TREM2 deficiency reprograms intestinal macrophages and microbiota to enhance anti-PD-1 tumor immunotherapy.

The gut microbiota and tumor-associated macrophages (TAMs) affect tumor responses to anti-programmed cell death protein 1 (PD-1) immune checkpoint blockade. Reprogramming TAM by either blocking or deleting the macrophage receptor triggering receptor on myeloid cells 2 (TREM2) attenuates tumor growth, and lack of functional TREM2 enhances tumor elimination by anti-PD-1. Here, we found that anti-PD-1 treatment combined with TREM2 deficiency in mice induces proinflammatory programs in intestinal macrophages and a concomitant expansion of Ruminococcus gnavus in the gut microbiota. Gavage of wild-type mice with R. gnavus enhanced anti-PD-1-mediated tumor elimination, recapitulating the effect occurring in the absence of TREM2. A proinflammatory intestinal environment coincided with expansion, increased circulation, and migration of TNF-producing CD4+ T cells to the tumor bed. Thus, TREM2 remotely controls anti-PD-1 immune checkpoint blockade through modulation of the intestinal immune environment and microbiota, with R. gnavus emerging as a potential probiotic agent for increasing responsiveness to anti-PD-1.

An On-Treatment Decreased Trend of Serum IL-6 and IL-8 as Predictive Markers Quickly Reflects Short-Term Efficacy of PD-1 Blockade Immunochemotherapy in Patients with Advanced Gastric Cancer.

Objective: Immunotherapy has proven effective in treating advanced gastric cancer (AGC), yet its benefits are limited to a subset of patients. Our aim is to swiftly identify prognostic biomarkers using cytokines to improve the precision of clinical guidance and decision-making for PD-1 inhibitor-based cancer immunotherapy in AGC. Materials and Methods: The retrospective study compared 36 patients with AGC who received combined anti-PD-1 immunotherapy and chemotherapy (immunochemotherapy) with a control group of 20 patients who received chemotherapy alone. The concentrations of TNF-α, IL-1ß, IL-2R, IL-6, IL-8, IL-10, and IL-17 in the serum were assessed using chemiluminescence immunoassay at three distinct time intervals following the commencement of immunochemotherapy. Results: When compared to controls, patients undergoing immunochemotherapy demonstrated a generalized rise in cytokine levels after the start of treatment. However, patients who benefited from immunochemotherapy showed a decrease in IL-6 or IL-8 concentrations throughout treatment (with varied trends observed for IL-1ß, IL-2R, IL-10, IL-17, and TNF-α) was evident in patients benefiting from immunochemotherapy but not in those who did not benefit. Among these markers, the combination of IL-6, IL-8, and CEA showed optimal predictive performance for short-term efficacy of immunochemotherapy in AGC patients. Conclusion: Reductions in IL-6/IL-8 levels observed during immunochemotherapy correlated with increased responsiveness to treatment effectiveness. These easily accessible blood-based biomarkers are predictive and rapid and may play a crucial role in identifying individuals likely to derive benefits from PD-1 blockade immunotherapy.

An NGS-based assay for accurate detection and quantification of immune gene expression in mouse tumor models.

Tumor microenvironment (TME) is a complex dynamic system with many tumor-interacting components including tumor-infiltrating leukocytes (TILs), cancer associated fibroblasts, blood vessels, and other stromal constituents. It intrinsically affects tumor development and pharmacology of oncology therapeutics, particularly immune-oncology (IO) treatments. Accurate measurement of TME is therefore of great importance for understanding the tumor immunity, identifying IO treatment mechanisms, developing predictive biomarkers, and ultimately, improving the treatment of cancer. Here, we introduce a mouse-IO NGS-based (NGSmIO) assay for accurately detecting and quantifying the mRNA expression of 1080 TME related genes in mouse tumor models. The NGSmIO panel was shown to be superior to the commonly used microarray approach by hosting 300 more relevant genes to better characterize various lineage of immune cells, exhibits improved mRNA and protein expression correlation to flow cytometry, shows stronger correlation with mRNA expression than RNAseq with 10x higher sequencing depth, and demonstrates higher sensitivity in measuring low-expressed genes. We describe two studies; firstly, detecting the pharmacodynamic change of interferon-γ expression levels upon anti-PD-1: anti-CD4 combination treatment in MC38 and Hepa 1-6 tumors; and secondly, benchmarking baseline TILs in 14 syngeneic tumors using transcript level expression of lineage specific genes, which demonstrate effective and robust applications of the NGSmIO panel.

