Co-stimulators CD40-CD40L, a potential immune-therapy target for atherosclerosis: A review.

The interaction between CD40 and CD40 ligand (CD40L) a crucial co-stimulatory signal for activating adaptive immune cells, has a noteworthy role in atherosclerosis. It is well-known that atherosclerosis is linked to immune inflammation in blood vessels. In atherosclerotic lesions, there is a multitude of proinflammatory cytokines, adhesion molecules, and collagen, as well as smooth muscle cells, macrophages, and T lymphocytes, particularly the binding of CD40 and CD40L. Therefore, research on inhibiting the CD40-CD40L system to prevent atherosclerosis has been ongoing for more than 30 years. However, it's essential to note that long-term direct suppression of CD40 or CD40L could potentially result in immunosuppression, emphasizing the critical role of the CD40-CD40L system in atherosclerosis. Thus, specifically targeting the CD40-CD40L interaction on particular cell types or their downstream signaling pathways may be a robust strategy for mitigating atherosclerosis, reducing potential side effects. This review aims to summarize the potential utility of the CD40-CD40L system as a viable therapeutic target for atherosclerosis.

Inhibition of CD40L with Frexalimab in Multiple Sclerosis.

BACKGROUND: The CD40-CD40L costimulatory pathway regulates adaptive and innate immune responses and has been implicated in the pathogenesis of multiple sclerosis. Frexalimab is a second-generation anti-CD40L monoclonal antibody being evaluated for the treatment of multiple sclerosis. METHODS: In this phase 2, double-blind, randomized trial, we assigned, in a 4:4:1:1 ratio, participants with relapsing multiple sclerosis to receive 1200 mg of frexalimab administered intravenously every 4 weeks (with an 1800-mg loading dose), 300 mg of frexalimab administered subcutaneously every 2 weeks (with a 600-mg loading dose), or the matching placebos for each active treatment. The primary end point was the number of new gadolinium-enhancing T1-weighted lesions seen on magnetic resonance imaging at week 12 relative to week 8. Secondary end points included the number of new or enlarging T2-weighted lesions at week 12 relative to week 8, the total number of gadolinium-enhancing T1-weighted lesions at week 12, and safety. After 12 weeks, all the participants could receive open-label frexalimab. RESULTS: Of 166 participants screened, 129 were assigned to a trial group; 125 participants (97%) completed the 12-week double-blind period. The mean age of the participants was 36.6 years, 66% were women, and 30% had gadolinium-enhancing lesions at baseline. At week 12, the adjusted mean number of new gadolinium-enhancing T1-weighted lesions was 0.2 (95% confidence interval [CI], 0.1 to 0.4) in the group that received 1200 mg of frexalimab intravenously and 0.3 (95% CI, 0.1 to 0.6) in the group that received 300 mg of frexalimab subcutaneously, as compared with 1.4 (95% CI, 0.6 to 3.0) in the pooled placebo group. The rate ratios as compared with placebo were 0.11 (95% CI, 0.03 to 0.38) in the 1200-mg group and 0.21 (95% CI, 0.08 to 0.56) in the 300-mg group. Results for the secondary imaging end points were generally in the same direction as those for the primary analysis. The most common adverse events were coronavirus disease 2019 and headaches. CONCLUSIONS: In a phase 2 trial involving participants with multiple sclerosis, inhibition of CD40L with frexalimab had an effect that generally favored a greater reduction in the number of new gadolinium-enhancing T1-weighted lesions at week 12 as compared with placebo. Larger and longer trials are needed to determine the long-term efficacy and safety of frexalimab in persons with multiple sclerosis. (Funded by Sanofi; ClinicalTrials.gov number, NCT04879628.).

A very long-acting IL-15: implications for the immunotherapy of cancer.

