CD161+CD127+CD8+ T cell subsets can predict the efficacy of anti-PD-1 immunotherapy in non-small cell lung cancer with diabetes mellitus.

The role of CD161+CD127+CD8+ T cells in non-small cell lung cancer (NSCLC) patients with diabetes remains unexplored. This study determined the prevalence, phenotype, and function of CD8+ T cell subsets in NSCLC with diabetes. We recruited NSCLC patients (n = 436) treated with anti-PD-1 immunotherapy as first-line treatment. The progression-free survival (PFS), overall survival (OS), T cells infiltration, and peripheral blood immunological characteristics were analyzed in NSCLC patients with or without diabetes. NSCLC patients with diabetes exhibited shorter PFS and OS (p = 0.0069 and p = 0.012, respectively) and significantly lower CD8+ T cells infiltration. Mass cytometry by time-of-flight (CyTOF) showed a higher percentage of CD161+CD127+CD8+ T cells among CD8+T cells in NSCLC with diabetes before anti-PD-1 treatment (p = 0.0071) than that in NSCLC without diabetes and this trend continued after anti-PD-1 treatment (p = 0.0393). Flow cytometry and multiple-immunofluorescence confirmed that NSCLC with diabetes had significantly higher CD161+CD127+CD8+ T cells to CD8+T cells ratios than NSCLC patients without diabetes. The RNA-sequencing analysis revealed immune-cytotoxic genes were reduced in the CD161+CD127+CD8+ T cell subset compared to CD161+CD127-CD8+ T cells in NSCLC with diabetes. CD161+CD127+CD8+ T cells exhibited more T cell-exhausted phenotypes in NSCLC with diabetes. NSCLC patients with diabetes with ≥ 6.3% CD161+CD127+CD8+ T cells to CD8+T cells ratios showed worse PFS. These findings indicate that diabetes is a risk factor for NSCLC patients who undergo anti-PD-1 immunotherapy.CD161+CD127+CD8+ T cells could be a key indicator of a poor prognosis in NSCLC with diabetes. Our findings would help in advancing anti-PD-1 therapy in NSCLC patients with diabetes.

Dual immunotherapy alternating with anti-PD-1 antibody plus liposomal doxorubicin show good efficacy in prostate epithelioid hemangioendothelioma: a case report.

Epithelioid hemangioendothelioma is a rare vascular malignancy, and currently, there is no standard treatment regimen for this disease and existing treatment options have limited efficacy. In this case report, we present a patient with lung and lymph node metastases from prostate epithelioid hemangioendothelioma who achieved a significant partial response. This was accomplished through alternating nivolumab therapy with ipilimumab and liposomal doxorubicin, resulting in a progression-free-survival more than 6 months to date. The treatment was well-tolerated throughout. Our report suggests that dual immunotherapy alternating with anti-PD-1antibody plus doxorubicin may be a potential treatment modality for epithelioid hemangioendothelioma. However, larger sample studies are necessary to ascertain the effectiveness of this treatment strategy and it is essential to continue monitoring this patient to sustain progression-free survival and overall survival.

Differences in the risk of immune-related pneumonitis between PD-1 and PD-L1 inhibitors: a meta-analysis according to the new mirror-principle and PRISMA guidelines.

PURPOSE: To compare the risk of immune-associated pneumonitis between PD-1 and PD-L1 inhibitors, the meta-analysis was designed. METHOD: The difference in risk of immune-associated pneumonitis between PD-1 and PD-L1 inhibitors was assessed by two different meta-analysis methods, the Mirror-pairing and the PRISMA guidelines. RESULTS: A total of eighty-eight reports were used for meta-analysis, while thirty-two studies were used for the Mirror-pairing. Both PD-1 and PD-L1 inhibitors (used alone or combined with chemotherapy) increased the risk of developing immune-related pneumonitis (P < 0.00001; P < 0.00001). Based on indirect analyses results (subgroup analyses), the risk of PD-L1-induced pneumonitis was weaker than that of PD-1 inhibitors when the control group was chemotherapy (OR = 3.33 vs. 5.43) or placebo (OR = 2.53 vs. 3.19), while no obvious significant differences were found (P = 0.17; P = 0.53). For the Mirror-pairing-based meta-analysis, the risk of PD-1-induced pneumonitis was significantly higher than that of PD-L1 inhibitors (OR = 1.46, 95%CI [1.08, 1.98], I2 = 0%, Z = 2.47 (P = 0.01)). However, this difference was not significant, when they were combined with chemotherapy (OR = 1.05, 95%CI [0.68, 1.60], I2 = 38%, Z = 0.21 (P = 0.84)). CONCLUSION: Both PD-1 and PD-L1 inhibitors increased the risk of immune-related pneumonitis, while the risk of PD-1-induced pneumonitis was significantly higher than that of PD-L1 inhibitors.

