Granulocyte Macrophage Research Articles

Cryptococcus gattii Infection as the Major Clinical Manifestation in Patients with Autoantibodies Against Granulocyte-Macrophage Colony-Stimulating Factor.

Anti-granulocyte-macrophage colony-stimulating factor autoantibodies (anti-GM-CSF Abs) are a predisposing factor for pulmonary alveolar proteinosis (PAP) and Cryptococcus gattii cryptococcosis. This study aimed to investigate clinical manifestations in anti-GM-CSF Ab-positive patients with C. gattii cryptococcosis and analyze the properties of anti-GM-CSF Abs derived from these patients and patients with PAP. Thirty-nine patients diagnosed with cryptococcosis (caused by C. neoformans or C. gattii) and 6 with PAP were enrolled in the present study. Clinical information was obtained from medical records. Blood samples were collected for analysis of autoantibody properties. We also explored the National Health Insurance Research Database (NHIRD) of Taiwan to investigate the epidemiology of cryptococcosis and PAP. High titers of neutralizing anti-GM-CSF Abs were identified in 15 patients with cryptococcosis (15/39, 38.5%). Most anti-GM-CSF Ab-positive cryptococcosis cases had central nervous system(CNS) involvement (14/15, 93.3%). Eleven out of 14 (78.6%) anti-GM-CSF Ab-positive CNS cryptococcosis patients were confirmed to be infected with C. gattii, and PAP did not occur synchronously or metachronously in a single patient from our cohort. Exploration of an association between HLA and anti-GM-CSF Ab positivity or differential properties of autoantibodies from cryptococcosis patients and PAP yielded no significant results. Anti-GM-CSF Abs can cause two diseases, C. gattii cryptococcosis and PAP, which seldom occur in the same subject. Current biological evidence regarding the properties of anti-GM-CSF Abs cannot provide clues regarding decisive mechanisms. Further analysis, including more extensive cohort studies and investigations into detailed properties, is mandatory to better understand the pathogenesis of anti-GM-CSF Abs.

Change in Ocular Surface Staining during Eyelid Warming Is Related to Tear Cytokine Levels.

Purpose To investigate the changes in the tear cytokine profile of patients with meibomian gland dysfunction (MGD) treated with eyelid warming and to correlate these changes with clinical parameters for dry eye disease (DED). Methods Seventy patients with MGD were included and treated with the warming of eyelids. Of these, 61 still used the treatment three months after baseline, while 48 completed the whole treatment period of six months. The concentrations of 39 cytokines in the tear fluid were measured at baseline and after three and six months of treatment. All participants were examined with tests for DED, including tear film break-up time (TBUT), ocular surface staining (OSS), and the self-reporting Ocular Surface Disease Index (OSDI). Changes in cytokine concentrations were assessed from baseline to three months, from three to six months, and from baseline to six months. Correlation analyses were performed between changes in the cytokine concentrations and changes in TBUT, OSS, and OSDI during the same time intervals. Results No significant changes were found in the concentrations of the 39 cytokines during any of the three treatment intervals. However, several correlations were detected between changes in the level of cytokines and OSS from baseline to three months of treatment. Decreasing concentrations of granulocyte chemotactic protein 2 (GCP-2/CXCL6, mean effect 2.36, p=0.042), interleukin 10 (IL-10, mean effect 1.04, p=0.045), and IL-16 (mean effect 1.36, p=0.035) were associated with decreasing OSS. Decreasing concentrations of granulocyte macrophage colony-stimulating factor (GM-CSF, mean effect −2.98, p=0.024), IL-8 (IL-8/CXCL8, mean effect −1.35, p=0.026), and macrophage migration inhibitory factor (MIF, mean effect −2.44, p=0.033) were related to increasing OSS. Conclusions Warming of eyelids did not change the concentration of cytokines in the tear fluid of patients with MGD significantly. However, alterations in the level of several cytokines were associated with changes in the OSS. This finding indicates a close connection between tear cytokines and OSS in MGD patients treated with eyelid warming.

GM-CSF derived from the inflammatory microenvironment potentially enhanced PD-L1 expression on tumor-associated macrophages in human breast cancer