BACKGROUND: Interleukin-15 (IL-15) is an important cytokine necessary for proliferation and maintenance of natural killer (NK) and CD8+ T cells, and with great promise as an immuno-oncology therapeutic. However, IL-15 has a very short half-life and a single administration does not provide the sustained exposure required for optimal stimulation of target immune cells. The purpose of this work was to develop a very long-acting prodrug that would maintain IL-15 within a narrow therapeutic window for long periods-similar to a continuous infusion. METHODS: We prepared and characterized hydrogel microspheres (MS) covalently attached to IL-15 (MS~IL-15) by a releasable linker. The pharmacokinetics and pharmacodynamics of MS~IL-15 were determined in C57BL/6J mice. The antitumor activity of MS~IL-15 as a single agent, and in combination with a suitable therapeutic antibody, was tested in a CD8+ T cell-driven bilateral transgenic adenocarcinoma mouse prostate (TRAMP)-C2 model of prostatic cancer and a NK cell-driven mouse xenograft model of human ATL (MET-1) murine model of adult T-cell leukemia. RESULTS: On subcutaneous administration to mice, the cytokine released from the depot maintained a long half-life of about 168 hours over the first 5 days, followed by an abrupt decrease to about ~30 hours in accordance with the development of a cytokine sink. A single injection of MS~IL-15 caused remarkably prolonged expansions of NK and ɣδ T cells for 2 weeks, and CD44hiCD8+ T cells for 4 weeks. In the NK cell-driven MET-1 murine model of adult T-cell leukemia, single-agent MS~IL-1550 µg or anti-CCR4 provided modest increases in survival, but a combination-through antibody-depedent cellular cytotoxicity (ADCC)-significantly extended survival. In a CD8+ T cell-driven bilateral TRAMP-C2 model of prostatic cancer, single agent subcutaneous MS~IL-15 or unilateral intratumoral agonistic anti-CD40 showed modest growth inhibition, but the combination exhibited potent, prolonged bilateral antitumor activity. CONCLUSIONS: Our results show MS~IL-15 provides a very long-acting IL-15 with low Cmax that elicits prolonged expansion of target immune cells and high anticancer activity, especially when administered in combination with a suitable immuno-oncology agent.

Anti-inflammatory, but not osteoprotective, effect of the TRAF6/CD40 inhibitor 6877002 in rodent models of local and systemic osteolysis.

NFκB plays a key role in inflammation and skeletal disorders. Previously, we reported that pharmacological inhibition of NFκB at the level of TRAF6 suppressed RANKL, CD40L and IL1ß-induced osteoclastogenesis and attenuated cancer-induced bone disease. TNFα is also known to regulate TRAF6/NFκB signalling, however the anti-inflammatory and osteoprotective effects associated with inhibition of the TNFα/TRAF6/NFκB axis have not been investigated. Here, we show that in vitro and ex vivo exposure to the verified small-molecule inhibitor of TRAF6, 6877002 prevented TNFα-induced NFκB activation, osteoclastogenesis and calvarial osteolysis, but it had no effects on TNFα-induced apoptosis or growth inhibition in osteoblasts. Additionally, 6877002 disrupted T-cells support for osteoclast formation and synoviocyte motility, without affecting the viability of osteoblasts in the presence of T-cells derived factors. Using the collagen-induced arthritis model, we show that oral and intraperitoneal administration of 6877002 in mice reduced joint inflammation and arthritis score. Unexpectedly, no difference in trabecular and cortical bone parameters were detected between vehicle and 6877002 treated mice, indicating lack of osteoprotection by 6877002 in the arthritis model described. Using two independent rodent models of osteolysis, we confirmed that 6877002 had no effect on trabecular and cortical bone loss in both osteoporotic rats or RANKL- treated mice. In contrast, the classic anti-osteolytic alendronate offered complete osteoprotection in RANKL- treated mice. In conclusion, TRAF6 inhibitors may be of value in the management of the inflammatory component of bone disorders, but may not offer protection against local or systemic bone loss, unless combined with anti-resorptive therapy such as bisphosphonates.

Resident Kupffer cells and neutrophils drive liver toxicity in cancer immunotherapy.

Immunotherapy is revolutionizing cancer treatment but is often restricted by toxicities. What distinguishes adverse events from concomitant antitumor reactions is poorly understood. Here, using anti-CD40 treatment in mice as a model of TH1-promoting immunotherapy, we showed that liver macrophages promoted local immune-related adverse events. Mechanistically, tissue-resident Kupffer cells mediated liver toxicity by sensing lymphocyte-derived IFN-γ and subsequently producing IL-12. Conversely, dendritic cells were dispensable for toxicity but drove tumor control. IL-12 and IFN-γ were not toxic themselves but prompted a neutrophil response that determined the severity of tissue damage. We observed activation of similar inflammatory pathways after anti-PD-1 and anti-CTLA-4 immunotherapies in mice and humans. These findings implicated macrophages and neutrophils as mediators and effectors of aberrant inflammation in TH1-promoting immunotherapy, suggesting distinct mechanisms of toxicity and antitumor immunity.

Clinical Implementation of Biologics and Small Molecules in the Treatment of Hidradenitis Suppurativa.