Immune checkpoint inhibitors induce acute interstitial nephritis in mice with increased urinary MCP1 and PD-1 glomerular expression.

INTRODUCTION: Immune checkpoint inhibitors (ICIs) induce acute interstitial nephritis (AIN) in 2-5% of patients, with a clearly higher incidence when they are combined with platinum derivatives. Unfortunately, suitable disease models and non-invasive biomarkers are lacking. To fill this gap in our understanding, we investigated the renal effects of cisplatin and anti-PD-L1 antibodies in mice, assessing PD-1 renal expression and cytokine levels in mice with AIN, and then we compared these findings with those in AIN-diagnosed cancer patients. METHODS: Twenty C57BL6J mice received 200 µg of anti-PD-L1 antibody and 5 mg/kg cisplatin intraperitoneally and were compared with those receiving cisplatin (n = 6), anti-PD-L1 (n = 7), or saline (n = 6). After 7 days, the mice were euthanized. Serum and urinary concentrations of TNFα, CXCL10, IL-6, and MCP-1 were measured by Luminex. The kidney sections were stained to determine PD-1 tissue expression. Thirty-nine cancer patients with AKI were enrolled (AIN n = 33, acute tubular necrosis (ATN) n = 6), urine MCP-1 (uMCP-1) was measured, and kidney sections were stained to assess PD-1 expression. RESULTS: Cisplatin and anti PD-L1 treatment led to 40% AIN development (p = 0.03) in mice, accompanied by elevated serum creatinine and uMCP1. AIN-diagnosed cancer patients also had higher uMCP1 levels than ATN-diagnosed patients, confirming our previous findings. Mice with AIN exhibited interstitial PD-1 staining and stronger glomerular PD-1 expression, especially with combination treatment. Conversely, human AIN patients only showed interstitial PD-1 positivity. CONCLUSIONS: Only mice receiving cisplatin and anti-PDL1 concomitantly developed AIN, accompanied with a more severe kidney injury. AIN induced by this drug combination was linked to elevated uMCP1, consistently with human AIN, suggesting that uMCP1 can be potentially used as an AIN biomarker.

Efficacy and safety of PD-1/PD-L1 inhibitors in elderly patients with advanced non-small cell lung cancer.

OBJECTIVE: This study aimed to investigate the efficacy and safety of PD-1/PD-L1 inhibitors in treatment of elderly patients with advanced non-small cell lung cancer (NSCLC). METHODS: Patients with advanced NSCLC ≥70 years old who received PD-1/PD-L1 inhibitors in our hospital were retrospectively analyzed. According to age, the patient were stratified as follows: 70-75 years old, 76-80 years old, and >80 years old. Kaplan-Meier method was used for survival analysis, and univariate and multivariate Cox proportional hazards regression models were used to analyze the correlation between different clinical characteristics and survival. RESULTS: A total of 58 elderly patients with advanced non-small cell cancer were enrolled in this study. Patients aged 70-75, 76-80, and >80 years old were 32, 19, and 7, respectively. For the all, median OS was 17.0 months, and PFS was 7.0 months. PFS and OS did not differ according to age (P = 0.396, 0.054, respectively). Univariate analysis showed that PS of 0-1, stage III, first-line therapy and irAEs were associated with longer PFS, and PS of 0-1, stage III, and first-line therapy were associated with longer OS. Multivariate analysis showed that patients with stage III had longer PFS. PFS and OS of patients with PS ≥ 2 were significantly shorter than those of patients with PS of 0-1. CONCLUSIONS: In the present real-world retrospective cohort, PD-1/PD-L1 inhibitors are effective and well tolerated in elderly patients with advanced NSCLC. Immunotherapy should be actively used as early as possible in older patients advanced NSCLC.

Probing the Immunoreceptor Tyrosine-Based Inhibition Motif Interaction Protein Partners with Proteomics.