Clinical Efficacy of Taxol Plus Platinum (TP) Chemotherapy Combined with Delayed Administration of PD-1 Inhibitors in Patients with Locally Advanced, Recurrent or Metastatic Esophageal Squamous Cell Carcinoma: A Retrospective Study.

Purpose: Immune checkpoint inhibitors (ICIs) combined with chemotherapy have become the first-line standard treatment for locally advanced or metastatic esophageal squamous cell carcinoma (ESCC). The evidence also demonstrates improved synergistic effects of chemotherapy when combined with delayed administration of ICIs. In this study, we conducted a retrospective investigation into the treatment efficacy of taxol plus platinum (TP) chemotherapy combined with delayed administration of PD-1 inhibitors for ESCC patients. Patients and Methods: Clinical data of ESCC patients who received PD-1 inhibitors 3-5 days after TP chemotherapy as first-line treatment was retrospectively reviewed between January 2019 and April 2023. Clinical outcomes and treatment safety were analyzed. The potential roles of neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), monocyte-to-lymphocyte ratio (MLR), and pan-immune-inflammation value (PIV) were investigated. Results: A total of 34 locally advanced, recurrent or metastatic ESCC patients received PD-1 inhibitors 3-5 days following TP chemotherapy were included. The objective response rate (ORR) and disease control rate (DCR) were 85.3% and 97.1% respectively. The median progression-free survival (PFS) and overall survival (OS) were 13.2 and 19.1 month respectively. Seven patients received radical surgery, 1 patient achieved pathologic complete response (pCR) and 3 patients achieved major pathologic response (MPR). Among the 27 patients without surgery, the median PFS and OS were 9.7 and 19.1 month respectively. A more favorable prognosis was correlated with NLR less than 3 at the 3rd and 4th cycle of immunochemotherapy. No significant correlations between other parameters (PLR, MLR and PIV) and prognosis were observed. A total of 22 patients developed grade 3-4 toxicity events. Conclusion: The optimized sequence of PD-1 inhibitors administered 3-5 days after TP chemotherapy as the first-line treatment of ESCC demonstrated favorable treatment efficacy. Pretreatment NLR of less than 3 at the 3rd and 4th cycle of immunochemotherapy is associated with a better prognosis.

A single-center, retrospective study-spring-evaluating the efficacy and safety of recombinant human vascular endothelial inhibitor combined with anti-PD-1 in elderly patients aged 80 and above with NSCLC.

Aim: To investigate the efficacy and safety of combining Recombinant Human Endostatin Injection (marketed as Endo) with anti-PD-1 in elderly patients aged 80 and above with non-small cell lung cancer (NSCLC). Methods: Retrospective analysis of 181 patients with NSCLC aged 80 and above treated in the Department of Respiratory and Critical Care Medicine at Chaohu Hospital, affiliated with Anhui Medical University, from June 2019 to January 2024. Patients who received at least one cycle of combined Endo with anti-PD-1 were included based on inclusion criteria. Clinical and pathological data were collected, including complete blood count, liver and kidney function, electrocardiogram, coagulation function, thyroid function, cardiac enzymes, and whole-body imaging. Adverse events were recorded with a final follow-up on January 25, 2024. The primary endpoints were progression-free survival (PFS) and overall survival (OS), with safety as a secondary endpoint. Results: This study involved 14 elderly patients with NSCLC aged over 80. Median progression-free survival (mPFS) was 102 days, and median overall survival (mOS) was 311 days. Subgroup analyses based on treatment cycles showed a non-significant 441-day mPFS increase in the long-term group (≥6 cycles, 5 patients) compared to the short-term group (<6 cycles, 9 patients). However, the mOS in the long-term group significantly exceeded the short-term group by 141 days, with statistical significance (P=0.048). Further categorization revealed a 204-day shorter mPFS in the monotherapy maintenance group (Endo or Immunol) compared to the combination maintenance group (Endo combined with Immunol, 441 days). The mOS of the monotherapy maintenance group was longer (686 days) than the combination maintenance group (311 days), but no statistical significance (P= 0.710, 0.920). Throughout the treatment, 77 adverse events were recorded, mainly grade 1-2, with no new treatment-related reactions occurred. Overall, the safety of Endo combined with anti-PD-1 was considered good and manageable. Conclusion: The combination of Endo and anti-PD-1 could be an effective treatment choice for patients with NSCLC aged 80 and above.

