Immune checkpoint blockade in infectious diseases.

Purpose: Regulatory T (Treg) cells perform the immune suppressive function in cancer, but their suppressive mechanism in tumor microenvironment (TME) has not been clearly elucidated. We aim to identify the phenotype and functional mechanism of Treg cells in TME from cancer patients. Experimental Design: We collected 72 malignant effusion (ME) and peripheral blood (PB) specimens from stage IV cancer patients and 10 tumor tissue (TM) and PB from lung cancer patients who underwent surgery. Lymphocytes from ME, TM, and PB were analyzed for subtype of Tconv and Treg cells, and their expressions of immune checkpoint (IC) molecules including programmed death (PD-1), cytotoxic T lymphocyte antigen-4 (CTLA-4), and T cell immunoglobulin containing molecule-3 (TIM-3) using by flow cytometry. To examine the functional role of PD-1 expressed Treg cells, TC-1 lung cancer mouse model was used. Result: Upregulation of PD-1 on Treg cells in TM and ME compared to PB was even more distinguishable than that on CD4+ and CD8+ Tconv cells. To clarify the characteristics of tumor infiltrating Treg cells, we comprehensively examined the characteristics of Treg cells from tumor, peri-tumor, and PB in lung cancer patients by analyzing the expression of IC molecules including PD-1, TIM-3, CTLA-4, and TIGIT, of which PD-1 is the highest expressed on tumor infiltrating Treg cells. To investigate the mechanism by which Treg cells mediate immune suppression, we compared the suppressive activity of Treg cells expressing high and low levels of PD-1 by co-culturing each population with naïve CD8+ T cells with or without αCD3/CD28 stimulation. This was more potently inhibited in co-cultures with PD-1high tumor infiltrating Treg cells than in those with PD-1low Treg cells. PD-1 blocked tumor infiltrating Treg cells demonstrated a significantly decreased suppressive function in the proliferation of CD8+ cells and their IFN-γ production. These results implicate that PD-1 expressed tumor infiltrating Treg confers the suppressive function of proliferation in CD8+ T cells through PD-1: PD-L1 interaction. Conclusion: Our study evidently demonstrated that upregulated PD-1 on tumor infiltrating Treg cells and their PD-1: PD-L1 interaction could be potential cause of T cell suppression, which might be helpful to completely understand their suppressive mechanism in cancer patients. Citation Format: Hye Ryun Kim, Hyo Jin Park, Jimin Son, Hyo Sup Shim, Byoung Chul Cho, Sun Young Rha, Sang-Jun Ha. Upregulation of immune checkpoint molecules on Treg cells in tumor microenvironment reinforces immune exhaustion in cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 713.

Antibodies that target immune checkpoints such as cytotoxic T lymphocyte antigen 4 (CTLA‐4) and the programmed cell death protein 1/ligand 1 (PD-1/PD-L1) are now a treatment option for multiple cancer types. However, as a monotherapy, objective responses only occur in a minority of patients. Chemotherapy is widely used in combination with immune checkpoint blockade (ICB). Although a variety of isolated immunostimulatory effects have been reported for several classes of chemotherapeutics, it is unclear which chemotherapeutics provide the most benefit when combined with ICB. We investigated 10 chemotherapies from the main canonical classes dosed at the clinically relevant maximum tolerated dose in combination with anti‐CTLA-4/anti-PD-L1 ICB. We screened these chemo-immunotherapy combinations in two murine mesothelioma models from two different genetic backgrounds, and identified chemotherapies that produced additive, neutral or antagonistic effects when combined with ICB. Using flow cytometry and bulk RNAseq, we characterized the tumor immune milieu in additive chemo-immunotherapy combinations. 5-fluorouracil (5-FU) or cisplatin were additive when combined with ICB while vinorelbine and etoposide provided no additional benefit when combined with ICB. The combination of 5-FU with ICB augmented an inflammatory tumor microenvironment with markedly increased CD8+ T cell activation and upregulation of IFNγ, TNFα and IL-1β signaling. The effective anti‐tumor immune response of 5-FU chemo-immunotherapy was dependent on CD8+ T cells but was unaffected when TNFα or IL-1β cytokine signaling pathways were blocked. Our study identified additive and non-additive chemotherapy/ICB combinations and suggests a possible role for increased inflammation in the tumor microenvironment as a basis for effective combination therapy.