Hidradenitis suppurativa (HS) is a chronic, recurrent, auto-inflammatory skin disease originating from the hair follicles. The typical inflammatory nodules, abscesses, and draining sinus tracts (tunnels) are characterized by a massive influx of neutrophils, macrophages, B-cells, plasma cells, T helper (Th)1, Th17 cells and upregulation of pro-inflammatory cytokines such as IL-1, IL-17, IL-12/23, and TNF-α. Over the last decades, several clinical trials evaluated the clinical efficacy of different biologics targeting these pro-inflammatory cytokines, in particular TNF-α and IL-1. However, adalimumab is still the only registered drug for HS. This review discusses biologics and small molecules with high level of evidence for their clinical application, provides guidance on when and how to use these biologics and small molecules in clinical practice, and elaborates on the combination with medical and surgical treatment options beyond the current guidelines. Furthermore this review provides an overview of potential biologics and small molecules currently under investigation for novel targets in HS such as IL-36, C5a, Janus kinase family members, CD-40, LTA4 and CXCR1/2.

Precision Medicine in Graves' Disease: CD40 Gene Variants Predict Clinical Response to an Anti-CD40 Monoclonal Antibody.

Background: CD40, a key co-stimulatory molecule expressed on antigen-presenting cells, is genetically associated with a number of autoimmune diseases including Graves' disease (GD). Therefore, recent therapies targeting CD40 have been developed, including the anti-CD40 monoclonal antibody Iscalimab. In a recent pilot study, Iscalimab was shown to induce clinical remission in ~ 50% of GD patients, but the reason why only 50% of GD patients responded is not known. The aim of our study was to test the hypothesis that specific CD40 single nucleotide polymorphism (SNP) genotypes and haplotypes are associated with clinical response of GD patients to Iscalimab. Methods: We extracted genomic DNA from the whole blood of 13 GD patients treated with Iscalimab, and genotyped seven CD40 single nucleotide polymorphisms (SNPs) associated with autoimmunity. Additionally, we analyzed CD40 mRNA expression levels in whole blood. The patients' CD40 SNP genotypes and mRNA levels were tested for association with clinical response to Iscalimab. Results: Three common haplotypes, designated haplotypes A, B, and C, were identified. Haplotypes B and C were associated with higher CD40 mRNA levels and clinical response to Iscalimab (i.e., patients achieving euthyroidism without need for additional medications), while haplotype A was associated with decreased CD40 mRNA levels and no response to Iscalimab. Conclusion: Our data suggest that genetic polymorphisms in the CD40 gene drive its expression levels and response to Iscalimab. Polymorphisms associated with higher CD40 levels are also associated with clinical response to CD40-targeted therapies. These results set the stage to implementing precision medicine in the therapeutic approach to GD.

Traumatic brain injury does not disrupt costimulatory blockade-induced immunological tolerance to glial-restricted progenitor allografts.

BACKGROUND: Cell transplantation-based treatments for neurological disease are promising, yet graft rejection remains a major barrier to successful regenerative therapies. Our group and others have shown that long-lasting tolerance of transplanted stem cells can be achieved in the brain with systemic application of monoclonal antibodies blocking co-stimulation signaling. However, it is unknown if subsequent injury and the blood-brain barrier breach could expose the transplanted cells to systemic immune system spurring fulminant rejection and fatal encephalitis. Therefore, we investigated whether delayed traumatic brain injury (TBI) could trigger graft rejection. METHODS: Glial-restricted precursor cells (GRPs) were intracerebroventricularly transplanted in immunocompetent neonatal mice and co-stimulation blockade (CoB) was applied 0, 2, 4, and 6 days post-grafting. Bioluminescence imaging (BLI) was performed to monitor the grafted cell survival. Mice were subjected to TBI 12 weeks post-transplantation. MRI and open-field test were performed to assess the brain damage and behavioral change, respectively. The animals were decapitated at week 16 post-transplantation, and the brains were harvested. The survival and distribution of grafted cells were verified from brain sections. Hematoxylin and eosin staining (HE) was performed to observe TBI-induced brain legion, and neuroinflammation was evaluated immunohistochemically. RESULTS: BLI showed that grafted GRPs were rejected within 4 weeks after transplantation without CoB, while CoB administration resulted in long-term survival of allografts. BLI signal had a steep rise following TBI and subsequently declined but remained higher than the preinjury level. Open-field test showed TBI-induced anxiety for all animals but neither CoB nor GRP transplantation intensified the symptom. HE and MRI demonstrated a reduction in TBI-induced lesion volume in GRP-transplanted mice compared with non-transplanted mice. Brain sections further validated the survival of grafted GRPs and showed more GRPs surrounding the injured tissue. Furthermore, the brains of post-TBI shiverer mice had increased activation of microglia and astrocytes compared to post-TBI wildtype mice, but infiltration of CD45+ leukocytes remained low. CONCLUSIONS: CoB induces sustained immunological tolerance towards allografted cerebral GRPs which is not disrupted following TBI, and unexpectedly TBI may enhance GRPs engraftment and contribute to post-injury brain tissue repair.