Phosphorylation of tyrosine is the basic mode of protein function and signal transduction in organisms. This process is regulated by protein tyrosine kinases (PTKs) and protein tyrosinases (PTPs). Immunoreceptor tyrosine-based inhibition motif (ITIM) has been considered as regulating the PTP activity through the interaction with the partner proteins in the cell signal pathway. The ITIM sequences need to be phosphorylated first to active the downstream signaling proteins. To explore potential regulatory mechanisms, the ITIM sequences of two transmembrane immunoglobulin proteins, myelin P0 protein-related protein (PZR) and programmed death 1 (PD-1), were analyzed to investigate their interaction with proteins involved in regulatory pathways. We discovered that phosphorylated ITIM sequences can selectively interact with the tyrosine phosphatase SHP2. Specifically, PZR-N-ITIM (pY) may be critical in the interaction between the ITIM and SH2 domains of SHP2, while PD1-C-ITSM (pY) may play a key role in the interaction between the ITIM and SH2 domains of SHP2. Quite a few proteins were identified containing the SH2 domain, exhibiting phosphorylation-mediated interaction with PZR-ITIM. In this study, 14 proteins with SH2 structural domains were identified by GO analysis on 339 proteins associated to the affinity pull-down of PZR-N-ITIM (pY). Through the SH2 domains, these proteins may interact with PZR-ITIM in a phosphorylation-dependent manner.

The efficacy and safety of adding PD-1 blockade to induction chemotherapy and concurrent chemoradiotherapy (IC-CCRT) for locoregionally advanced nasopharyngeal carcinoma: an observational, propensity score-matched analysis.

BACKGROUND: Despite the success of PD-1 blockade in recurrent/metastatic nasopharyngeal carcinoma (NPC), its effect for locoregionally advanced NPC (LANPC) remains unclear. This study aimed to evaluate the benefit of adding PD-1 blockade to the current standard treatment (gemcitabine and cisplatin IC  plus cisplatin CCRT ) for LANPC patients. METHODS: From January 2020 to November 2022, 347 patients with non-metastatic high-risk LANPC (stage III-IVA, excluding T3-4N0) were included. Of the 347 patients, 268 patients were treated with standard treatment (IC-CCRT), and 79 received PD-1 blockade plus IC-CCRT (PD-1 group). For the PD-1 group, PD-1 blockade was given intravenously once every 3 weeks for up to 9 cycles (3 induction and 6 adjuvant). The primary endpoint was disease-free survival (DFS) (i.e. freedom from local/regional/distant failure or death). The propensity score matching (PSM) with the ratio of 1:2 was performed to control confounding factors. RESULTS: After PSM analysis, 150 patients receiving standard treatment and 75 patients receiving additional PD-1 blockade remained in the current analysis. After three cycles of IC, the PD-1 group had significantly higher rates of complete response (defined as disappearance of all target lesions; 24% vs. 9%; P = 0.006) and complete biological response (defined as undetectable cell-free Epstein-Barr virus DNA, cfEBV DNA; 79% vs. 65%; P = 0.046) than that in the standard group. And the incidence of grade 3-4 toxicity during IC was 47% in the PD-1 group and 41% in the standard group, with no significant difference (P = 0.396). During follow-up period, additional PD-1 blockade to standard treatment improved 3-year DFS from 84 to 95%, with marginal statistical significance (HR, 0.28; 95%CI, 0.06-1.19; P = 0.064). CONCLUSION: Additiaonl PD-1 blockade to gemcitabine and cisplatin IC and adjuvant treatment results in significant improvement in tumor regression, cfEBV DNA clearance, superior DFS, and comparable toxicity profiles in high-risk LANPC patients.

Expression of CD39 is associated with T cell exhaustion in ovarian cancer and its blockade reverts T cell dysfunction.

Immune exhaustion is a hallmark of ovarian cancer. Using multiparametric flow cytometry, the study aimed to analyze protein expression of novel immunological targets on CD3+ T cells isolated from the peripheral blood (n = 20), malignant ascites (n = 16), and tumor tissue (n = 6) of patients with ovarian cancer (OVCA). The study revealed an increased proportion of effector memory CD8+ T cells in OVCA tissue and malignant ascites. An OVCA-characteristic PD-1high CD8+ T cell population was detected, which differed from PD-1lowCD8+ T cells by increased co-expression of TIGIT, CD39, and HLA-DR. In addition, these OVCA-characteristic CD8+ T cells showed reduced expression of the transcription factor TCF-1, which may also indicate reduced effector function and memory formation. On the contrary, the transcription factor TOX, which significantly regulates terminal T cell-exhaustion, was found more frequently in these cells. Further protein and gene analysis showed that CD39 and CD73 were also expressed on OVCA tumor cells isolated from solid tumors (n = 14) and malignant ascites (n = 9). In the latter compartment, CD39 and CD73 were also associated with the expression of the "don't eat me" molecule CD24 on tumor cells. Additionally, ascites-derived CD24+EpCAM+ tumor cells showed a higher frequency of CD39+ or CD73+ cells. Furthermore, CD39 expression was associated with unfavorable clinical parameters. Expression of CD39 on T cells was upregulated through CD3/CD28 stimulation and its blockade by a newly developed nanobody construct resulted in increased proliferation (eFluor), activation (CD25 and CD134), and production of cytotoxic cytokines (IFN-γ, TNF-α, and granzyme-B) of CD8+ T cells.