Spatiotemporal single-cell analysis decodes cellular dynamics underlying different responses to immunotherapy in colorectal cancer.

Expanding the efficacy of immune checkpoint blockade (ICB) in colorectal cancer (CRC) presses for a comprehensive understanding of treatment responsiveness. Here, we analyze multiple sequential single-cell samples from 22 patients undergoing PD-1 blockade to map the evolution of local and systemic immunity of CRC patients. In tumors, we identify coordinated cellular programs exhibiting distinct response associations. Specifically, exhausted T (Tex) or tumor-reactive-like CD8+ T (Ttr-like) cells are closely related to treatment efficacy, and Tex cells show correlated proportion changes with multiple other tumor-enriched cell types following PD-1 blockade. In addition, we reveal the less-exhausted phenotype of blood-associated Ttr-like cells in tumors and find that their higher abundance suggests better treatment outcomes. Finally, a higher major histocompatibility complex (MHC) II-related signature in circulating CD8+ T cells at baseline is linked to superior responses. Our study provides insights into the spatiotemporal cellular dynamics following neoadjuvant PD-1 blockade in CRC.

Short-term cultured tumor fragments to study immunotherapy combinations based on CD137 (4-1BB) agonism.

Biomarkers for cancer immunotherapy are an unmet medical need. The group of Daniela Thommen at the NKI recently reported on novel methodologies based on short-term cultures of patient-derived tumor fragments whose cytokine concentrations in the supernatants and activation markers on infiltrating T cells were associated with clinical response to PD-1 blockade. We set up a similar culture technology with tumor-derived fragments using mouse tumors transplanted into syngeneic immunocompetent mice to test an agonist anti-CD137 mAb and its combinations with anti-PD-1 and/or anti-TGF-ß. Increases in IFNγ concentrations in the tissue culture supernatants were detected upon in-culture activation with the anti-CD137 and anti-PD-1 mAb combinations or concanavalin A as a positive control. No other cytokine from a wide array was informative of stimulation with these mAbs. Interestingly, increases in Ki67 and other activation markers were substantiated in lymphocytes from cell suspensions gathered at the end of 72 h cultures. In mice bearing bilateral tumors in which one was excised prior to in vivo anti-CD137 + anti-PD-1 treatment to perform the fragment culture evaluation, no association was found between IFNγ production from the fragments and the in vivo therapeutic outcome in the non-resected contralateral tumors. The experimental system permitted freezing and thawing of the fragments with similar functional outcomes. Using a series of patient-derived tumor fragments from excised solid malignancies, we showed IFNγ production in a fraction of the studied cases, that was conserved in frozen/thawed fragments. The small tumor fragment culture technique seems suitable to preclinically explore immunotherapy combinations.

Design, synthesis, pharmacological evaluation, and computational study of benzo[d] isothiazol-based small molecule inhibitors targeting PD-1/PD-l1 interaction.