Homotypic aggregates contribute to heterogeneity in B cell fates due to an intrinsic gradient of stimulant exposure.

Monocultures of several cell types result in the formation of robust clusters called homotypic aggregates (HAs). How this physical aggregation affects cell fates in immune cell cultures, is poorly understood. We studied anti-CD40-stimulated primary B cell cultures, where cells assembled into large three-dimensional LFA1-driven HAs by 72 h. The dense packing in these aggregates restricts the infiltration of stimulants, such as antibodies, to cells inside the clusters. This creates a concentration gradient of stimulant availability across the cross-section of HAs. We describe a method to retain this positional information even after the disruption of HAs, for analysis by flow cytometry. Comparison of stage-specific cell-surface markers showed that the extent of stimulant-binding affected multiple fates non-uniformly. While germinal center and lineage markers were moderately upregulated, immunoglobulins and markers associated with memory were more than doubled in the peripheral cells binding more anti-CD40. These cells also experienced a strong repression of the plasma cell regulator Prdm1 and an upregulation of the oncogene Myc. Thus, cells at different locations in HAs are subjected to unequal doses of stimulants, leading to a hitherto unreported source of heterogeneity in cell fates. These findings can be extrapolated to understand the dose-dependent effects of stimulants in other three-dimensional cell clusters.

Acute Hemolysis and Heme Suppress Anti-CD40 Antibody-Induced Necro-Inflammatory Liver Disease.

Clearance of red blood cells and hemoproteins is a key metabolic function of macrophages during hemolytic disorders and following tissue injury. Through this archetypical phagocytic function, heme is detoxified and iron is recycled to support erythropoiesis. Reciprocal interaction of heme metabolism and inflammatory macrophage functions may modify disease outcomes in a broad range of clinical conditions. We hypothesized that acute hemolysis and heme induce acute anti-inflammatory signals in liver macrophages. Using a macrophage-driven model of sterile liver inflammation, we showed that phenylhydrazine (PHZ)-mediated acute erythrophagocytosis blocked the anti-CD40 antibody-induced pathway of macrophage activation. This process attenuated the inflammatory cytokine release syndrome and necrotizing hepatitis induced by anti-CD40 antibody treatment of mice. We further established that administration of heme-albumin complexes specifically delivered heme to liver macrophages and replicated the anti-inflammatory effect of hemolysis. The anti-inflammatory heme-signal was induced in macrophages by an increased intracellular concentration of the porphyrin independently of iron. Overall, our work suggests that induction of heme-signaling strongly suppresses inflammatory macrophage function, providing protection against sterile liver inflammation.

The Role of CD40 in Allergic Rhinitis and Airway Remodelling.

BACKGROUND: Allergic rhinitis (AR) affects millions of people and is lack of effective treatment. CD40 is an important costimulatory molecule in immunity. However, few studies have focused on the role of CD40 in AR. METHODS: In this study, we built mouse model of chronic AR. The mice were divided into the AR, control, intravenous CD40 siRNA, and nasal CD40 siRNA groups (n = 6 each). We detected OVA-sIgE, IL-4, IL-5, IL-13, IL-10, IFN-γ, and TGF-ß levels in serum and supernatant by ELISA, CD40+ splenic DCs, and Foxp3+ Tregs by flow cytometry and CD40 mRNA by RT2-PCR. We also used PAS and MT stains to assess tissue remodelling. RESULTS: (1) The OVA-sIgE, IL-4, IL-5, and IL-13 levels in the serum or supernatant of nasal septal membrane of AR mice were significantly higher than control. After treated with CD40 siRNA, those indicators were significantly decreased. The IFN-γ, IL-10, and TGF-ß levels in AR mice were significantly lower than that in control and were increased by administration of CD40 siRNA. (2) AR mice had significantly fewer Foxp3+ Tregs in the spleen than control mice. After treated with CD40 siRNA, AR mice had significantly more Foxp3+ Tregs. (3) AR mice exhibited a significantly higher CD40 mRNA levels than control. Administration of CD40 siRNA significantly reduced the CD40 mRNA level. (4) The AR mice showed significantly greater collagen deposition than the control in MT staining. Applications of CD40 siRNA significantly reduced the collagen deposition in AR mice. CONCLUSION: CD40 siRNA therapy shows promise for chronic AR as it significantly attenuated allergic symptoms and Th2-related inflammation and upregulated Foxp3+ Tregs. CD40 plays a role in tissue remodelling in AR, which can be inhibited by CD40 siRNA application.