PD-1/CD80+ small extracellular vesicles from immunocytes induce cold tumours featured with enhanced adaptive immunosuppression.

Only a minority of cancer patients benefit from immune checkpoint blockade therapy. Sophisticated cross-talk among different immune checkpoint pathways as well as interaction pattern of immune checkpoint molecules carried on circulating small extracellular vesicles (sEV) might contribute to the low response rate. Here we demonstrate that PD-1 and CD80 carried on immunocyte-derived sEVs (I-sEV) induce an adaptive redistribution of PD-L1 in tumour cells. The resulting decreased cell membrane PD-L1 expression and increased sEV PD-L1 secretion into the circulation contribute to systemic immunosuppression. PD-1/CD80+ I-sEVs also induce downregulation of adhesion- and antigen presentation-related molecules on tumour cells and impaired immune cell infiltration, thereby converting tumours to an immunologically cold phenotype. Moreover, synchronous analysis of multiple checkpoint molecules, including PD-1, CD80 and PD-L1, on circulating sEVs distinguishes clinical responders from those patients who poorly respond to anti-PD-1 treatment. Altogether, our study shows that sEVs carry multiple inhibitory immune checkpoints proteins, which form a potentially targetable adaptive loop to suppress antitumour immunity.

Deep learning based digital pathology for predicting treatment response to first-line PD-1 blockade in advanced gastric cancer.

BACKGROUND: Advanced unresectable gastric cancer (GC) patients were previously treated with chemotherapy alone as the first-line therapy. However, with the Food and Drug Administration's (FDA) 2022 approval of programmed cell death protein 1 (PD-1) inhibitor combined with chemotherapy as the first-li ne treatment for advanced unresectable GC, patients have significantly benefited. However, the significant costs and potential adverse effects necessitate precise patient selection. In recent years, the advent of deep learning (DL) has revolutionized the medical field, particularly in predicting tumor treatment responses. Our study utilizes DL to analyze pathological images, aiming to predict first-line PD-1 combined chemotherapy response for advanced-stage GC. METHODS: In this multicenter retrospective analysis, Hematoxylin and Eosin (H&E)-stained slides were collected from advanced GC patients across four medical centers. Treatment response was evaluated according to iRECIST 1.1 criteria after a comprehensive first-line PD-1 immunotherapy combined with chemotherapy. Three DL models were employed in an ensemble approach to create the immune checkpoint inhibitors Response Score (ICIsRS) as a novel histopathological biomarker derived from Whole Slide Images (WSIs). RESULTS: Analyzing 148,181 patches from 313 WSIs of 264 advanced GC patients, the ensemble model exhibited superior predictive accuracy, leading to the creation of ICIsNet. The model demonstrated robust performance across four testing datasets, achieving AUC values of 0.92, 0.95, 0.96, and 1 respectively. The boxplot, constructed from the ICIsRS, reveals statistically significant disparities between the well response and poor response (all p-values < = 0.001). CONCLUSION: ICIsRS, a DL-derived biomarker from WSIs, effectively predicts advanced GC patients' responses to PD-1 combined chemotherapy, offering a novel approach for personalized treatment planning and allowing for more individualized and potentially effective treatment strategies based on a patient's unique response situations.

Vaccimel immunization is associated with enhanced response to treatment with anti-PD-1 monoclonal antibodies in cutaneous melanoma patients - a case reports study.