To date, clinical trials of various vaccine therapies using autologous tumor antigens or tumor-associated/specific antigen peptide with adjuvants have been performed to treat patients with high-grade gliomas (HGG). Furthermore, immune checkpoint pathway-targeted therapies including anti- programmed cell death 1 (PD-1) antibody have been remarkably effective in other neoplasms, and various clinical trials with anti-PD-1 antibody in patients with HGG have started to date. It is possible that up-regulation of immune checkpoint molecules in tumor tissues after vaccine therapy may be one of the mechanisms of vaccine failure. Multiple preclinical studies indicate that combination therapy with vaccination and immune checkpoint blockade is effective for the treatment of malignant tumors including HGG. Thus, immunotherapy, especially combination therapy with vaccine and immune checkpoint inhibitors, may be a promising strategy for treatment of patients with HGG.

Immune checkpoint inhibitor-based therapy for advanced clear cell renal cell carcinoma: A narrative review

'Immune checkpoint blockade' for cancer describes the use of therapeutic antibodies that disrupt negative immune regulatory checkpoints and unleash pre-existing antitumour immune responses. Antibodies targeting the checkpoint molecules cytotoxic T lymphocyte antigen 4 (CTLA4), programmed cell death 1 (PD1) and PD1 ligand 1 (PD-L1) have had early success in the clinic, which has led to approval by the US Food and Drug Administration of multiple agents in several cancer types. Yet, clinicians still have very limited tools to discriminate a priori patients who will and will not respond to treatment. This has fuelled a wave of research into the molecular mechanisms of tumour-intrinsic resistance to immune checkpoint blockade, leading to the rediscovery of biological processes critical to antitumour immunity, namely interferon signalling and antigen presentation. Other efforts have shed light on the immunological implications of canonical cancer signalling pathways, such as WNT-β-catenin signalling, cell cycle regulatory signalling, mitogen-activated protein kinase signalling and pathways activated by loss of the tumour suppressor phosphoinositide phosphatase PTEN. Here we review each of these molecular mechanisms of resistance and explore ongoing approaches to overcome resistance to immune checkpoint blockade and expand the spectrum of patients who can benefit from immune checkpoint blockade.

Background: Immune checkpoint blockade (ICB) immunotherapy improves survival for many cancer patients, but those with immunologically ‘cold’ tumors do not derive benefit. Radiation therapy (RT) has been demonstrated to augment antitumor immunity in preclinical and clinical studies and may enhance response to ICB in immunologically cold tumors. We have developed DNA-based, cost-effective, and versatile ICB immunotherapies. Here, we test the use of these alone and in combination with RT as a novel treatment approach in syngeneic murine melanoma models. Materials and methods: We constructed ICB DNA vaccines targeting cytotoxic T lymphocyte antigen 4 (CTLA-4) and programmed cell death 1 (PD-1), CTLA-4 Vax and PD-1 Vax, respectively. These were delivered to mice by intramuscular or intratumoral electroporation. The effects of combinations of ICB DNA vaccines and RT were monitored for generation of endogenous anti-CTLA-4 and anti-PD-1 antibodies, modification of tumor microenvironment (TME), and tumor response in mice bearing syngeneic B78 or B16 melanoma. Results: Intramuscular vaccination with CTLA-4 Vax and PD-1 Vax induced endogenous anti-CTLA-4 and anti-PD-1 antibodies, respectively, and the titers increased with additional vaccinations. Vaccination with CTLA-4 Vax and PD-1 Vax alone or in combination (Dual Vax) did not inhibit the murine B16 melanoma model; however, the combination of CTLA-4 Vax and RT (CTLA-4 Vax + RT) led to enhanced infiltration of the TME with effector T cells. Dual Vax combined with RT enabled systemic anti-tumor immunity in mice bearing two B16 melanoma tumors in which only one was radiated. With intra-tumoral route of vaccination, the Dual Vax + RT treatment induced a trend toward systemic anti-tumor response in the immunologically cold B78 tumor model. Conclusions: DNA vaccines that stimulate endogenous production of antibodies against immune checkpoints may serve as an alternative form of ICB. The combination of ICB DNA vaccine and RT enabled anti-tumor immunity and tumor response in preclinical models of melanoma. Citation Format: Keng-Hsueh Lan, Ying-Chun Sheng, Keng-Li Lan, KyungMann Kim, Sung-Hsin Kuo, Zachary Morris. Combination of a DNA vaccine-induced immune checkpoint blockade and radiation therapy induces anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1098.