Blockade of the programmed cell death-1 (PD-1)/programmed cell death ligand 1 (PD-L1) pathway is an attractive strategy for immunotherapy, but the clinical application of small molecule PD-1/PD-L1 inhibitors remains unclear. In this work, based on BMS-202 and our previous work YLW-106, a series of compounds with benzo[d]isothiazol structure as scaffold were designed and synthesized. Their inhibitory activity against PD-1/PD-L1 interaction was evaluated by a homogeneous time-resolved fluorescence (HTRF) assay. Among them, LLW-018 (27c) exhibited the most potent inhibitory activity with an IC50 value of 2.61 nM. The cellular level assays demonstrated that LLW-018 exhibited low cytotoxicity against Jurkat T and MDA-MB-231. Further cell-based PD-1/PD-L1 blockade bioassays based on PD-1 NFAT-Luc Jurkat cells and PD-L1 TCR Activator CHO cells indicated that LLW-018 could interrupt PD-1/PD-L1 interaction with an IC50 value of 0.88 µM. Multi-computational methods, including molecular docking, molecular dynamics, MM/GBSA, MM/PBSA, Metadynamics, and QM/MM MD were utilized on PD-L1 dimer complexes, which revealed the binding modes and dissociation process of LLW-018 and C2-symmetric small molecule inhibitor LCH1307. These results suggested that LLW-018 exhibited promising potency as a PD-1/PD-L1 inhibitor for further investigation.

Establishment of an animal model of immune-related adverse events induced by immune checkpoint inhibitors.

OBJECTIVE: Immunotherapy, specifically immune checkpoint inhibitors (ICIs), has revolutionized cancer treatment. However, it can also cause immune-related adverse events (irAEs). This study aimed to develop a clinically practical animal model of irAEs using BALB/c mice. METHODS: Subcutaneous tumors of mouse breast cancer 4T1 cells were generated in inbred BALB/c mice. The mice were treated with programmed death-1 (PD-1) and cytotoxic t-lymphocyte antigen 4 (CTLA-4) inhibitors once every 3 days for five consecutive administration cycles. Changes in tumor volume and body weight were recorded. Lung computed tomography (CT) scans were conducted. The liver, lungs, heart, and colon tissues of the mice were stained with hematoxylin-eosin (H&E) staining to observe inflammatory infiltration and were scored. Serum samples were collected, and enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of ferritin, glutamic-pyruvic transaminase (ALT), tumor necrosis factor-α (TNF-α), interferon-gamma (IFN-γ), and interleukin-6 (IL-6). Mouse liver and lung cell suspensions were prepared, and changes in macrophages, T cells, myeloid-derived suppressor cells (MDSCs), and regulatory (Treg) cells were detected by flow cytometry. RESULTS: Mice treated with PD-1 and CTLA-4 inhibitors showed significant reductions in tumor volume and body weight. The tissue inflammatory scores in the experimental group were significantly higher than those in the control group. Lung CT scans of mice in the experimental group showed obvious inflammatory spots. Serum levels of ferritin, IL-6, TNF-α, IFN-γ, and ALT were significantly elevated in the experimental group. Flow cytometry analysis revealed a substantial increase in CD3+T cells, Treg cells, and macrophages in the liver and lung tissues of mice in the experimental group compared with the control group, and the change trend of MDSCs was opposite. CONCLUSIONS: The irAE-related animal model was successfully established in BALB/c mice using a combination of PD-1 and CTLA-4 inhibitors through multiple administrations with clinical translational value and practical. This model offers valuable insights into irAE mechanisms for further investigation.

[Analysis of the efficacy and safety of preoperative programmed death protein-1 inhibitor combined with chemotherapy in immunotherapy-sensitive patients with locally advanced gastric cancer or adenocarcinoma of the esophagogastric junction].

