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.

A Holistic Approach to Hard-to-Treat Cancers: The Future of Immunotherapy for Glioblastoma, Triple Negative Breast Cancer, and Advanced Prostate Cancer.

Immunotherapy represents an attractive avenue for cancer therapy due to its tumour specificity and relatively low frequency of adverse effects compared to other treatment modalities. Despite many advances being made in the field of cancer immunotherapy, very few immunotherapeutic treatments have been approved for difficult-to-treat solid tumours such as triple negative breast cancer (TNBC), glioblastoma multiforme (GBM), and advanced prostate cancer (PCa). The anatomical location of some of these cancers may also make them more difficult to treat. Many trials focus solely on immunotherapy and have failed to consider or manipulate, prior to the immunotherapeutic intervention, important factors such as the microbiota, which itself is directly linked to lifestyle factors, diet, stress, social support, exercise, sleep, and oral hygiene. This review summarises the most recent treatments for hard-to-treat cancers whilst factoring in the less conventional interventions which could tilt the balance of treatment in favour of success for these malignancies.

Robust formation of optimal single spheroids towards cost-effective in vitro three-dimensional tumor models.

While useful for fundamental in vitro studies, monolayer cell cultures are not physiologically relevant. Spheroids, a complex three-dimensional (3D) structure, more closely resemble in vivo tumor growth. Spheroids allow the results obtained relating to proliferation, cell death, differentiation, metabolism, and various antitumor therapies to be more predictive of in vivo outcomes. In the protocol herein, a rapid and high-throughput method is discussed for the generation of single spheroids using various cancer cell lines, including brain cancer cells (U87 MG, SEBTA-027, SF188), prostate cancer cells (DU-145, TRAMP-C1), and breast cancer cells (BT-549, Py230) in 96-round bottom-well plates. The proposed method is associated with significantly low costs per plate without requiring refining or transferring. Homogeneous compact spheroid morphology was evidenced as early as 1 day after following this protocol. Proliferating cells were traced in the rim, while dead cells were found to be located inside the core region of the spheroid using confocal microscopy and the Incucyte® live imaging system. H&E staining of spheroid sections was utilized to investigate the tightness of the cell packaging. Through western blotting analyses, it was revealed that a stem cell-like phenotype was adopted by these spheroids. This method was also used to obtain the EC50 of the anticancer dipeptide carnosine on U87 MG 3D culture. This affordable, easy-to-follow five-step protocol allows for the robust generation of various uniform spheroids with 3D morphology characteristics.

What are the odds? Identifying factors related to competitive success in powerlifting.

BACKGROUND: The ability for athletes to gain a competitive advantage over their opponents is well recognised. At times, this advantage may be considered a marginal gain. However, in the context of competition, marginal advantages may be the difference between winning and losing. This investigation explores how competition factors influence the odds of competitive success (i.e. winning) in powerlifting (PL) to assist athletes and coaches in achieving a competitive advantage. METHODS: A cross-sectional, retrospective analysis of competition data from raw/classic, Australian powerlifting competitions 2010-2019 was conducted. Data included 10,599 competition entries (males: n = 6567 [62%], females: n = 4032 [38%]). Independent t-tests were used to compare continuous data between sexes or winners and non-winners at an event. Cohen's d and the 95% confidence interval (d [95% CI]) were calculated. Univariate odds of winning an event based on independent variables (age [irrespective of category], sex, body weight and weight of first lift attempt [regardless of success]), were assessed by separate simple logistic regression. RESULTS: When compared to males, the odds of winning for females were 50% greater (OR [95% CI] 1.500 [1.384, 1.625]; P < 0.001). Athletes who had larger first lift attempts (Squat: + 7.0 kg P < 0.001, Bench Press: + 3.2 kg P < 0.001, and Deadlift: + 6.1 kg P < 0.001and competed for a longer period (winners: 401 vs non-winners: 304 days, P < 0.001) had an increased likelihood winning. Age was associated with increased odds of success for males (OR [95% CI] 1.014 [1.009, 1.019], P < 0.001) per additional year of age for males, but not females (P = 0.509). CONCLUSIONS: Multiple factors appear to contribute to the likelihood of winning a PL competition. These results may help coaches to develop competition and training strategies that optimise athletes' likelihood of competitive success in PL.

Impacts of squat attempt weight selection and success on powerlifting performance.