Mannan-BAM, TLR ligands, and anti-CD40 immunotherapy in established murine pancreatic adenocarcinoma: understanding therapeutic potentials and limitations.

Pancreatic adenocarcinoma is one of the leading causes of cancer-related deaths, and its therapy remains a challenge. Our proposed therapeutic approach is based on the intratumoral injections of mannan-BAM, toll-like receptor ligands, and anti-CD40 antibody (thus termed MBTA therapy), and has shown promising results in the elimination of subcutaneous murine melanoma, pheochromocytoma, colon carcinoma, and smaller pancreatic adenocarcinoma (Panc02). Here, we tested the short- and long-term effects of MBTA therapy in established subcutaneous Panc02 tumors two times larger than in previous study and bilateral Panc02 models as well as the roles of CD4+ and CD8+ T lymphocytes in this therapy. The MBTA therapy resulted in eradication of 67% of Panc02 tumors with the development of long-term memory as evidenced by the rejection of Panc02 cells after subcutaneous and intracranial transplantations. The initial Panc02 tumor elimination is not dependent on the presence of CD4+ T lymphocytes, although these cells seem to be important in long-term survival and resistance against tumor retransplantation. The resistance was revealed to be antigen-specific due to its inability to reject B16-F10 melanoma cells. In the bilateral Panc02 model, MBTA therapy manifested a lower therapeutic response. Despite numerous combinations of MBTA therapy with other therapeutic approaches, our results show that only simultaneous application of MBTA therapy into both tumors has potential for the treatment of the bilateral Panc02 model.

Ginkgolide C attenuates lipopolysaccharide­induced acute lung injury by inhibiting inflammation via regulating the CD40/NF­κB signaling pathway.

Excessive lung inflammation caused by endotoxins, including lipopolysaccharide (LPS), mediates the detrimental effects of acute lung injury (ALI), as evidenced by severe alveolar epithelial cell injury. CD40, a member of the tumor necrosis factor receptor superfamily, serves as a central activator in triggering and transducing a series of severe inflammatory events during the pathological processes of ALI. Ginkgolide C (GC) is an efficient and specific inhibitor of CD40. Therefore, the present study aimed to investigate whether GC alleviated LPS­induced ALI, as well as the potential underlying mechanisms. LPS­injured wild­type and CD40 gene conditional knockout mice, and primary cultured alveolar epithelial cells isolated from these mice served as in vivo and in vitro ALI models, respectively. In the present study, histopathological assessment, polymorphonuclear neutrophil (PMN) infiltration, lung injury score, myeloperoxidase activity, wet­to­dry (W/D) weight ratio and hydroxyproline (Hyp) activity were assessed to evaluate lung injury. In addition, immunohistochemistry was performed to evaluate intracellular adhesion molecule­1, vascular cell adhesion molecule­1 and inducible nitric oxide synthase expression levels, and TNF­α, IL­1ß, IL­6 ELISAs and western blotting were conducted to elucidate the signaling pathway. The results demonstrated that GC alleviated LPS­induced lung injury, as evidenced by improvements in ultrastructural characteristics and histopathological alterations of lung tissue, inhibited PMN infiltration, as well as reduced lung injury score, W/D weight ratio and hydroxyproline content. In LPS­injured alveolar epithelial cells, GC significantly reduced IκBα phosphorylation, IKKß activity and NF­κB p65 subunit translocation via downregulating CD40, leading to a significant decrease in downstream inflammatory cytokine levels and protein expression levels. In conclusion, the results of the present study demonstrated that GC displayed a protective effect against LPS­induced ALI via inhibition of the CD40/NF­κB signaling pathway; therefore, the present study suggested that the CD40/NF­κB signaling pathway might serve as a potential therapeutic target for ALI.

What Therapeutic Regimen Will Be Optimal for Initial Clinical Trials of Pig Organ Transplantation?