Objective: To evaluate the short-term efficacy and safety of a preoperative combination of programmed cell death protein-1 (PD-1) inhibitor with either oxaliplatin + capecitabine (CapeOx) or oxaliplatin + tegafur gimeracil oteracil potassium (SOX) in the treatment of locally advanced immunotherapy-sensitive gastric cancer (LAGC) or adenocarcinoma of the esophagogastric junction (AEG). Methods: The cohort of this retrospective descriptive case series comprised patients with LAGC or AEG whose cancers had been determined to be immunotherapy- sensitive by endoscopic biopsy before treatment in the Gastrointestinal Cancer Center, Unit III, Peking University Cancer Hospital and Institute from 1 August 1 2021 to 31 January 2024. Patients with any one of the following three characteristics were immunotherapy-sensitive: (i) PD-L1 combined positive score (CPS) ≥5; (ii) microsatellite instability-high (MSI-H) / mismatch repair deficiency (dMMR); or (iii) Epstein-Barr virus-encoded RNA (EBER) positivity. All study patients received PD-1 inhibitors combined with CapeOx or SOX as a neoadjuvant or conversion treatment strategy before surgery. Patients with immune system diseases, distant metastases, or human epidermal growth factor receptor 2 positivity were excluded. Factors analyzed included pathological complete response, clinical complete response, major pathological response, R0 resection rate, surgical conversion rate, and safety of the treatment, including immune-related adverse events (irAEs) and surgical complications. Results: The study cohort comprised 39 patients (28 men and 11 women) of median age 62 (range 44-79) years. After the above-described preoperative treatment, radical resection of the 14 tumors that were initially considered unresectable was achieved (surgical conversion rate: 14/14). Twenty-three of the remaining 25 patients underwent radical resection. The last two patients achieved clinical complete responses and opted for a "non-surgical strategy" (watch and wait). Overall, 37 patients (94.9%) underwent radical resection, with an R0 resection rate of 100% (37/37), pathological complete response rate of 48.6% (18/37), and major pathological response rate of 62.2% (23/37). Of the 24 patients with CPS ≥ 5 (non-MSI-H/dMMR and non-EBER positive), 11 achieved pathological complete responses and one with CPS=95 achieved a clinical complete response. Of the eight patients with MSI-H/dMMR, six achieved pathological complete responses and one a clinical complete response. Of the seven patients with EBER positivity, one achieved a pathological complete response. After excluding patients with major pathological complete responses, there was a statistically significant difference in CPS scores between preoperative biopsy specimens and postoperative surgical specimens in 13 patients (7.769±5.570 vs. 15.538±16.870, t=2.287, P=0.041). All patients tolerated preoperative immunotherapy well; nine patients (9/39, 23.1%) had Grade I-II irAEs. There were no Grade III-IV irAEs. The five patients with pyloric obstruction before treatment tolerated normal diets after treatment. The incidence of postoperative complications among all patients who underwent surgery was 18.9% (7/37), including one case of Grade IIIA anastomotic leakage, one of Grade IIIA intestinal obstruction, one of Grade II abdominal hemorrhage, two of Grade II abdominal infection, one of Grade I intestinal obstruction. Additionally, one patient developed COVID-19 postoperatively. All patients recovered with symptomatic treatment. Conclusion: We found that preoperative treatment of patients with LAGC or AEG of one of three types (CPS≥5, dMMR+MSI-H, and EBER positivity) with a PD-1 inhibitor combined with CapeOx or SOX chemotherapy achieved promising effectiveness and safety, with high surgical conversion, R0 resection, and complete response rates.

Immunomic longitudinal profiling of the NeoPembrOv trial identifies drivers of immunoresistance in high-grade ovarian carcinoma.

PD-1/PD-L1 blockade has so far shown limited survival benefit for high-grade ovarian carcinomas. By using paired samples from the NeoPembrOv randomized phase II trial (NCT03275506), for which primary outcomes are published, and by combining RNA-seq and multiplexed immunofluorescence staining, we explore the impact of NeoAdjuvant ChemoTherapy (NACT) ± Pembrolizumab (P) on the tumor environment, and identify parameters that correlated with response to immunotherapy as a pre-planned exploratory analysis. Indeed, i) combination therapy results in a significant increase in intraepithelial CD8+PD-1+ T cells, ii) combining endothelial and monocyte gene signatures with the CD8B/FOXP3 expression ratio is predictive of response to NACT + P with an area under the curve of 0.93 (95% CI 0.85-1.00) and iii) high CD8B/FOXP3 and high CD8B/ENTPD1 ratios are significantly associated with positive response to NACT + P, while KDR and VEGFR2 expression are associated with resistance. These results indicate that targeting regulatory T cells and endothelial cells, especially VEGFR2+ endothelial cells, could overcome immune resistance of ovarian cancers.

Circulating T cell status and molecular imaging may predict clinical benefit of neoadjuvant PD-1 blockade in oral cancer.