Cancer vaccines are gaining ground as immunotherapy options. We have previously demonstrated in cutaneous melanoma (CM) patients that adjuvant treatment with VACCIMEL, a mixture of four irradiated CM cell lines co-adjuvanted with BCG and GM-CSF, increases the cellular immune response to melanocyte differentiation antigens, cancer-testis antigens and neoantigens, with respect to basal levels. On the other hand, it is also known that treatment with anti-PD-1 monoclonal antibodies (MAbs), acting on pre-existing tumor-reactive lymphocytes, induces clinical responses in CM patients, albeit in a fraction of treated patients. A combination of both treatments would appear therefore desirable. In this paper, we describe CM patients who, having progressed even years after vaccination, were treated with anti-PD-1 MAbs. In 5/5 of such progressor patients, complete responses were obtained which lasted between 3 and 65+ months. Three of the patients remain disease-free and two recurred. One of the patients passed away after a recurrence of brain metastases. We suggest that clonally expanded reactive lymphocytes induced by VACCIMEL partially remain as memory cells, which may be recalled after tumor recurrence and may foster ulterior activity of anti-PD-1 MAbs.

Engineering PD-1-targeted small protein variants for in vitro diagnostics and in vivo PET imaging.

BACKGROUND: Programmed cell death 1 (PD-1) belongs to immune checkpoint proteins ensuring negative regulation of the immune response. In non-small cell lung cancer (NSCLC), the sensitivity to treatment with anti-PD-1 therapeutics, and its efficacy, mostly correlated with the increase of tumor infiltrating PD-1+ lymphocytes. Due to solid tumor heterogeneity of PD-1+ populations, novel low molecular weight anti-PD-1 high-affinity diagnostic probes can increase the reliability of expression profiling of PD-1+ tumor infiltrating lymphocytes (TILs) in tumor tissue biopsies and in vivo mapping efficiency using immune-PET imaging. METHODS: We designed a 13 kDa ß-sheet Myomedin scaffold combinatorial library by randomization of 12 mutable residues, and in combination with ribosome display, we identified anti-PD-1 Myomedin variants (MBA ligands) that specifically bound to human and murine PD-1-transfected HEK293T cells and human SUP-T1 cells spontaneously overexpressing cell surface PD-1. RESULTS: Binding affinity to cell-surface expressed human and murine PD-1 on transfected HEK293T cells was measured by fluorescence with LigandTracer and resulted in the selection of most promising variants MBA066 (hPD-1 KD = 6.9 nM; mPD-1 KD = 40.5 nM), MBA197 (hPD-1 KD = 29.7 nM; mPD-1 KD = 21.4 nM) and MBA414 (hPD-1 KD = 8.6 nM; mPD-1 KD = 2.4 nM). The potential of MBA proteins for imaging of PD-1+ populations in vivo was demonstrated using deferoxamine-conjugated MBA labeled with 68Galium isotope. Radiochemical purity of 68Ga-MBA proteins reached values 94.7-99.3% and in vitro stability in human serum after 120 min was in the range 94.6-98.2%. The distribution of 68Ga-MBA proteins in mice was monitored using whole-body positron emission tomography combined with computerized tomography (PET/CT) imaging up to 90 min post-injection and post mortem examined in 12 mouse organs. The specificity of MBA proteins was proven by co-staining frozen sections of human tonsils and NSCLC tissue biopsies with anti-PD-1 antibody, and demonstrated their potential for mapping PD-1+ populations in solid tumors. CONCLUSIONS: Using directed evolution, we developed a unique set of small binding proteins that can improve PD-1 diagnostics in vitro as well as in vivo using PET/CT imaging.

Efficacy and safety of hepatic arterial infusion chemotherapy combined with lenvatinib and PD-1 inhibitors for advanced hepatocellular carcinoma with macrovascular invasion.