BACKGROUND: To date, there is no evidence to support the optimal competition strategy for success in powerlifting competitions. The purpose of this study was to analyze powerlifting (PL) competition data to assess the relationship between squat attempts, the success of each lift attempt, and weight increase between attempts, with winning. METHODS: The analysis from 'raw' Powerlifting Australia sanctioned competitions held between 2008 and 2019 included 10,672 individual competition entries (males: N.=6617, females: N.=4055). We reported Cohen's d, statistical significance, 95% confidence intervals and the univariate odds of winning an event. Factors were assessed by separate simple logistic regression and reported as an odds ratio. RESULTS: Overall, first squat attempt weight for those who won was on average 7.0 kg greater (P<0.001, d=0.14 [0.10, 0.18]) than for non-winners. In the total sample, athletes selected opening attempts which were an average of 92% of their achieved maximum on the day with 93.5% of competitors improving on this weight in subsequent attempts. Winners had a 0.5 kg (P<0.001, d=0.10 [0.06, 0.14]) greater absolute increase in weight between first and second lift attempts than non-winners. Overall, ~68% of winners successfully lifted their third attempt weight compared to ~64% of non-winners (P<0.001). CONCLUSIONS: A powerlifting athlete's odds of winning a competition overall are significantly increased by selecting a larger opening squat attempt weight than competitors and completing the attempt successfully. The opening squat may be one of the most important lifts during competition.

Helicase antigen (HAGE)-derived vaccines induce immunity to HAGE and ImmunoBody®-HAGE DNA vaccine delays the growth and metastasis of HAGE-expressing tumors in vivo.

The management of patients with triple-negative breast cancer (TNBC) continues to pose a significant clinical challenge. Less than 30% of women with metastatic TNBC survive 5 years, despite adjuvant chemotherapy and the initial higher rates of clinical response that can be achieved with neoadjuvant chemotherapy. ImmunoBody is a plasmid DNA designed to encode a human antibody molecule with complementarity-determining regions engineered to express cytotoxic and helper T-cell epitopes derived from the cancer antigen of interest. The helicase antigen (HAGE) is a cancer testis antigen, which is expressed in TNBC. Herein, we have identified a 30-amino-acid-long HAGE-derived sequence containing human leukocyte antigen (HLA)-A2- and HLA-DR1-restricted epitopes and demonstrated that the use of this sequence as a peptide (with CpG/incomplete Freund's adjuvant) or incorporated into an ImmunoBody vaccine can generate specific interferon-γ-secreting splenocytes in HHDII+ DR1+ mice. T-cell responses elicited by the ImmunoBody-HAGE vaccine were superior to peptide immunization. Moreover, splenocytes from ImmunoBody-HAGE-vaccinated mice stimulated in vitro could recognize HAGE+ tumor cells and the human TNBC cell line MDA-MB-231. More importantly, the growth of implanted HHDII+ DR1+ HAGE+ Luc+ B16 cells.

AMG 757, a Half-Life Extended, DLL3-Targeted Bispecific T-Cell Engager, Shows High Potency and Sensitivity in Preclinical Models of Small-Cell Lung Cancer.

PURPOSE: Small-cell lung cancer (SCLC) is an aggressive neuroendocrine tumor with a high relapse rate, limited therapeutic options, and poor prognosis. We investigated the antitumor activity of AMG 757, a half-life extended bispecific T-cell engager molecule targeting delta-like ligand 3 (DLL3)-a target that is selectively expressed in SCLC tumors, but with minimal normal tissue expression. EXPERIMENTAL DESIGN: AMG 757 efficacy was evaluated in SCLC cell lines and in orthotopic and patient-derived xenograft (PDX) mouse SCLC models. Following AMG 757 administration, changes in tumor volume, pharmacodynamic changes in tumor-infiltrating T cells (TILs), and the spatial relationship between the appearance of TILs and tumor histology were examined. Tolerability was assessed in nonhuman primates (NHPs). RESULTS: AMG 757 showed potent and specific killing of even those SCLC cell lines with very low DLL3 expression (<1,000 molecules per cell). AMG 757 effectively engaged systemically administered human T cells, induced T-cell activation, and redirected T cells to lyse tumor cells to promote significant tumor regression and complete responses in PDX models of SCLC and in orthotopic models of established primary lung SCLC and metastatic liver lesions. AMG 757 was well tolerated with no AMG 757-related adverse findings up to the highest tested dose (4.5 mg/kg weekly) in NHP. AMG 757 exhibits an extended half-life in NHP, which is projected to enable intermittent administration in patients. CONCLUSIONS: AMG 757 has a compelling safety and efficacy profile in preclinical studies making it a viable option for targeting DLL3-expressing SCLC tumors in the clinical setting.

Immune Escape in Glioblastoma Multiforme and the Adaptation of Immunotherapies for Treatment.