Ever since immune checkpoint inhibitors have been approved for anti-cancer therapy in several cancers, including triple-negative breast cancer, the significance of programmed death-1 ligand 1 (PD-L1) expression in the tumor immune microenvironment has been a topic of interest. In the present study, we investigated the detailed mechanisms of PD-L1 overexpression on tumor-associated macrophages (TAMs) in breast cancer. In in vitro culture studies using human monocyte-derived macrophages, lymphocytes, and breast cancer cell lines, PD-L1 overexpression on macrophages was induced by the conditioned medium (CM) of activated lymphocytes, but not that of cancer cells. Granulocyte–macrophage colony-stimulating factor (GM-CSF) derived from activated lymphocytes was found to be involved in PD-L1 overexpression, in addition to interferon (IFN)-γ, via STAT3 pathway activation. Macrophages suppressed lymphocyte activation, and this inhibition was impaired by PD-1 blocking. The CM of activated lymphocytes also induced the overexpression of PD-L2, but GM-CSF did not affect PD-L2 expression. In the murine E0771 breast cancer model, anti-GM-CSF therapy did not affect PD-L1 expression on TAMs, and the mechanisms of PD-L1 expression on TAMs might differ between humans and mice. However, not only PD-L1, but also PD-L2 was overexpressed on TAMs in the E0771 tumor model, and their expression levels were significantly lower in the tumors in nude mice than in wild-type mice. Anti-PD-L1 antibody and anti-PD-L2 antibody synergistically inhibited E0771 tumor development. In conclusion, PD-L1 and PD-L2 were overexpressed on TAMs, and they potentially contributed to immunosuppression. The GM-CSF-STAT3 pathway is thought to represent a new mechanism of PD-L1 overexpression on TAMs in human breast cancer microenvironment.

Antenatal maternal depression, early life inflammation and neurodevelopment in a South African birth cohort

BackgroundAntenatal exposure to maternal psychological adversity, including depression, increases the risk of impaired neurodevelopment in children. The underlying biological mechanisms remain unclear, especially in early life during critical windows of development and maturation. This study investigated the association of antenatal maternal depression, maternal and early life inflammatory markers and neurodevelopmental outcomes in children at 2 years of age. MethodsA subgroup of mothers and their children (n = 255) that were enrolled in a South African birth cohort study, the Drakenstein Child Health Study, were followed from the antenatal period through to 2 years of child age. Maternal depressive symptoms were measured by the Beck Depression Inventory (BDI-II) at 26 weeks gestation. Serum inflammatory markers [granulocyte–macrophage colony-stimulating factor (GM-CSF), interferon-γ (IFN-γ), interleukin IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12p70, IL-13, tumour necrosis factor-α (TNF-α), neutrophil gelatinase-associated lipocalin (NGAL) and metalloproteinase-9 (MMP-9)] were measured in mothers at enrolment and in their children at 6–10 weeks and at 2 years. Neurodevelopment was assessed at 2 years using the Bayley Scales of Infant and Toddler Development III. ResultsAntenatal depressive symptoms (present in 25% of the mothers) were significantly associated with higher levels of IL-7 (p = 0.008), IL-8 (p = 0.019) and TNF-α (p = 0.031) in the mothers after correcting for sociodemographic and lifestyle factors. Serum IL-1β and NGAL levels were significantly elevated over time in children born to mothers with depressive symptoms compared to those without depression, after controlling for maternal and child health and sociodemographic factors. Elevated infant IL-1β at 6–10 weeks of age partially mediated the association of maternal depressive symptoms with poorer language scores at 2 years. ConclusionAlterations in early life immunity, as reflected by elevated IL-1β, is a potential pathway through which antenatal maternal depressive symptoms may impact language development in young children.

C reactive protein utilisation, a biomarker for early COVID-19 treatment, improves lenzilumab efficacy: results from the randomised phase 3 ‘LIVE-AIR’ trial

ObjectiveCOVID-19 severity is correlated with granulocyte macrophage colony-stimulating factor (GM-CSF) and C reactive protein (CRP) levels. In the phase three LIVE-AIR trial, lenzilumab an anti-GM-CSF monoclonal antibody, improved the likelihood...

Mechanism of human airway epithelial cell injury induced by Candida albicans infection

Objective: To investigate the immune mechanism of human airway epithelial cell injury induced by invasion of Candida albicans with different biofilm formation abilities. Methods: Twenty-five strains of Candida albicans isolated and cultured in General Hospital of Ningxia Medical University from June to December 2019 were selected, and quality control strain SC5314 was used as the standard strain. An in vitro model of Candida albicans biofilm was established, and the biofilm formation ability of different Candida albicans was detected by crystal violet staining and enzyme plate method. The absorbance value at 570 nm (A570) was determined by enzyme plate method. A570≥0.5, 0.25<A570<0.5 and A570≤0.25 indicated strong biofilm Candida albicans form (SBF), moderate biofilm Candida albicans form (DRF) and weak biofilm Candida albicans form (WBF), respectively. The gas-liquid phase culture model of human airway epithelial cells was isolated and established in vitro and divided into five groups, including blank control group (n=20), standard strain group (n=20), strong biofilm group (n=19), weak biofilm group (n=17) and fluconazole-resistant group (n=18).The morphology of the epithelial cells was observed by scanning electron microscope (SEM), and the expression of marker protein in the model was detected by immunofluorescence in vitro. The level of lactate dehydrogenase (LDH) in cells was detected by microplate method, and the secretion of β-defensin (hBD2), granulocyte macrophage colony stimulating factor(GM-CSF) and granulocyte colony-stimulating factor (G-CSF) was detected by enzyme-linked immunosorbent assay (ELISA). Results: The strong biofilm strains grew with interlacing mycelia, and very few yeast cells could be seen wrapped in them.SEM observed that the mycelia of epithelial cells in gas-liquid phase culture could actively invade epithelial cells, and the expression of acetylated tubulin and keratin in cilia were significantly reduced, while the expression of Ki67 was down-regulated.The LDH levels in the blank control group, standard strain group, strong biofilm group, weak biofilm group, and fluconazole-resistant group were (12.21±5.68), (46.35±6.35), (18.69±4.38), (12.56±3.69), and (13.48±4.28) U/L, respectively, with statistically significant differences (P<0.001). Compared with standard strain group, LDH level in strong biofilm group, weak biofilm group and fluconazole resistant group were significantly decreased (all P<0.01). The hBD2 levels of the five groups were (26.14±0.77), (56.18±0.83), (30.66±2.59), (29.22±0.48), (28.28±1.56) ng/L, respectively, with statistically significant differences(P<0.001). Compared with the blank control group, SC5314-treated epithelial cells induced an increase of intracellular hBD2 expression (P<0.001). The differences in the expression of GM-CSF and G-CSF between different groups were not statistically significant(all P>0.05). Conclusion: Strong biofilm Candida albican can inhibit cell proliferation, disrupt the integrity of epithelial cells and induce cell damage by down-regulating the expression of cell proliferation-related protein.