BACKGROUND AND AIMS: The prognosis of hepatocellular carcinoma (HCC) with macrovascular invasion(MaVI)is poor, and the treatment is limited. This study aims to explore the efficacy and safety of hepatic arterial infusion chemotherapy (HAIC), combined with lenvatinib and programmed cell death-1(PD-1) inhibitor in the first-line treatment of HCC with MaVI. METHODS: From July 2020 to February 2022, we retrospectively analyzed consecutive patients with HCC with MaVI who received hepatic arterial infusion FOLFOX(oxaliplatin, 5-fluorouracil, and leucovorin)combined with lenvatinib and PD-1 inhibitor. The efficacy was evaluated by RECIST 1.1. Kaplan-Meier was used to explore the overall survival and progression-free survival (PFS), and the COX regression model was used to analyze the risk factors of PFS. Adverse events (AEs) were evaluated according to CTCAE5.0. RESULTS: Thirty-two patients with HCC complicated with MaVI were recruited from the Second Affiliated Hospital of Nanchang University. Among the patients treated with HAIC combined with lenvatinib and PD-1 inhibitor, ten patients (31.25%) got partial response, eighteen patients (56.25%) maintained stable disease and four patients (12.50%) suffered progressive disease during follow-up; and objective response rate was 31.25%, and disease control rate was 87.5%. The median PFS was 179 days. Univariate and multivariate Cox analysis showed that the extrahepatic metastases and Child-Pugh score were independent prognostic factors of PFS. Twenty-two (68.75%) patients suffered adverse reactions. The main AEs were elevated transaminase (46.87%), thrombocytopenia (40.63%), hypoalbuminemia (28.13%), nausea and vomiting (21.88%), leukopenia (18.76%), abdominal pain (15.63%), hypertension (15.63%) and fever (15.63%). There were seven cases (21.88%) that had grade 3 or above AEs; Among them, two cases with elevated transaminase (6.25%), leukopenia, thrombocytopenia, nausea and vomiting, abdominal pain, and diarrhea occurred in one case respectively. Moreover, no treatment-related death was observed. CONCLUSIONS: Hepatic arterial infusion of FOLFOX combined with lenvatinib and PD-1 inhibitor as the first-line treatment for HCC complicated with MaVI is effective, and adverse reactions are tolerable.

Systematic analysis of mucosal-associated invariant T cells in haematological malignancies.

Mucosal-associated invariant T-cells (MAIT) are unconventional T-cells with cytotoxic and pro-inflammatory properties. Previous research has reported contradictory findings on their role in cancerogenesis with data being even scarcer in haematological malignancies. Here, we report the results of a systematic analysis of MAIT cells in treatment-naïve patients with a broad range of haematological malignancies. We analysed peripheral blood of 204 patients and 50 healthy subjects. The pool of haematological patients had a statistically significant lower both the absolute value (median values, 0.01 × 109/L vs. 0.05 × 109/L) of MAIT cells and their percentage (median values 0.94% vs. 2.56%) among T-cells compared to the control group. Separate analysis showed that the decrease in the absolute number of MAIT cells is significant in patients with acute myeloid leukaemia, myeloproliferative neoplasms, plasma cell myeloma, B-cell non-Hodgkin lymphomas, otherwise not specified, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone lymphoma compared to the control population. Furthermore, in haematological malignancies, MAIT cells overexpress PD-1 (average values, 51.7% vs. 6.7%), HLA-DR (average values, 40.2% vs. 7%), CD38 (average values, 25.9% vs. 4.9%) and CD69 (average values, 40.2% vs. 9.2%). Similar results were obtained when comparing patients with individual malignancies to the control population. Our data show that the depletion of circulating MAIT cells is a common observation in a broad spectrum of haematological malignancies. In addition to their reduced numbers, MAIT cells acquire an activated/exhausted phenotype.

Artificial neural network identified a 20-gene panel in predicting immunotherapy response and survival benefits after anti-PD1/PD-L1 treatment in glioblastoma patients.

BACKGROUND: Immune checkpoint inhibitors (ICIs) are a promising immunotherapy approach, but glioblastoma clinical trials have not yielded satisfactory results. OBJECTIVE: To screen glioblastoma patients who may benefit from immunotherapy. METHODS: Eighty-one patients receiving anti-PD1/PD-L1 treatment from a large-scale clinical trial and 364 patients without immunotherapy from The Cancer Genome Atlas (TCGA) were included. Patients in the ICI-treated cohort were divided into responders and nonresponders according to overall survival (OS), and the most critical responder-relevant features were screened using random forest (RF). We constructed an artificial neural network (ANN) model and verified its predictive value with immunotherapy response and OS. RESULTS: We defined two groups of ICI-treated glioblastoma patients with large differences in survival benefits as nonresponders (OS ≤6 months, n = 18) and responders (OS ≥17 months, n = 8). No differentially mutated genes were observed between responders and nonresponders. We performed RF analysis to select the most critical responder-relevant features and developed an ANN with 20 input variables, five hidden neurons and one output neuron. Receiver operating characteristic analysis and the DeLong test demonstrated that the ANN had the best performance in predicting responders, with an AUC of 0.97. Survival analysis indicated that ANN-predicted responders had significantly better OS rates than nonresponders. CONCLUSION: The 20-gene panel developed by the ANN could be a promising biomarker for predicting immunotherapy response and prognostic benefits in ICI-treated GBM patients and may guide oncologists to accurately select potential responders for the preferential use of ICIs.