BACKGROUND: Addition of neoadjuvant immune checkpoint inhibition to standard-of-care interventions for locally advanced oral cancer could improve clinical outcome. METHODS: In this study, 16 evaluable patients with stage III/IV oral cancer were treated with one dose of 480 mg nivolumab 3 weeks prior to surgery. Primary objectives were safety, feasibility, and suitability of programmed death receptor ligand-1 positron emission tomography (PD-L1 PET) as a biomarker for response. Imaging included 18F-BMS-986192 (PD-L1) PET and 18F-fluorodeoxyglucose (FDG) PET before and after nivolumab treatment. Secondary objectives included clinical and pathological response, and immune profiling of peripheral blood mononuclear cells (PBMCs) for response prediction. Baseline tumor biopsies and postnivolumab resection specimens were evaluated by histopathology. RESULTS: Grade III or higher adverse events were not observed and treatment was not delayed in relation to nivolumab administration and other study procedures. Six patients (38%) had a pathological response, of whom three (19%) had a major (≥90%) pathological response (MPR). Tumor PD-L1 PET uptake (quantified using standard uptake value) was not statistically different in patients with or without MPR (median 5.3 vs 3.4). All major responders showed a significantly postnivolumab decreased signal on FDG PET. PBMC immune phenotyping showed higher levels of CD8+ T cell activation in MPR patients, evidenced by higher baseline expression levels of PD-1, TIGIT, IFNγ and lower levels of PD-L1. CONCLUSION: Together these data support that neoadjuvant treatment of advanced-stage oral cancers with nivolumab was safe and induced an MPR in a promising 19% of patients. Response was associated with decreased FDG PET uptake as well as activation status of peripheral T cell populations.

Exploring prognostic factors for survival in patients with advanced pancreatic cancer undergoing PD-1 inhibitor immunotherapy.

Immunotherapy, led by programmed cell death protein-1 (PD-1) inhibitors, has emerged as a prominent antitumor therapy, yet prognostic challenges persist in pancreatic cancer (PC). This retrospective, single-center study evaluated prognostic factors in advanced PC patients treated with PD-1 inhibitors at the PLA General Hospital's Oncology Department from 2015-2022. With ethics approval by the Ethics Committee of the General Hospital of the People's Liberation Army (S2021-228-03), we analyzed 126 patients using Kaplan-Meier and Cox models. p < .05 was considered a statistically significant difference. Median overall survival (mOS) and progression-free survival (mPFS) were 12.1 and 4.6 months, respectively. Significant mOS predictors were surgery history (44.2 months vs. 10 months, *p = .022), absence of liver metastases (44.2 months vs. 6.4 months, *p = .034), and baseline CA19-9 ≤ 216.15 U/ml (18.5 months vs. 9.2 months, *p = .049). For mPFS, histologic differentiation (5.5 months vs. 3.2 months, *p = .022) and first-line PD-1 inhibitor use (5.1 months vs. 1.5 months, ***p = .001) were key. Subgroup analyses highlighted early progression in low histologic differentiation and earlier death without surgery. History of surgery, absence of liver metastases, baseline CA19-9 level, and histologic intermediate/high differentiation may predict PD-1 inhibitor efficacy in advanced PC, pending validation in prospective trials.

TRP-2 / gp100 DNA vaccine and PD-1 checkpoint blockade combination for the treatment of intracranial tumors.

Intracranial tumors present a significant therapeutic challenge due to their physiological location. Immunotherapy presents an attractive method for targeting these intracranial tumors due to relatively low toxicity and tumor specificity. Here we show that SCIB1, a TRP-2 and gp100 directed ImmunoBody® DNA vaccine, generates a strong TRP-2 specific immune response, as demonstrated by the high number of TRP2-specific IFNγ spots produced and the detection of a significant number of pentamer positive T cells in the spleen of vaccinated mice. Furthermore, vaccine-induced T cells were able to recognize and kill B16HHDII/DR1 cells after a short in vitro culture. Having found that glioblastoma multiforme (GBM) expresses significant levels of PD-L1 and IDO1, with PD-L1 correlating with poorer survival in patients with the mesenchymal subtype of GBM, we decided to combine SCIB1 ImmunoBody® with PD-1 immune checkpoint blockade to treat mice harboring intracranial tumors expressing TRP-2 and gp100. Time-to-death was significantly prolonged, and this correlated with increased CD4+ and CD8+ T cell infiltration in the tissue microenvironment (TME). However, in addition to PD-L1 and IDO, the GBM TME was found to contain a significant number of immunoregulatory T (Treg) cell-associated transcripts, and the presence of such cells is likely to significantly affect clinical outcome unless also tackled.