Macrophage Distribution Affected by Virus-Encoded Granulocyte Macrophage Colony Stimulating Factor Combined with Lactate Oxidase.

Oncolytic virotherapy was approved as a localized treatment for advanced melanoma by the US Food and Drug Administration (FDA) in 2015. Granulocyte macrophage colony stimulating factor (GM-CSF) encoded by clinical virus-infected tumor cells, acting as a pro-inflammatory cytokine or growth factor, increases tumor antigen presentation, leading to the activation of macrophages and T cells. Notably, tumor-secreted lactate can promote the suppressive functions of M2-polarized tumor-associated macrophages and subsequently promote tumor growth. Furthermore, the consumption of tumor-secreted lactate has been implicated in the beneficial polarization of macrophages. Here, we report that GM-CSF-encoded recombinant adeno-associated virus (AAV2-GM-CSF) infection in B16-F10 mouse melanoma cells combined with lactate oxidase (LOX) leads to the recruitment of M1 macrophages for the inhibition of cancer cell growth. This study suggests that GM-CSF combined with LOX has potential as cancer virotherapy.

Granulocyte-macrophage colony-stimulating factor-triggered innate immune tolerance against chronic stress-induced behavioral abnormalities in mice

Pre-stimulation of the innate immune is considered a potential strategy to prevent chronic stress-induced behavioral abnormalities in animals. In this study, we investigated whether granulocyte–macrophage colony-stimulating factor (GM-CSF), an immunostimulant used in the clinic to treat diseases of the hematopoietic system, can prevent chronic stress-induced behavioral abnormalities in mice. Our results showed that a single intraperitoneal injection of GM-CSF (100 μg/kg) one day before stress exposure prevented the depression- and anxiety-like behaviors induced by chronic social defeat stress (CSDS) in mice, including preventing the CSDS-induced increase in the immobility time in the tail suspension test and forced swimming test and decrease in the time spent in the interaction zone in the social interaction test, as well as preventing the CSDS-induced decrease in the time spent (i) in open arms in the elevated plus maze test, (ii) on the illuminated side in the light–dark test, and (iii) in the central region of the open field test. The single GM-CSF preinjection (100 μg/kg) also prevented the CSDS-induced increase in the expression levels of interleukin-6 (IL-6), IL-1β, and tumor necrosis factor-α (TNF-α) mRNA in the hippocampus and medial prefrontal cortex of the mice. Further analysis showed that the preventive effect of GM-CSF on CSDS-induced depression- and anxiety-like behaviors and neuroinflammatory responses was abolished by pretreatment with minocycline (an innate immune inhibitor). These results indicate that a single low dose of GM-CSF before injection could be a potential way to prevent behavioral abnormalities induced by chronic stress in mice.

A randomized, controlled, open-label, phase 2 study of cemiplimab ± RP1 in patients with advanced cutaneous squamous cell carcinoma (CERPASS).