Emerging role of MMR, MSI, TMB and neoantigen testing in cancer patients receiving immunotherapy

T cells have a central role in immune system balance. When activated, they may lead to autoimmune diseases. When too anergic, they contribute to infection spread and cancer proliferation. Immune checkpoint proteins regulate T cell function, including cytotoxic T lymphocyte antigen-4 (CTLA-4) and programmed cell death-1 (PD-1) and its ligand (PD-L1). These nodes of self-tolerance may be exploited pharmacologically to downregulate (CTLA-4 agonists) and activate [CTLA-4 and PD-1/PD-L1 antagonists, also called immune checkpoint inhibitors (ICIs)] the immune system.CTLA-4 agonists are used to treat rheumatologic immune disorders and graft rejection. CTLA-4, PD-1, and PD-L1 antagonists are approved for multiple cancer types and are being investigated for chronic viral infections. Notably, ICIs may be associated with immune-related adverse events (irAEs), which can be highly morbid or fatal. CTLA-4 agonism has been a promising method to reverse such life-threatening irAEs. Herein, we review the clinical pharmacology of these immune checkpoint agents with a focus on their interplay in human diseases.

Colorectal cancer (CRC) is a common cancer in China and worldwide [1, 2]. Immune checkpoint blockade (ICB) has been proven effective for DNA mismatch repair-deficient (dMMR)/microsatellite instability-high (MSI-H) CRC [3-10] but not for mismatch repair-proficient (pMMR)/microsatellite stable (MSS) CRC in clinical trials [3]. No published data on the real-world application of ICB in CRC exist, and thus, whether the response to ICB in unselected patients is similar to that in patients from published trials remains unclear. In this study, we reported results from the real-world application of ICB in off-trial CRC patients (irrespective of stage), treated at the Sun Yat-sen University Cancer Center from March 1, 2017, to October 1, 2019. We analyzed the mismatch repair (MMR) and microsatellite instability (MSI) status, demographic characteristics, treatment regimens, response to ICB, and adverse events of CRC patients who received ICB in a real-world off-trial, to help devise effective immunotherapy strategies for routine clinical practice. We identified 69 CRC patients who received ICB using an off-trial protocol. The detailed methods are provided in the Supplementary Materials. The inclusion and exclusion criteria are shown in Supplementary Figure S1. Fifty-two patients were classified according to their MMR status; 27 (51.9%) as dMMR and 25 (48.1%) as pMMR. Fifty patients were classified according to their MSI status; 29 (58.0%) as MSI-H, 2 (4.0%) as MSI-low (MSI-L), and 19 (38.0%) as MSS. In this study, dMMR and/or MSI-H patients were further classified as dMMR/MSI-H, and patients who were neither dMMR nor MSI-H were classified as pMMR/MSI-L/MSS. Finally, 36 (52.2%) patients were classified as dMMR/MSI-H, and 30 (43.5%) as pMMR/MSI-L/MSS. The MMR/MSI status of 3 (4.3%) patients was unknown. The patients' characteristics are shown in Supplementary Table S1. Their median age was 45 (range, 16-67) years at ICB initiation. Thirty-nine (56.5%) patients were male. Fifty-six (81.2%) patients had stage IV disease when ICB was initiated. The most common primary tumor site was the colon (n = 48; 69.6%). Twenty-one (30.4%) patients had a family history of CRC. Before immunotherapy, the number of patients who received chemotherapy, targeted therapy, surgery, and radiotherapy were 55 (79.7%), 30 (43.5%), 38 (55.1%), and 11 (15.9%), respectively (Supplementary Table S1 and S2). In addition, 38 patients underwent surgery and among them, 21 patients had stage II/III disease and 17 patients had stage IV disease when first diagnosed. The 21 patients with stage II/III disease all underwent primary tumor resection, and two also underwent