We discuss what therapeutic regimen might be acceptable/successful in the first clinical trial of genetically engineered pig kidney or heart transplantation. As regimens based on a calcineurin inhibitor or CTLA4-Ig have proved unsuccessful, the regimen we administer to baboons is based on induction therapy with antithymocyte globulin, an anti-CD20 mAb (Rituximab), and cobra venom factor, with maintenance therapy based on blockade of the CD40/CD154 costimulation pathway (with an anti-CD40 mAb), with rapamycin, and a corticosteroid. An anti-inflammatory agent (etanercept) is administered for the first 2 wk, and adjuvant therapy includes prophylaxis against thrombotic complications, anemia, cytomegalovirus, and pneumocystis. Using this regimen, although antibody-mediated rejection certainly can occur, we have documented no definite evidence of an adaptive immune response to the pig xenograft. This regimen could also form the basis for the first clinical trial, except that cobra venom factor will be replaced by a clinically approved agent, for example, a C1-esterase inhibitor. However, none of the agents that block the CD40/CD154 pathway are yet approved for clinical use, and so this hurdle remains to be overcome. The role of anti-inflammatory agents remains unproven. The major difference between this suggested regimen and those used in allotransplantation is the replacement of a calcineurin inhibitor with a costimulation blockade agent, but this does not appear to increase the complications of the regimen.

IL-1 and CD40/CD40L platelet complex: elements of induction of Crohn's disease and new therapeutic targets.

Ulcerative colitis (UC) and Crohn's disease (CD) are chronic and multifactorial diseases that affect the intestinal tract, both characterized by recurrent inflammation of the intestinal mucosa, resulting in abdominal pain, diarrhea, vomiting and, rectal bleeding. Inflammatory bowel diseases (IBD) regroup these two disorders. The exact pathological mechanism of IBD remains ambiguous and poorly known. In genetically predisposed patients, defects in intestinal mucosal barrier are due to an uncontrolled inflammatory response to normal flora. In addition to the genetic predisposition, these defects could be triggered by environmental factors or by a specific lifestyle which is widely accepted as etiological hypothesis. The involvement of the CD40/CD40L platelet complex in the development of IBD has been overwhelmingly demonstrated. CD40L is climacteric in cell signalling in innate and adaptive immunity, the CD40L expression on the platelet cell surface gives them an immunological competence. The IL-1, a major inflammation mediator could be involved in different ways in the development of IBD. Here, we provide a comprehensive review regarding the role of platelet CD40/CD40L in the pathophysiological effect of IL-1 in the development of Crohn's disease (CD). This review could potentially help future approaches aiming to target these two pathways for therapeutic purposes and elucidate the immunological mechanisms driving gut inflammation.

CD40 agonistic monoclonal antibody APX005M (sotigalimab) and chemotherapy, with or without nivolumab, for the treatment of metastatic pancreatic adenocarcinoma: an open-label, multicentre, phase 1b study.

BACKGROUND: Standard chemotherapy remains inadequate in metastatic pancreatic adenocarcinoma. Combining an agonistic CD40 monoclonal antibody with chemotherapy induces T-cell-dependent tumour regression in mice and improves survival. In this study, we aimed to evaluate the safety of combining APX005M (sotigalimab) with gemcitabine plus nab-paclitaxel, with and without nivolumab, in patients with pancreatic adenocarcinoma to establish the recommended phase 2 dose. METHODS: This non-randomised, open-label, multicentre, four-cohort, phase 1b study was done at seven academic hospitals in the USA. Eligible patients were adults aged 18 years and older with untreated metastatic pancreatic adenocarcinoma, Eastern Cooperative Oncology Group performance status score of 0-1, and measurable disease by Response Evaluation Criteria in Solid Tumors version 1.1. All patients were treated with 1000 mg/m2 intravenous gemcitabine and 125 mg/m2 intravenous nab-paclitaxel. Patients received 0·1 mg/kg intravenous APX005M in cohorts B1 and C1 and 0·3 mg/kg in cohorts B2 and C2. In cohorts C1 and C2, patients also received 240 mg intravenous nivolumab. Primary endpoints comprised incidence of adverse events in all patients who received at least one dose of any study drug, incidence of dose-limiting toxicities (DLTs) in all patients who had a DLT or received at least two doses of gemcitabine plus nab-paclitaxel and one dose of APX005M during cycle 1, and establishing the recommended phase 2 dose of intravenous APX005M. Objective response rate in the DLT-evaluable population was a key secondary endpoint. This trial (PRINCE, PICI0002) is registered with ClinicalTrials.gov, NCT03214250 and is ongoing. FINDINGS: Between Aug 22, 2017, and July 10, 2018, of 42 patients screened, 30 patients were enrolled and received at least one dose of any study drug; 24 were DLT-evaluable with median follow-up 17·8 months (IQR 16·0-19·4; cohort B1 22·0 months [21·4-22·7], cohort B2 18·2 months [17·0-18·9], cohort C1 17·9 months [14·3-19·7], cohort C2 15·9 months [12·7-16·1]). Two DLTs, both febrile neutropenia, were observed, occurring in one patient each for cohorts B2 (grade 3) and C1 (grade 4). The most common grade 3-4 treatment-related adverse events were lymphocyte count decreased (20 [67%]; five in B1, seven in B2, four in C1, four in C2), anaemia (11 [37%]; two in B1, four in B2, four in C1, one in C2), and neutrophil count decreased (nine [30%]; three in B1, three in B2, one in C1, two in C2). 14 (47%) of 30 patients (four each in B1, B2, C1; two in C2) had a treatment-related serious adverse event. The most common serious adverse event was pyrexia (six [20%] of 30; one in B2, three in C1, two in C2). There were two chemotherapy-related deaths due to adverse events: one sepsis in B1 and one septic shock in C1. The recommended phase 2 dose of APX005M was 0·3 mg/kg. Responses were observed in 14 (58%) of 24 DLT-evaluable patients (four each in B1, C1, C2; two in B2). INTERPRETATION: APX005M and gemcitabine plus nab-paclitaxel, with or without nivolumab, is tolerable in metastatic pancreatic adenocarcinoma and shows clinical activity. If confirmed in later phase trials, this treatment regimen could replace chemotherapy-only standard of care in this population. FUNDING: Parker Institute for Cancer Immunotherapy, Cancer Research Institute, and Bristol Myers Squibb.