A novel anti-LAG-3/TIGIT bispecific antibody exhibits potent anti-tumor efficacy in mouse models as monotherapy or in combination with PD-1 antibody.

We report the generation of a novel anti-LAG-3/TIGIT bispecific IgG4 antibody, ZGGS15, and evaluated its anti-tumor efficacy in mouse models as monotherapy or in combination with a PD-1 antibody. ZGGS15 exhibited strong affinities for human LAG-3 and TIGIT, with KDs of 3.05 nM and 2.65 nM, respectively. ZGGS15 has EC50s of 0.69 nM and 1.87 nM for binding to human LAG-3 and TIGIT on CHO-K1 cells, respectively. ZGGS15 competitively inhibited the binding of LAG-3 to MHC-II (IC50 = 0.77 nM) and the binding of TIGIT to CD155 (IC50 = 0.24 nM). ZGGS15 does not induce ADCC, CDC, or obvious cytokine production. In vivo results showed that ZGGS15 had better anti-tumor inhibition than single anti-LAG-3 or anti-TIGIT agents and demonstrated a synergistic effect when combined with nivolumab, with a significantly higher tumor growth inhibition of 95.80% (p = 0.001). The tumor volume inhibition rate for ZGGS15 at 2 mg/kg was 69.70%, and for ZGGS15 at 5 mg/kg plus nivolumab at 1 mg/kg, it was 94.03% (p < 0.001). Our data reveal that ZGGS15 exhibits potent anti-tumor efficacy without eliciting ADCC or CDC or causing cytokine production, therefore having a safe profile.

GNUV201, a novel human/mouse cross-reactive and low pH-selective anti-PD-1 monoclonal antibody for cancer immunotherapy.

BACKGROUND: Several PD-1 antibodies approved as anti-cancer therapies work by blocking the interaction of PD-1 with its ligand PD-L1, thus restoring anti-cancer T cell activities. These PD-1 antibodies lack inter-species cross-reactivity, necessitating surrogate antibodies for preclinical studies, which may limit the predictability and translatability of the studies. RESULTS: To overcome this limitation, we have developed an inter-species cross-reactive PD-1 antibody, GNUV201, by utilizing an enhanced diversity mouse platform (SHINE MOUSE™). GNUV201 equally binds to human PD-1 and mouse PD-1, equally inhibits the binding of human PD-1/PD-L1 and mouse PD-1/PD-L1, and effectively suppresses tumor growth in syngeneic mouse models. The epitope of GNUV201 mapped to the "FG loop" of hPD-1, distinct from those of Keytruda® ("C'D loop") and Opdivo® (N-term). Notably, the structural feature where the protruding epitope loop fits into GNUV201's binding pocket supports the enhanced binding affinity due to slower dissociation (8.7 times slower than Keytruda®). Furthermore, GNUV201 shows a stronger binding affinity at pH 6.0 (5.6 times strong than at pH 7.4), which mimics the hypoxic and acidic tumor microenvironment (TME). This phenomenon is not observed with marketed antibodies (Keytruda®, Opdivo®), implying that GNUV201 achieves more selective binding to and better occupancy on PD-1 in the TME. CONCLUSIONS: In summary, GNUV201 exhibited enhanced affinity for PD-1 with slow dissociation and preferential binding in TME-mimicking low pH. Human/monkey/mouse inter-species cross-reactivity of GNUV201 could enable more predictable and translatable efficacy and toxicity preclinical studies. These results suggest that GNUV201 could be an ideal antibody candidate for anti-cancer drug development.

Clinical and Biologic Correlates of ADORA2A Transcriptomic Expression in Cancer.