TPS9593 Background: RP1 is an oncolytic virus (HSV-1) that expresses a fusogenic glycoprotein (GALV-GP R-) and granulocyte macrophage colony stimulating factor (GM-CSF). In preclinical studies, RP1 induced immunogenic tumor cell death and provided potent systemic anti-tumor activity, which is further improved by combining anti-PD-1 therapy. The prognosis for advanced and metastatic cutaneous squamous cell carcinoma (CSCC) remains poor for many patients despite the adoption of cemiplimab and pembrolizumab as a standard treatment. Preliminary results from IGNYTE, a phase I/II clinical study of RP1 in combination with nivolumab showed a high rate of deep and durable responses in patients (pts) with CSCC. This study is evaluating the efficacy and safety of cemiplimab ± RP1 versus cemiplimab alone in advanced CSCC. Methods: This global, multicenter, randomized phase 2 study is enrolling pts with metastatic or unresectable, locally advanced CSCC who are not candidates for/refuse surgery and/or radiation therapy. Key eligibility criteria include no prior treatment with anti-PD1/PD-L1 antibodies or oncolytic viruses. The clinical trial is enrolling approximately 180 pts from centers in the EU, Australia, Canada, and USA. Pts are randomized in a 2:1 ratio to receive combination therapy or monotherapy respectively. Pts receive 350 mg of cemiplimab intravenously (IV) Q3W for up to 108 weeks. In the RP1 + cemiplimab arm, RP1 is injected intratumorally at a starting RP1 dose of 1 × 106 plaque-forming units (PFU)/mL alone, followed by up to 7 doses of RP1 at 1 × 107 PFU/mL Q3W together with the same dose of cemiplimab. Pts in the combination arm may receive up to 8 additional RP1 doses if protocol specific criteria are met. No crossover is allowed. Pts are stratified by disease status (nodal or distant metastatic or locally advanced CSCC) and prior systemic therapy. Tumor assessments are performed every 9 weeks. The dual independent primary endpoints are overall response rate and complete response rate, both by a blinded independent review. Secondary endpoints include safety, progression-free survival, duration of response, and overall survival. Exploratory endpoints include quality of life, and immune biomarker analyses. This trial is currently enrolling pts. Clinical trial information: NCT04050436.

Naxitamab and granulocyte macrophage colony stimulating factor (GM-CSF) in combination with irinotecan and temozolomide in patients with high-risk neuroblastoma with primary refractory disease or in first relapse: An international, single-arm, multicenter phase 2 trial.

TPS10061 Background: Neuroblastoma (NB) is the commonest extracranial solid tumor in children. At diagnosis, &gt; 50% have high-risk (HR) disease with poor prognosis despite intensive therapy; thus, there is a high unmet medical need. Naxitamab is an anti-GD2 monoclonal antibody indicated under accelerated approval in the US with GM-CSF for the treatment of relapsed/refractory HR NB in the bone and/or bone marrow in patients (pts) ≥1 year of age with a partial response, minor response, or stable disease to prior therapy. Irinotecan and temozolomide (IT) are commonly used salvage chemotherapies following failure of frontline therapy. The objective of this study is to investigate the efficacy and safety of naxitamab plus IT in pts with HR NB with primary refractory disease or first relapse. Methods: Trial 203 (NCT04560166) is an international, single-arm, multicenter phase 2 trial for HR NB patients ≥12 mo of age, with either evaluable primary refractory disease or first relapse following induction and consolidation therapy. The primary endpoint of the study is the centrally-assessed overall response rate (ORR) (complete [CR] or partial response) at or before completion of 4 cycles of treatment. Key secondary endpoints include ORR after 2 cycles of treatment, duration of response (DoR), CR rate, time to first subsequent therapy or death, progression-free survival (PFS), and overall survival (OS). 52 pts will be enrolled to provide 82% power to demonstrate that response rate is significantly higher than 20%. Key inclusion criteria are documented HR NB at time of diagnosis; received frontline induction/consolidation therapy; active disease despite previous aggressive chemotherapy; and measurable tumor (CT/MRI) that is either MIBG-avid/PET-positive or have MIBG scan with uptake at ≥1 site. Key exclusion criteria include prior treatment with duration ≤3 wks (chemotherapy), ≤6wks (autologous stem cell transplant or therapeutic 131I-MIBG), ≤4wks (radiation therapy), or progressed while on anti-GD2; residual NB in the bone marrow only; and CNS/leptomeningeal disease in the prior 6 mo. In addition, there should not be &lt; 50% performance status per Lansky or Karnosky scale and life expectancy &lt; 6 mo. Pts will receive irinotecan 50 mg/m2/day IV and temozolomide 100 mg/m2/day orally (both on Days 1-5) in combination with naxitamab 2.25 mg/kg/day IV (Days 2, 4, 8 and 10) and GM-CSF 250 ug/m2/day sc (Days 6-10) in 21 days cycle. Response rates will be assessed using the 2-sided binomial test at the 5% significance level; DoR, PFS, and OS will be estimated using Kaplan-Meier analysis. Adverse events will be graded per CTCAE. Clinical trial information: NCT04560166.

A first-in-human, phase I, open-label study of a novel cancer vaccine labvax 3(22)-23 and adjuvant GM-CSF in patients with advanced stage adenocarcinomas.