Systemic inflammation is a determinant of outcomes of CD40 agonist-based therapy in pancreatic cancer patients.

Agonistic anti-CD40 monoclonal antibody (mAb) therapy in combination with chemotherapy (chemoimmunotherapy) shows promise for the treatment of pancreatic ductal adenocarcinoma (PDA). To gain insight into immunological mechanisms of response and resistance to chemoimmunotherapy, we analyzed blood samples from patients (n = 22) with advanced PDA treated with an anti-CD40 mAb (CP-870,893) in combination with gemcitabine. We found a stereotyped cellular response to chemoimmunotherapy characterized by transient B cell, CD56+CD11c+HLA-DR+CD141+ cell, and monocyte depletion and CD4+ T cell activation. However, these cellular pharmacodynamics did not associate with outcomes. In contrast, we identified an inflammatory network in the peripheral blood consisting of neutrophils, cytokines (IL-6 and IL-8), and acute phase reactants (C-reactive protein and serum amyloid A) that was associated with outcomes. Furthermore, monocytes from patients with elevated plasma IL-6 and IL-8 showed distinct transcriptional profiles, including upregulation of CCR2 and GAS6, genes associated with regulation of leukocyte chemotaxis and response to inflammation. Patients with systemic inflammation, defined by neutrophil/lymphocyte ratio (NLR) greater than 3.1, had a shorter median overall survival (5.8 vs. 12.3 months) as compared with patients with NLR less than 3.1. Taken together, our findings identify systemic inflammation as a potential resistance mechanism to a CD40-based chemoimmunotherapy and suggest biomarkers for future studies.

Skin dendritic cells in melanoma are key for successful checkpoint blockade therapy.

BACKGROUND: Immunotherapy with checkpoint inhibitors has shown impressive results in patients with melanoma, but still many do not benefit from this line of treatment. A lack of tumor-infiltrating T cells is a common reason for therapy failure but also a loss of intratumoral dendritic cells (DCs) has been described. METHODS: We used the transgenic tg(Grm1)EPv melanoma mouse strain that develops spontaneous, slow-growing tumors to perform immunological analysis during tumor progression. With flow cytometry, the frequencies of DCs and T cells at different tumor stages and the expression of the inhibitory molecules programmed cell death protein-1 (PD-1) and T-cell immunoglobulin and mucin-domain containing-3 (TIM-3) on T cells were analyzed. This was complemented with RNA-sequencing (RNA-seq) and real-time quantitative PCR (RT-qPCR) analysis to investigate the immune status of the tumors. To boost DC numbers and function, we administered Fms-related tyrosine 3 ligand (Flt3L) plus an adjuvant mix of polyI:C and anti-CD40. To enhance T cell function, we tested several checkpoint blockade antibodies. Immunological alterations were characterized in tumor and tumor-draining lymph nodes (LNs) by flow cytometry, CyTOF, microarray and RT-qPCR to understand how immune cells can control tumor growth. The specific role of migratory skin DCs was investigated by coculture of sorted DC subsets with melanoma-specific CD8+ T cells. RESULTS: Our study revealed that tumor progression is characterized by upregulation of checkpoint molecules and a gradual loss of the dermal conventional DC (cDC) 2 subset. Monotherapy with checkpoint blockade could not restore antitumor immunity, whereas boosting DC numbers and activation increased tumor immunogenicity. This was reflected by higher numbers of activated cDC1 and cDC2 as well as CD4+ and CD8+ T cells in treated tumors. At the same time, the DC boost approach reinforced migratory dermal DC subsets to prime gp100-specific CD8+ T cells in tumor-draining LNs that expressed PD-1/TIM-3 and produced interferon γ (IFNγ)/tumor necrosis factor α (TNFα). As a consequence, the combination of the DC boost with antibodies against PD-1 and TIM-3 released the brake from T cells, leading to improved function within the tumors and delayed tumor growth. CONCLUSIONS: Our results set forth the importance of skin DC in cancer immunotherapy, and demonstrates that restoring DC function is key to enhancing tumor immunogenicity and subsequently responsiveness to checkpoint blockade therapy.

