the TME through NK cell activation. In vivo, the combination of radiotherapy and anti-TIM-3 antibodies effectively controlled the growth of subcutaneously transplanted colon cancer tumors in C57BL/6 mice. However, this combined treatment effect was significantly diminished after NK cells were depleted by the anti-NK1.1 antibody. Conclusions: This study elucidates a novel mechanism by which radiation activates NK cells in the tumor microenvironment through the MYB/TIM-3 pathway. It provides new insights for enhancing the efficacy of radiotherapy and offers a theoretical basis for the potential clinical application of these cells in future research.

Targeting immune checkpoint molecules PD1 and CTLA-4 by activating the immune system have improved the lives of many cancer patients. However, this type of immunotherapy is not effective for all patients, suggesting that there are additional mechanisms of immune evasion. T-cell immunoglobulin and mucin-domain containing-3 (TIM-3) has been reported to be expressed on exhausted T and NK cells within the tumor microenvironment. The data in many preclinical models suggest that targeting TIM-3 pathway might provide an additional immune modulation of this anti-tumor axis in cancer patients. We have developed a novel monoclonal antibody NB002 against TIM-3,which forms a high-affinity interaction with a unique epitope. NB002 can enhance IFN-gamma secretion and proliferation of these exhausted T cells that has increased TIM-3 expression. Moreover, TIM-3 is constitutively expressed on NK cells. TIM-3 blockade could enhance in vitro cytotoxic activity of NK cells. Altogether, our data shows that NB002 is capable of targeting exhausted T cells and NK cells resulting activation of antitumor immunity. Further preclinical assessment in vivo and in vitro for solid tumors will support clinical development of NB002. Citation Format: Xin Dong, Jannie Dong, Jun Ma, Lianqi Zhao, Haojie Wang, Binbin Wang, Qian Gao, Hui Pan, Dong Wang, Liegang Shao. Pre-clinical characterization of TIM-3 antibody NB002 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 933.

Hepatocellular carcinoma (HCC) is the most common liver cancer, with poor survival rates in advanced stages due to late diagnosis, tumor heterogeneity, and therapy resistance. The tumor microenvironment (TME) in HCC has a crucial role in tumor progression, characterized by a complex interaction of immune cells, stromal components, and immunosuppressive signaling pathways. Chronic inflammation driven by viral infections, metabolic dysfunction, and alcohol consumption triggers an immunosuppressive TME, promoting immune evasion and tumor growth. Immune cell populations, such as myeloid-derived suppressor cells, regulatory T cells, and tumor-associated macrophages, contribute to immunosuppression, while cytotoxic T lymphocytes and natural killer cells exert anti-tumor effects. Recent advances in immunotherapy, mainly immune checkpoint inhibitors (ICIs) targeting programmed death-ligand 1 and programmed cell death protein 1 and cytotoxic T-lymphocyte-associated protein 4, have revolutionized HCC treatment, though response rates remain limited. Combined therapies using tyrosine kinase inhibitors, anti-angiogenic agents, and ICIs improve patient outcomes. This review discusses the immunological mechanisms contributing to HCC progression, the role of immune cell subsets in tumor evasion, and therapeutic interventions, from conventional treatments to advanced immunotherapies. Ongoing clinical trials, barriers to effective treatment, and future directions to enhance HCC management and patient survival will also be overviewed.