metastasectomy when metastatic disease progression was observed. Among the 17 patients with stage IV disease who underwent surgery, 16 underwent primary tumor resection, and 13 underwent metastasectomy (7 received radical surgery, and 6 received palliative surgery). Three of the 17 patients also underwent metastasectomy when disease progression with new metastasis was observed. The median time from initial diagnosis to first ICB treatment was 6.2 months (range, 0.2-124.1 months, Supplementary Figure S2). ICB was administered as neoadjuvant therapy to 13 (18.8%) patients, as first-line therapy to 16 (23.2%) patients, and as second-line or later therapy to 40 (58.0%) patients. The median duration of immunotherapy was 68 days (range, 1-939 days, Supplementary Figure S2), and the median number of immunotherapy cycles was 4 (range, 1-39). All patients received one or two types of programmed cell death protein-1 (PD-1) blockade. The cytotoxic T lymphocyte antigen-4 (CTLA-4) inhibitor ipilimumab was administered to only 8 (11.6%) patients (Supplementary Table S3). Twenty-three (33.3%) patients received only mono-immunotherapy, and 46 (66.7%) patients received combined therapy, including combined chemotherapy (32, 46.4%), targeted therapy (24, 34.8%), and/or radiotherapy (8, 11.6%). The chemotherapy and targeted therapy agents administered during immunotherapy are listed in Supplementary Table S4. Regarding treatment effects, the best overall response was considered as the short-term treatment effect, while overall survival (OS) and progression-free survival (PFS) as long-term effects. Of the 69 patients, an objective response to ICB was noted in 22 (31.9%) patients, including complete response (CR) in 9 (13.0%) and partial response (PR) in 13 (18.8%) patients. Stable disease (SD) was observed in 18 (26.1%) patients. The remaining 29 patients (42.0%) experienced progressive disease (PD), with a median PFS duration of 2.1 (range, 0.3-30.6) months. The best overall response of all patients, dMMR/MSI-H patients, and pMMR/MSI-L/MSS patients are described in Supplementary Tables S5-7. Univariate analysis showed that MMR/MSI status and immunotherapy setting were significantly associated with the objective response rate (ORR) and disease control rate (DCR). Similar to that reported in clinical trials, in this study, dMMR/MSI-H patients had a higher ORR and DCR than pMMR/MSI-L/MSS patients (50.0% vs. 13.3%, P = 0.002; 75.0% vs. 43.3%, P = 0.003) (Supplementary Table S5). In addition to MMR/MSI status, immunotherapy setting was also associated with ORR and DCR. The ORRs in patients who received ICB as neoadjuvant therapy, first-line therapy, and second-line or later therapy were 84.6%, 50.0%, and 7.5%, respectively (P < 0.001), and the DCRs were 92.3%, 87.5%, and 35.0%, respectively (P < 0.001). When the two factors were subjected to multivariate analysis, only the immunotherapy setting was independently associated with ORR and DCR (Supplementary Table S5). We also separately analyzed the best overall response of dMMR/MSI-H and pMMR/MSI-L/MSS patients. Patients who received ICB early, as neoadjuvant therapy or first-line therapy, had higher ORRs and DCRs in both the dMMR/MSI-H (Supplementary Table S6) and pMMR/MSI-L/MSS patient groups (Supplementary Table S7). For all patients, by January 4, 2021, after a median follow-up time of 15.1 (range, 0.3-38.0) months, the median OS had not been reached. The median PFS was 6.3 months, and the one-year OS and PFS rates were 72.2% and 46.4% (Supplementary Table S8, Figure 1A-B). The OS of each patient is shown in Supplementary Figure S2. Univariate analysis for investigating the association between clinical factors and survival showed that dMMR/MSI-H status and early immunotherapy use were significantly associated with favorable OS (P < 0.001, P < 0.001) and PFS (P < 0.001, P < 