TPS2700 Background: Adenocarcinoma is the most common histologic type of solid tumors and can arise from almost anywhere in the body. Labyrinthin is a novel tumor-specific protein expressed on the cell surface of the majority of adenocarcinomas of various cancer types. Several therapeutic strategies targeting labyrinthin are under development. We hypothesize that vaccination against labyrinthin can elicit strong immune responses against the labyrinthin-positive adenocarcinomas in cancer patients. LabVax 3(22)-23 is a novel anti-tumor vaccine that contains 4 synthetic labyrinthin-based peptides designed to elicit both B-cell and T-cell responses. Sargramostim, a recombinant granulocyte macrophage colony-stimulating factor (GM-CSF), is given as an immunostimulator with LabVax 3(22)-23 to boost the antitumor immune response. Preclinical studies showed that LabVax 3(22)-23 significantly inhibited tumor growth that was augmented by GM-CSF without any significant toxicity in C57/BL6 transgenic mice expressing human PD-1/PD-L1 implanted with the murine colon adenocarcinoma cell line MC-38-huPD-L1. This single institution, first-in-human, phase I trial (UCDCC#296) evaluates LabVax 3(22)-23 and adjuvant sargramostim in patients with labyrinthin-positive metastatic or recurrent adenocarcinoma of any primary tumor site after all standard-of-care therapies. Methods: The primary objective is to assess the toxicity of LabVax 3(22)-23 and adjuvant sargramostim according to NCI CTCAE V5.0. The primary endpoint is dose limiting toxicity (DLT), which is defined as grade ≥ 2 allergic and autoimmune reaction, grade ≥ 3 injection site reaction, any grade 3 toxicities lasting &gt;1 week, or any grade ≥ 4 toxicities. With a sample size of 10 patients, there is 89-97% of chance to observe ≥1 DLT if the true event rate is 20-30%. A secondary endpoint is the preliminary assessment of tumor response rate by Response Evaluation Criteria in Solid Tumors (RECIST) v1.1. Exploratory objectives measure the effect of LabVax 3(22)-23 on various immune responses (cytokines, anti-labyrinthin antibody production) and the correlation between the level of labyrinthin expression and the efficacy of LabVax 3(22)-23. Eligible patients are required to have labyrinthin expression on their tumor cells by immunohistochemistry and adequate organ function. Patients receive sargramostim subcutaneously and LabVax 3(22)-23 intradermally on weeks 1, 2, 4, 8, and 12 in the absence of disease progression or unacceptable toxicity. After completion of study treatment, patients are followed every 3 months for 1 year. Two patients are enrolled at the time of submission. Clinical trial information: NCT05101356.

An open-label, multicenter, phase 1b/2 study of RP1, a first-in-class, enhanced potency oncolytic virus in solid organ transplant recipients with advanced cutaneous malignancies (ARTACUS).

TPS9597 Background: RP1 is an oncolytic virus (HSV-1) that expresses a fusogenic glycoprotein (GALV-GP R-) and granulocyte macrophage colony stimulating factor (GM-CSF). In preclinical studies, RP1 induced immunogenic tumor cell death and provided potent systemic anti-tumor activity. Clinical data in combination with nivolumab has demonstrated a high rate of deep and durable response in patients with advanced skin cancer. Solid organ transplantation (SOT) is an important lifesaving procedure for patients with a wide range of end-organ diseases, but requires patients (pts) to undergo lifelong immunosuppression to prevent allograft rejection, and skin cancers (SCs) – including cutaneous squamous cell carcinoma (CSCC) – are common post-transplant malignancies. SC in SOT pts is generally managed with surgical resection, radiation therapy, and chemotherapy or targeted therapy. The use of immune checkpoint inhibitors in SOT recipients has improved outcomes but is associated with a high risk of allograft rejection. Thus, there is a high unmet need for a safe and effective treatment that also protects pts from allograft rejection. The objective of this study is to assess the safety and efficacy of single-agent RP1 in SOT patients with SCs, with a focus on CSCC. Methods: This study will enroll up to 65 evaluable SOT pts with locally advanced or metastatic SCs. The study has two parts. In Part A, pts will receive an initial dose of 1 x 106 plaque-forming units (PFU) of RP1. Two weeks later they will receive 1 x 107 PFU of RP1 and continue every two weeks until pre-specified study endpoints are met. In Part B, after determining the safety and tolerability in the initial cohort with kidney and liver transplants, the study may also enroll heart and lung transplant recipients. RP1 will be administered by intra-tumoral injection, utilizing image guidance as clinically appropriate. Key inclusion criteria are pts with confirmed recurrent, locally advanced or metastatic CSCC and up to 10 pts with non-CSCC SC, stable allograft function and ECOG performance status of ≤1. Pts with prior systemic anti-cancer treatment are allowed. Key exclusion criteria are prior treatment with an oncolytic therapy, active herpetic infections or prior complications of HSV-1 infection and a history of organ graft rejection within 12 months. The primary objective of the trial is to assess efficacy determined by objective response rate and safety of single agent RP1. Additional secondary endpoints include duration of response, complete response rate, disease control rate, progression-free survival and overall survival. Clinical trial information: NCT04349436.