In vitro T cell responses to PD-1 blockade are reduced by IFN-α but do not predict therapy response in melanoma patients.

PD-1 blockade therapy has revolutionized melanoma treatment, but still not all patients benefit and pre-treatment identification of those patients is difficult. Increased expression of inflammatory markers such as interleukin (IL)-6 in blood of patients correlates with poor treatment response. We set out to study the effect of inflammatory cytokines on PD-1 blockade in vitro. For this, we studied the effect of IL-6 and type I interferon (IFN) in vitro on human T cells in a mixed leukocyte reaction (MLR) in the absence or presence of PD-1 blockade. While IL-6 reduced IFN-γ secretion by T cells in both the presence and absence of PD-1 blockade, IFN-α specifically reduced the IFN-γ secretion only in the presence of PD-1 blockade. IFN-α reduced T cell proliferation independent of PD-1 blockade and reduced the percentage of cells producing IFN-γ only in the presence of PD-1 blockade. Next we determined the type I IFN score in a cohort of 22 melanoma patients treated with nivolumab. In this cohort, we did not find a correlation between clinical response and type I IFN score, nor between clinical response and IFN-γ secretion in vitro in a MLR in the presence of PD-1 blockade. We conclude that IFN-α reduces the effectiveness of PD-1 blockade in vitro, but that in this cohort, type I IFN score in vivo, nor IFN-γ secretion in vitro in a MLR in the presence of PD-1 blockade correlated to decreased therapy responses in patients.

Construction of Hierarchically Biomimetic Iron Oxide Nanosystems for Macrophage Repolarization-Promoted Immune Checkpoint Blockade of Cancer Immunotherapy.

Cancer immunotherapy is developing as the mainstream strategy for treatment of cancer. However, the interaction between the programmed cell death protein-1 (PD-1) and the programmed death ligand 1 (PD-L1) restricts T cell proliferation, resulting in the immune escape of tumor cells. Recently, immune checkpoint inhibitor therapy has achieved clinical success in tumor treatment through blocking the PD-1/PD-L1 checkpoint pathway. However, the presence of M2 tumor-associated macrophages (TAMs) in the tumor microenvironment (TME) will inhibit antitumor immune responses and facilitate tumor growth, which can weaken the effectiveness of immune checkpoint inhibitor therapy. The repolarization of M2 TAMs into M1 TAMs can induce the immune response to secrete proinflammatory factors and active T cells to attack tumor cells. Herein, hollow iron oxide (Fe3O4) nanoparticles (NPs) were prepared for reprogramming M2 TAMs into M1 TAMs. BMS-202, a small-molecule PD-1/PD-L1 inhibitor that has a lower price, higher stability, lower immunogenicity, and higher tumor penetration ability compared with antibodies, was loaded together with pH-sensitive NaHCO3 inside hollow Fe3O4 NPs, followed by wrapping with macrophage membranes. The formed biomimetic FBN@M could produce gaseous carbon dioxide (CO2) from NaHCO3 in response to the acidic TME, breaking up the macrophage membranes to release BMS-202. A series of in vitro and in vivo assessments revealed that FBN@M could reprogram M2 TAMs into M1 TAMs and block the PD-1/PD-L1 pathway, which eventually induced T cell activation and the secretion of TNF-α and IFN-γ to kill the tumor cells. FBN@M has shown a significant immunotherapeutic efficacy for tumor treatment.

Engineered reversible inhibition of SpyCatcher reactivity enables rapid generation of bispecific antibodies.