0.001) (Supplementary Table S8, Figure 1C-F). Multivariate analyses showed that MMR/MSI status was significantly associated with OS and PFS, while the immunotherapy setting was only significantly associated with PFS (Supplementary Table S8). Long-term survival analysis for dMMR/MSI-H patients (Supplementary Table S9) showed that the OS and PFS of patients who received ICB early were longer than those who received ICB as second-line or later therapy, although no significant differences were observed. In pMMR/MSI-L/MSS patients (Supplementary Table S10), early ICB and combined therapy were associated with longer OS (P = 0.020, P = 0.011) and PFS (P = 0.004, P < 0.001). When the top two factors were subjected to multivariate analysis, immunotherapy setting and combination therapy were independently associated with PFS but not with OS. Treatment-related adverse events (TRAEs) were reported for 65 (94.2%) patients (Supplementary Table S11). Eleven (15.9%) patients experienced grade 3 TRAEs, and five patients (7.2%) experienced grade 4 TRAEs. All the TRAEs were successfully treated. Only two patients discontinued ICB treatment due to the TRAEs. Despite the study limitations (small cohort and non-uniform ICB regimens), to our knowledge, this is the first study to explore the efficacy and safety of immunotherapy in a real-world cohort of CRC patients. Our results demonstrated that ICB was more effective for dMMR/MSI-H CRC than pMMR/MSI-L/MSS, as previously reported in clinical trials, and the earlier use of ICB resulted in better tumor response, especially in pMMR/MSI-L/MSS CRC patients. In real-world settings, combined therapy could prolong the OS and PFS of pMMR/MSI-L/MSS CRC patients but not dMMR/MSI-H CRC patients. Study concept and design: CJZ, WWX, YHG. Data acquisition: CJZ, TJ, RZL. Data analysis and interpretation: CJZ, WHX, XXH, YY, QXW, HC. Manuscript writing: CJZ, WWX. Critical revision: All authors. Final approval of manuscript: All authors. The study was approved by the Ethics Committee of Sun Yat-sen University Cancer Center (No. B2020-134-01). Informed consent was waived as this was a noninterventional study using routinely collected data. This study was funded by the National Natural Science Foundation of China (81672987, 82073329) and the Natural Science Foundation of Guangdong Province (2020A1515011286). The authors declare there are no conflicts of interest. The authors wish to thank the medical staff and patients for their contribution to this study. The raw data have been deposited into the Research Data Deposit (http://www.researchdata.org.cn), with the RDD number RDDA2021001951 and the datasets used in this study are publicly available. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Targeting immune checkpoints such as programmed cell death protein 1 (PD1), programmed cell death 1 ligand 1 (PDL1) and cytotoxic T lymphocyte antigen 4 (CTLA4) has achieved noteworthy benefit in multiple cancers by blocking immunoinhibitory signals and enabling patients to produce an effective antitumour response. Inhibitors of CTLA4, PD1 or PDL1 administered as single agents have resulted in durable tumour regression in some patients, and combinations of PD1 and CTLA4 inhibitors may enhance antitumour benefit. Numerous additional immunomodulatory pathways as well as inhibitory factors expressed or secreted by myeloid and stromal cells in the tumour microenvironment are potential targets for synergizing with immune checkpoint blockade. Given the breadth of potential targets in the immune system, critical questions to address include which combinations should move forward in development and which patients will benefit from these treatments. This Review discusses the leading drug targets that are expressed on tumour cells and in the tumour microenvironment that allow enhancement of the antitumour immune response.