ADORA2A (adenosine A2a receptor) and ADORA2B propagate immunoregulatory signals, including restricting both innate and adaptive immunity, though recent data also suggest a tumor suppressor effect in certain settings. We evaluated the RNA expression from 514 tumors in a clinical-grade laboratory; 489 patients with advanced/metastatic disease had clinical outcome correlates. Transcript expression was standardized to internal housekeeping genes and ranked (0-100 scale) relative to 735 specimens from 35 different cancer types. Transcript abundance rank values were defined as "low/moderate" (0-74) or "high" (75-100) percentile RNA expression ranks. Overall, 20.8% of tumors had high ADORA2A (≥75 percentile RNA rank). The greatest proportion of high ADORA2A expressors was found in neuroendocrine and breast cancers and sarcomas, whereas the lowest was found in colorectal and ovarian cancers, albeit with patient-to-patient variability. In multivariable logistic regression analysis, there was a significant positive correlation between high ADORA2A RNA expression and a high expression of the immune checkpoint-related molecules PD-1 (p = 0.015), VISTA (p ≤ 0.001), CD38 (p = 0.031), and CD39 (p ≤ 0.001). In 217 immunotherapy-treated patients, high ADORA2A did not correlate significantly with progression-free (p = 0.51) or overall survival (OS) (p = 0.09) from the initiation of the checkpoint blockade. However, high versus not-high ADORA2A transcript expression correlated with longer OS from the time of advanced/metastatic disease (N = 489 patients; (HR 0.69 (95% CI 0.51-0.95) (p = 0.02)). Therefore, high ADORA2A transcript levels may be a favorable prognostic factor, unrelated to immunotherapy. Importantly, ascertaining co-expression patterns of ADORA2A with PD-1 and VISTA in individual tumors as a basis for the precision co-targeting of ADORA2A and these other checkpoint-related molecules warrants investigation in clinical trials.

Predicting effect of anti-PD-1/PD-L1 inhibitors therapy for hepatocellular carcinoma by detecting plasma metabolite based on UHPLC-MS.

Introduction: Anti-PD-1/PD-L1 inhibitors therapy has become a promising treatment for hepatocellular carcinoma (HCC), while the therapeutic efficacy varies significantly among effects for individual patients are significant difference. Unfortunately, specific predictive biomarkers indicating the degree of benefit for patients and thus guiding the selection of suitable candidates for immune therapy remain elusive.no specific predictive biomarkers are available indicating the degree of benefit for patients and thus screening the preferred population suitable for the immune therapy. Methods: Ultra-high-pressure liquid chromatography-mass spectrometry (UHPLC-MS) considered is an important method for analyzing biological samples, since it has the advantages of high rapid, high sensitivity, and high specificity. Ultra-high-pressure liquid chromatography-mass spectrometry (UHPLC-MS) has emerged as a pivotal method for analyzing biological samples due to its inherent advantages of rapidity, sensitivity, and specificity. In this study, potential metabolite biomarkers that can predict the therapeutic effect of HCC patients receiving immune therapy were identified by UHPLC-MS. Results: A partial least-squares discriminant analysis (PLS-DA) model was established using 14 glycerophospholipid metabolites mentioned above, and good prediction parameters (R2 = 0.823, Q2 = 0.615, prediction accuracy = 0.880 and p < 0.001) were obtained. The relative abundance of glycerophospholipid metabolite ions is closely related to the survival benefit of HCC patients who received immune therapy. Discussion: This study reveals that glycerophospholipid metabolites play a crucial role in predicting the efficacy of immune therapy for HCC.

Challenges in autoimmune polyendocrine syndrome type 2 with the full triad induced by anti-programmed cell death 1: a case report and review of the literature.

Background: Immune checkpoint inhibitors (ICPis) induce autoimmune diseases, including autoimmune polyendocrine syndrome type 2 (APS-2), which is defined as a combination of at least two of the following endocrinopathies: autoimmune thyroid disease, type 1 diabetes, and Addison's disease. Cases with the full triad are rare. We present a case of an elderly woman who developed APS-2 with the complete triad shortly after starting anti-programmed cell death 1 (anti-PD1) treatment and review the related literature. Case: A 60-year-old woman, without any personal or family history of autoimmune and endocrine diseases, started the immunotherapy of anti-PD1 (camrelizumab) for squamous cell carcinoma of the urethral meatus. She developed primary hypothyroidism with elevated antibodies to thyroid peroxidase and thyroglobulin after 25 weeks of treatment, and developed primary adrenal insufficiency with adrenal crisis and fulminant type 1 diabetes with ketoacidosis after 45 weeks. Therefore, this patient met the diagnosis of APS-2 and was given multiple hormone replacement including glucocorticoid, levothyroxine and insulin therapy. Continuous improvement was achieved through regular monitoring and titration of the dosage. Conclusions: Different components of APS-2 may appear at different time points after anti-PD1 administration, and can be acute and life-threatening. A good prognosis can be obtained by appropriate replacement with multiple hormones. Insights: With the clinical application of ICPis to APS-2, the complexity of its treatment should be paid enough attention.