The precise regulation of protein function is essential in biological systems and a key goal in chemical biology and protein engineering. Here, we describe a straightforward method to engineer functional control into the isopeptide bond-forming SpyTag/SpyCatcher protein ligation system. First, we perform a cysteine scan of the structured region of SpyCatcher. Except for two known reactive and catalytic residues, none of these mutations abolish reactivity. In a second screening step, we modify the cysteines with disulfide bond-forming small molecules. Here we identify 8 positions at which modifications strongly inhibit reactivity. This inhibition can be reversed by reducing agents. We call such a reversibly inhibitable SpyCatcher "SpyLock". Using "BiLockCatcher", a genetic fusion of wild-type SpyCatcher and SpyLock, and SpyTagged antibody fragments, we generate bispecific antibodies in a single, scalable format, facilitating the screening of a large number of antibody combinations. We demonstrate this approach by screening anti-PD-1/anti-PD-L1 bispecific antibodies using a cellular reporter assay.

An in-situ peptide-antibody self-assembly to block CD47 and CD24 signaling enhances macrophage-mediated phagocytosis and anti-tumor immune responses.

Targeted immunomodulation for reactivating innate cells, especially macrophages, holds great promise to complement current adaptive immunotherapy. Nevertheless, there is still a lack of high-performance therapeutics for blocking macrophage phagocytosis checkpoint inhibitors in solid tumors. Herein, a peptide-antibody combo-supramolecular in situ assembled CD47 and CD24 bi-target inhibitor (PAC-SABI) is described, which undergoes biomimetic surface propagation on cancer cell membranes through ligand-receptor binding and enzyme-triggered reactions. By simultaneously blocking CD47 and CD24 signaling, PAC-SABI enhances the phagocytic ability of macrophages in vitro and in vivo, promoting anti-tumor responses in breast and pancreatic cancer mouse models. Moreover, building on the foundation of PAC-SABI-induced macrophage repolarization and increased CD8+ T cell tumor infiltration, sequential anti-PD-1 therapy further suppresses 4T1 tumor progression, prolonging survival rate. The in vivo construction of PAC-SABI-based nano-architectonics provides an efficient platform for bridging innate and adaptive immunity to maximize therapeutic potency.

Novel insights into paclitaxel's role on tumor-associated macrophages in enhancing PD-1 blockade in breast cancer treatment.

BACKGROUND: Triple-negative breast cancer (TNBC) poses unique challenges due to its complex nature and the need for more effective treatments. Recent studies showed encouraging outcomes from combining paclitaxel (PTX) with programmed cell death protein-1 (PD-1) blockade in treating TNBC, although the exact mechanisms behind the improved results are unclear. METHODS: We employed an integrated approach, analyzing spatial transcriptomics and single-cell RNA sequencing data from TNBC patients to understand why the combination of PTX and PD-1 blockade showed better response in TNBC patients. We focused on toll-like receptor 4 (TLR4), a receptor of PTX, and its role in modulating the cross-presentation signaling pathways in tumor-associated macrophages (TAMs) within the tumor microenvironment. Leveraging insights obtained from patient-derived data, we conducted in vitro experiments using immunosuppressive bone marrow-derived macrophages (iBMDMs) to validate if PTX could augment the cross-presentation and phagocytosis activities. Subsequently, we extended our study to an in vivo murine model of TNBC to ascertain the effects of PTX on the cross-presentation capabilities of TAMs and its downstream impact on CD8+ T cell-mediated immune responses. RESULTS: Data analysis from TNBC patients revealed that the activation of TLR4 and cross-presentation signaling pathways are crucial for the antitumor efficacy of PTX. In vitro studies showed that PTX treatment enhances the cross-presentation ability of iBMDMs. In vivo experiments demonstrated that PTX activates TLR4-dependent cross-presentation in TAMs, improving CD8+ T cell-mediated antitumor responses. The efficacy of PTX in promoting antitumor immunity was elicited when combined with PD-1 blockade, suggesting a complementary interaction. CONCLUSIONS: This study reveals how PTX boosts the effectiveness of PD-1 inhibitors in treating TNBC. We found that PTX activates TLR4 signaling in TAMs. This activation enhances their ability to present antigens, thereby boosting CD8+ T cell antitumor responses. These findings not only shed light on PTX's immunomodulatory role in TNBC but also underscore the potential of targeting TAMs' antigen presentation capabilities in immunotherapy approaches.