Study to evaluate intraperitoneal (IP) ONCOS-102 with systemic durvalumab in patients with peritoneal disease who have epithelial ovarian (OC) or metastatic colorectal cancer (CRC): Phase 2 results.

2600 Background: Locoregional treatment with oncolytic viruses may be used to improve the efficacy of immune checkpoint inhibitors at both treated and distant tumor sites. This study evaluated the combination of IP-administered ONCOS-102, an oncolytic adenovirus encoding for granulocyte macrophage colony stimulating factor (GM-CSF), with systemic durvalumab (durva) in patients (pts) with advanced OC or CRC who have failed prior chemotherapy. Methods: This open-label study (NCT02963831) evaluated ONCOS-102 (IP 3 x 1011 VP in 500ml saline [recommended phase 2 dose] weekly x 6) + durva (IV 1500 mg every 4 weeks x 12). One dose of cyclophosphamide was given prior to first ONCOS-102 dose. Phase 2 evaluated the activity of the combination using Simon’s 2-stage MINIMAX design. In MINIMAX stage 1, if ≥ 5 of 18 OC pts or ≥ 1 of 13 CRC pts met the efficacy criteria (progression free at end of week 24), 15 additional OC pts or 14 additional CRC pts were to be enrolled in stage 2. The efficacy endpoint would be met if ≥ 11 OC pts or ≥ 4 CRC pts remained progression free at 24 weeks. Safety, response rate and progression-free survival (PFS) by RECIST 1.1, overall survival (OS), and immunologic effects in tumors were evaluated. ITT population = all pts who received at least one dose of durva or ONCOS-102; per protocol (PP) population = all pts who received at least 60% of ONCOS-102 doses and at least 1 durva dose in the first 2 cycles. Results: In MINIMAX stage 1, the OC cohort did not meet the efficacy criteria and was closed. For CRC, stage 1 efficacy criteria were achieved and the cohort was opened for stage 2. As of the 14 Dec 2021 cutoff, CRC enrollment was complete, and all pts were followed for 24 weeks or until progression or off study. Two pts were progression free at 24 weeks (see table). Treatment-related adverse events (TRAEs) occurring in &gt; 30% pts were vomiting, nausea, fatigue, chills, and pyrexia. There were no grade 4 or 5 TRAEs. Grade 3 TRAEs were reported in 8 pts, 2 in the OC cohort and 6 in CRC. All grade 3 TRAEs occurred in no more than 1 pt for each AE except abdominal pain, which occurred in 2 pts. Conclusions: The combination of IP ONCOS-102 and durva was well tolerated. The study did not meet its efficacy endpoint. Evaluation of pre- and on-therapy translational parameters is ongoing. Clinical trial information: NCT02963831. [Table: see text]

A phase Ia/Ib study evaluating the safety and efficacy of intratumorally administrated OH2, an oncolytic herpes simplex virus 2, in unresected stage IIIC to IV melanoma patients.

e21537 Background: OH2 is a herpes simplex virus type 2-derived oncolytic virus. It was engineered to selectively replicate within tumors and express granulocyte macrophage colony-stimulating factor (GM-CSF) to enhance tumor-specific immune responses. We conducted a phase Ia/Ib clinical trial to evaluate the safety and preliminary efficacy in unresected stage IIIC to IV melanoma patients (pts). Methods: In this phase Ia/Ib trial, we enrolled pts aged between 18-75 with histologically confirmed unresectable stage III or distant metastatic melanoma failed with prior therapies. In phase Ia, nine pts were in the single-dose group, where 3 dose levels (106, 107, and 108 CCID50/mL) of OH2 were assessed. And six pts were in the multi-dose group testing multiple administration regimens. The recommended doses were then expanded in phase Ib. The primary objective was the safety and tolerability of OH2 injection as defined by the dose-limiting toxicities (DLTs) and the maximum-tolerated dose (MTD) in phase Ia. Antitumor activity was assessed per Response Evaluation Criteria in Solid Tumors (RECIST version 1.1) and immune-RECIST in phase Ib. The clinical trial information: NCT04386967. Results: 35 pts were enrolled between November, 2018 and December, 2021. Among this, 37.1% were stageIVM1b-M1c; 88.6% had received of previous treatments: In the phase Ia study, OH2 was well-tolerated, neither serious AEs (SAEs) nor death were reported. No patient experienced TEAE leading to early withdrawal. Most TRAEs were Grade 1 in severity. There were no Grade 3 or above TRAEs. No DLTs were recorded, and the most frequent TRAEs were pyrexia (80%), Skin depigmentation (26.7), Oedema peripheral (26.7%), which were similar in three different doses of the single-dose group. The one-year survival rate was 93.3 % with median overall survival (mOS) being 20.8 months (still ongoing). In the phase Ib study, five pts achieved immune-partial response/partial response. The overall response rate (ORR) was 25.0 % and the five pts had ongoing responses. Six pts experienced stable disease (SD) with a disease control rate (DCR) being 55 %. The durable response rate (partial or complete response for ≥ 6 months) was 60 %. There were seven III-IV1a pts prior treated with anti-PD-1 antibodies. Three of the seven pts achieved iPR/PR (42.9 %), with DCR being 85.7 %. Conclusions: OH2 oncolytic virotherapy showed a favorable safety profile with no DLTs. And exhibited durable antitumor activity in pts. It warrants further investigation for the treatment of pts with unresectable melanoma. Clinical trial information: NCT04386967.