As representatives of immune checkpoint blockades (ICBs), antibodies targeting cytotoxic T-lymphocyte antigen-4 (CTLA-4) and programmed cell death-1 (PD-1)/programmed death-ligand 1 (PD-L1) have significantly improved the treatment efficacies of a variety of malignant tumors. However, ICBs can also produce a wide range of immune-related adverse reactions, forcing patients to stop treatment and even affect the survival of the patients. Therefore, with the wide application of ICBs in clinical practice, clinical oncologists need to fully understand the possible side effects of ICBs therapy and the appropriate treatment strategies to improve the survival rate and therapeutic effect of patients receiving ICBs. DOI:10.3781/j.issn.1000-7431.2018.55.503

Hyponatremia in a Patient With Cancer

Almost all patients with prostate cancer progress to metastatic castration-resistant prostate cancer (mCRPC) despite initial responses. In cases where traditional first-line treatments prove ineffective, the potential of immune checkpoint blockade (ICB) therapy emerges as a promising approach for managing mCRPC. However, while immune checkpoint inhibitor monotherapy or combination therapy targeting cytotoxic T lymphocyte antigen 4 (CTLA-4) and/or programmed cell death-1 (PD-1)/PD-1 ligand 1 (PD-L1) axis has been regarded as the standard therapy in many solid tumours, mCRPC as ‘cold’ tumours are considered to be relatively resistant to ICB treatment. Encouragingly, recent evidence suggests that ICB therapy may be particularly beneficial in specific subgroups of patients with high PD-L1 tumour expression, high tumour mutational burden or high tumour microsatellite instability/mismatch repair deficiency. Better understanding of these predictive biomarkers could screen which patients are most likely to benefit. This review article examines biomarkers for screening patients potentially effective in immune checkpoint inhibitor therapy.

Background: Chronic lymphocytic leukemia (CLL) is characterized by the proliferation of dysfunctional B cells, resulting in significant immune dysregulation. Patients with CLL exhibit varied responses to B cell receptor (BCR) targeted therapies, emphasizing the need for tailored immunotherapy approaches. This study investigated B cell function in untreated patients with CLL, and we further explored the effects of ex vivo protein kinase C activation on immune checkpoint expression and B cell profiles. Methods: Peripheral blood samples were collected from 21 untreated patients with CLL at King Edward Hospital in South Africa, between 2019 and 2022. B cells were stimulated with phorbol myristate acetate (PMA) and ionomycin. Using flow cytometry, the study explored the levels of B cell subsets and immune checkpoint proteins programmed cell death 1 (PD-1), programmed cell death-ligand 1 (PD-L1), programmed cell death-ligand 2 (PD-L2) and cytotoxic T-lymphocyte associated protein 4 (CTLA-4) expression on various B cell subsets. Results: PMA and ionomycin B cell stimulation upregulated PD-1, CTLA-4 and PD-L2 expression on B cell subsets (p < 0.01). As expected, monoclonal antibodies targeting PD-1, PD-L1 and CTLA-4 significantly downregulated the CTLA-4 expression of B cell subsets (p < 0.05), while PD-L2 exhibited varied responses in different B cell subsets. Moreover, PD-1 and PD-L1 expression on total B cells significantly declined following their blockage (p < 0.01). In addition, these monoclonal antibodies increased the levels of CD19+CD27+ B cells (p < 0.0128) and activated CD19+CD27+ B cells (p < 0.01). Conclusions: Protein kinase C activation on B cells stimulates immune checkpoint expression. The use of monoclonal antibodies on B cells plays a critical role in the B cell function through the reduction in CD38 expressing activated B cells and upregulation of CD19+CD27+ B cells. Moreover, the monoclonal antibody targeting PD-1, PD-L1 and CTLA-4 are effective in reducing the expression of CTLA-4 on B cell subsets, while PD-1 and PD-L1 blockage may be effective in reducing the expression of these immune checkpoints on total B cells.

Keeping Tumors in Check: A Mechanistic Review of Clinical Response and Resistance to Immune Checkpoint Blockade in Cancer