Different regulatory effects of CD40 ligand and B-cell activating factor on the function of B cells.

CD40 ligand (CD40L) and B-cell activating factor (BAFF) play important roles in the function of B cells. However, the difference of their regulatory effects remains obscure. In this study, we used anti-CD40 to imitate CD40L and investigated the different regulatory effects of CD40L and BAFF on the function of B cells. In the functional analyses, both anti-CD40 and BAFF significantly enhanced the survival and differentiation of B cells, and slightly increased the activation and proliferation. However, in the transcriptome analysis, anti-CD40 and BAFF exerted very different regulation on the gene expression profile of B cells. Anti-CD40 upregulated the expression of genes related to the adaptive immune function of B cells, but BAFF enhanced the genes associated with the innate immune function. Furthermore, the effect analysis of the combination of anti-CD40 or BAFF with anti-IgM also demonstrated that anti-CD40 could cooperate with anti-IgM to promote the proliferation of B cells, but BAFF could not do it. The mechanism study revealed that the different effects of anti-CD40 and BAFF on B cells were resulting from the different modulation on NF-кB, ERK1/2, and PI3K-AKT signaling pathways. Collectively, the results suggest that CD40L mainly promotes adaptive immune function of B cells, but BAFF primarily enhances innate immune function.

Targeting of CD40 and PD-L1 Pathways Inhibits Progression of Oral Premalignant Lesions in a Carcinogen-induced Model of Oral Squamous Cell Carcinoma.

We have previously demonstrated that PD-1 blockade decreased the incidence of high-grade dysplasia in a carcinogen-induced murine model of oral squamous cell carcinoma (OSCC). It remains unknown, however, whether there are additional factors involved in escape from immune surveillance that could serve as additional targets for immunoprevention. We performed this study to further characterize the immune landscape of oral premalignant lesions (OPL) and determine the impact of targeting of the PD-1, CTLA-4, CD40, or OX40 pathways on the development of OPLs and oral carcinomas in the 4-nitroquinoline 1-oxide model. The immune pathways were targeted using mAbs or, in the case of the PD-1/PD-L1 pathway, using PD-L1-knockout (PD-L1ko) mice. After intervention, tongues and cervical lymph nodes were harvested and analyzed for malignant progression and modulation of the immune milieu, respectively. Targeting of CD40 with an agonist mAb was the most effective treatment to reduce transition of OPLs to OSCC; PD-1 alone or in combination with CTLA-4 inhibition, or PD-L1ko, also reduced progression of OPLs to OSCC, albeit to a lesser extent. Distinct patterns of immune system modulation were observed for the CD40 agonists compared with blockade of the PD-1/PD-L1 axis with or without CTLA-4 blockade; CD40 agonist generated a lasting expansion of experienced/memory cytotoxic T lymphocytes and M1 macrophages, whereas PD-1/CTLA-4 blockade resulted in a pronounced depletion of regulatory T cells among other changes. These data suggest that distinct approaches may be used for targeting different steps in the development of OSCC, and that CD40 agonists merit investigation as potential immunoprevention agents in this setting. PREVENTION RELEVANCE: PD-1/PD-L1 pathway blockade, as well as activation of the CD40 pathway, were able to prevent OPL progression into invasive OSCC in a murine model. A distinct pattern of immune modulation was observed when either the CD40 or the PD-1/PD-L1 pathways were targeted.