Magmas Inhibition in Prostate Cancer: A Novel Target for Treatment-Resistant Disease.

Simple SummaryMetastatic and treatment-resistant prostate cancer remains a life-threatening disease despite recent therapeutic advances. Literature suggests that treatment resistance and prostate cancer progression is associated with prostate cancer stem cells. In this study, we evaluated the role of the mitochondria-associated granulocyte–macrophage colony-stimulating factor signaling (Magmas) protein as a molecular target and applied a novel Magmas inhibitor, BT#9, on prostate cancer cells and normal control cells. We found that Magmas was overexpressed in human prostate cancers and its expression was linked to the aggressiveness of the disease. BT#9 downregulated Magmas expression, reduced viability and induced apoptotic cell death in prostate cancer cells. The mechanism of cell death by BT#9 is mainly caspase-independent and via a Reactive Oxygen Species (ROS)-mediated pathway. This is the first study that has evaluated targeting the Magmas protein in prostate cancer and, to our knowledge, the first to elucidate the potential molecular mechanism of BT#9 activity in prostate cancer, including the mode of cell death and the critical role of ROS accumulation. Our work may provide a potential clinical application for a novel prostate cancer treatment that can overcome cancer stem cell and therapeutic resistance.The purpose of our study was to evaluate Magmas as a potential target in prostate cancer. In addition, we evaluated our synthetic Magmas inhibitor (BT#9) effects on prostate cancer and examined the molecular mechanism of BT#9. A cell viability assay showed that treatment with BT#9 caused a significant decrease in the viability of DU145 and PC3 prostate cancer cells with little effect on the viability of WPMY-1 normal prostate cells. Western blot proved that BT#9 downregulated the Magmas protein and caspase-3 activation. Flow cytometry studies demonstrated increased apoptosis and disturbed mitochondrial membrane potential. However, the main mode of cell death was caspase-independent necrosis, which was correlated with the accumulation of mitochondrial and intra-cellular Reactive Oxygen Species (ROS). Taken together, our data suggest Magmas is a potential molecular target for the treatment of prostate cancer and that Magmas inhibition results in ROS-dependent and caspase-independent necrotic cell death.

Monocyte Chemotactic Protein-1 (MCP1) Accumulation in Human Osteoclast Precursor Cultures.

In vitro osteoclast methods require constant treatment with macrophage colony stimulating factor (M-CSF) to support precursor survival and addition of the differentiation agent receptor activator of NF-κB ligand (RANKL). Constant exposure to granulocyte macrophage colony stimulating factor (GM-CSF) suppresses human osteoclast formation in vitro. Addition of the chemokine monocyte chemotactic protein-1 (MCP1) to such cultures dramatically increases osteoclast formation and overcomes GM-CSF mediated suppression. We investigated the effect of M-CSF, GM-CSF and the combination of M-CSF and GM-CSF treatment on the expression of chemokines in human CD14+ cells in culture. Of assayed chemokines, MCP1 was the most abundant in terms of mRNA transcript and protein in M-CSF treated cultures and was suppressed by GM-CSF. MCP1 protein accumulated up to 50 ng/mL in culture medium, greatly exceeding other assayed chemokines. C-C chemokine receptor-2 (CCR2) is the receptor for MCP1: the formation of osteoclast-like cells was inhibited by constant exposure to the CCR2 antagonist RS102895, in part by decreasing expression of RANK, the receptor for RANKL.

Ameliorative effect of flavocoxid on cyclophosphamide-induced cardio and neurotoxicity via targeting the GM-CSF/NF-κB signaling pathway.

Cyclophosphamide (Cyclo) is a chemotherapeutic agent used as an immunosuppressant and as a treatment for many cancerous diseases. Many previous pieces of literature proved the marked cardio and neurotoxicity of the drug. Thus, this research provides evidence on the alleviative effect of flavocoxid on the cardiac and brain toxicity of cyclophosphamide in mice and determines its underlying mechanisms. Flavocoxid (Flavo) is a potent antioxidant and anti-inflammatory agent that inhibits the peroxidase activity of cyclooxygenase (COX-1 and COX-2) enzymes and 5-lipooxygenase (5-LOX). Flavo was administered orally (20 mg/kg) for 2 weeks, followed by Cyclo (100 mg/kg, i.p.) on day 14. Higher heart and brain weight indices, serum lactate dehydrogenase (LDH), creatine kinase (CK-MB), and nitric oxide (NO) were mitigated following Flavo administration. Flavo modulated oxidative stress biomarkers (malonaldehyde (MDA), glutathione (GSH), and superoxide dismutase (SOD)), tumor necrosis factor-α (TNF-α), and interleukin (IL)-1β. Additionally, cardiac troponin I (cTn-I), nuclear factor kappa B (NF-κB), brain amyloid precursor protein (APP), and granulocyte macrophage colony-stimulating factor (GM-CSF) were decreased by Flavo administration. Moreover, Flavo ameliorated heart and brain histopathological changes and caspase-3 levels. Collectively, Flavo (20 mg/kg) for 14 days showed significant cardio and neuroprotective effects due to its antioxidant, anti-inflammatory, and antiapoptotic activities via modulation of oxidative stress, inflammation, and the GM-CSF/NF-κB signaling pathway.

Evaluation of the α-casein (CSN1S1) locus as a potential target for a site-specific transgene integration

Transgenic animals are an important tool in biotechnology, including the production of recombinant proteins in the milk. Traditionally, expression constructs are based on hybrid vectors bearing mammary gland specific regulatory elements from the α-casein (Csn1s1), β-casein (Csn2), whey acidic protein (WAP), or β-lactoglobulin (BLG) genes. Overexpression from the randomly integrated vectors typically provides high levels of expression, but has drawbacks due to unpredictable genome localization. CRISPR-Cas9 targeted transgene integration into the endogenous casein locus could alleviate the need for extensive animal screening to achieve high and reproducible expression levels. We decided to evaluate such a “precise” integration approach, placing the human granulocyte–macrophage colony-stimulating factor (hGMCSF) gene under control of the mouse endogenous alpha-S1-casein (Csn1s1) promoter. We designed two types of transgene integrations: a knock-in in the second exon of the Csn1s1 (INS-GM) and a full-size Csn1s1 replacement with hGMCSF (REP-GM) which was never tested before. The INS-GM approach demonstrated low transgene expression and milk protein levels (0.4% of Csn2 transcripts; 2–11 µg/ml hGMCSF). This was probably caused by the absence of the 3’-polyadenylation signal in the hGMCSF transgene. REP-GM animals displayed high transgene expression, reaching and slightly exceeding the level of the endogenous Csn1s1 (30–40% of Csn2 transcripts), but yielded less hGMCSF protein than expected (0.2–0.5 mg/ml vs 25 mg/ml of Csn1s1), indicating that translation of the protein is not optimal. Homozygous inserts leading to the Csn1s1 knock-out did not have any long standing effects on the animals’ health. Thus, in our experimental design, site-specific transgene integration into the casein locus did not provide any significant advantage over the overexpression approach.

Characterization of structural features driving promiscuous ligand binding in GMCSF

Granulocyte macrophage colony stimulating factor (GMCSF) is a cytokine that is involved in myelopoiesis as well as immunomodulation. GMCSF has been shown to worsen conditions such as rheumatoid arthritis (RA) and multiple sclerosis (MS) while ameliorating diseases such as Type I Diabetes. GMCSF has also been used clinically to combat neutropenia based on its immunostimulatory activity. Pathophysiological properties of GMCSF have been highlighted by the incidence of the global pandemic, COVID‐19 (caused by SARS‐CoV‐2), where elevated levels of GMCSF were identified in COVID‐19 patients in cases related to development of systemic hyperinflammation, pneumonia and acute respiratory distress syndrome (ARDS). A key feature of signalling molecules like GMCSF is the selective and nuanced way in which responses can be tuned to external stimuli. The mechanism of how these signals are modulated are unknown and of particular interest. GMCSF and heparin have been shown to interact under low pH conditions. This binding interaction is important in two ways. Firstly, this pH sensitive interaction can serve as a basis to understand how cytokines can selectively interact with binding partners under variable physiological conditions. Secondly, in clinical settings where both anticoagulatory and immunostimulatory drugs are required, clear understanding of the binding interactions between the two drugs will help inform treatment decisions. Previously we identified heparin dependent structural changes as well as a striking dynamic change in GMCSF that was dependent on heparin chain length and pH using solution NMR studies which we believe to be dependent on a histidine triad. Here we have characterized GMCSF mutants using sited‐directed mutagenesis to exchange critical histidine residues for alternate amino acid residues (including Ala, Phe, Tyr and Asp) to probe the contributions of histidine residues to the increased flexibility of GMCSF observed in acidic conditions. We find that the histidine residues are critical for stability and as little as one amino acid difference will alter flexibility and stability of the protein. In some instances, mutations of all three histidine residues can lead to partial protein unfolding. Our results highlight the critical chemical properties at the amino acid level that allow GMCSF to interact with heparin in an environmentally dependent way.

Features of cellular and molecular mechanisms of re-gulation of reparative processes in chronic wounds using photobiomodulation therapy.

Treatment of chronic wounds is an urgent worldwide problem for medicine. Among the many efforts in wound healing techniques, photobiomodulation therapy has shown promising results.