Glioblastoma multiforme (GBM) is the most frequently occurring primary brain tumor and has a very poor prognosis, with only around 5% of patients surviving for a period of 5 years or more after diagnosis. Despite aggressive multimodal therapy, consisting mostly of a combination of surgery, radiotherapy, and temozolomide chemotherapy, tumors nearly always recur close to the site of resection. For the past 15 years, very little progress has been made with regards to improving patient survival. Although immunotherapy represents an attractive therapy modality due to the promising pre-clinical results observed, many of these potential immunotherapeutic approaches fail during clinical trials, and to date no immunotherapeutic treatments for GBM have been approved. As for many other difficult to treat cancers, GBM combines a lack of immunogenicity with few mutations and a highly immunosuppressive tumor microenvironment (TME). Unfortunately, both tumor and immune cells have been shown to contribute towards this immunosuppressive phenotype. In addition, current therapeutics also exacerbate this immunosuppression which might explain the failure of immunotherapy-based clinical trials in the GBM setting. Understanding how these mechanisms interact with one another, as well as how one can increase the anti-tumor immune response by addressing local immunosuppression will lead to better clinical results for immune-based therapeutics. Improving therapeutic delivery across the blood brain barrier also presents a challenge for immunotherapy and future therapies will need to consider this. This review highlights the immunosuppressive mechanisms employed by GBM cancers and examines potential immunotherapeutic treatments that can overcome these significant immunosuppressive hurdles.

Effect of Competition Frequency on Strength Performance of Powerlifting Athletes.

Pearson, J, Spathis, JG, van den Hoek, DJ, Owen, PJ, Weakley, J, and Latella, C. Effect of competition frequency on strength performance of powerlifting athletes. J Strength Cond Res 34(5): 1213-1219, 2020-Powerlifting (PL) requires athletes to achieve the highest possible "total" weight lifted across squat, bench press, and deadlift. Athletes compete multiple times per year; however, it is not well understood how often PL athletes should compete to facilitate maximal strength performance. This study investigated the effect of competition frequency on strength (relative and absolute) in PL athletes over a 12-month period. Results across all male (n = 563, mean ± SD; age; 28 ± 10 years, body mass; 89.3 ± 19.3 kg) and female (n = 437, age; 31 ± 11 years, body mass; 70.1 ± 15.8 kg) PL athletes were collated. Total competition scores were used to calculate absolute and relative strength for each competition. Linear mixed models with random effects, and effect sizes ± 95% confidence intervals compared competition frequency and total score for (a) all, (b) male, and (c) female competition entries, respectively. The association between total score at each competition was assessed with Pearson's correlation coefficient for the same independent variables. Results demonstrate greater absolute strength at competition 2 for all athletes (5.1%: p = 0.043: d = 0.16) and males (2.9%: p = 0.049: d = 0.15). For females, absolute strength was greater at competition 5 compared to 1 (12.0%: p = 0.001: d = 0.65) and 2 (9.6%: p = 0.007: d = 0.50). Weak positive correlations for relative strength and number of times competed for males were evident between competitions 1 to 4 (r = 0.070-0.085, p = 0.003-0.043). For females, 3 competitions weakly correlated with absolute strength (r = 0.106, p = 0.016). PL athletes who compete multiple times per year are more likely to achieve higher totals; however, there is an upper limit to the number of competitions (4 per year) that seem to allow a performance increase.

Two algorithms for the reorganisation of the problem list by organ system.

OBJECTIVE: Long problem lists can be challenging to use. Reorganisation of the problem list by organ system is a strategy for making long problem lists more manageable. METHODS: In a small-town primary care setting, we examined 4950 unique problem lists over 5 years (24 033 total problems and 2170 unique problems) from our electronic health record. All problems were mapped to the International Classification of Diseases, 10th Revision, Clinical Modification (ICD-10-CM) and SNOMED CT codes. We developed two different algorithms for reorganising the problem list by organ system based on either the ICD-10-CM or the SNOMED CT code. RESULTS: The mean problem list length was 4.9±4.6 problems. The two reorganisation algorithms allocated problems to one of 15 different categories (12 aligning with organ systems). 26.2% of problems were assigned to a more general category of 'signs and symptoms' that did not correspond to a single organ system. The two algorithms were concordant in allocation by organ system for 90% of the unique problems. Since ICD-10-CM is a monohierarchic classification system, problems coded by ICD-10-CM were assigned to a single category. Since SNOMED CT is a polyhierarchical ontology, 19.4% of problems coded by SNOMED CT were assigned to multiple categories. CONCLUSION: Reorganisation of the problem list by organ system is feasible using algorithms based on either ICD-10-CM or SNOMED CT codes, and the two algorithms are highly concordant.

PYK2 promotes HER2-positive breast cancer invasion.

BACKGROUND: Metformin, a biguanide, is one of the most commonly prescribed treatments for type 2 diabetes and has recently been recommended as a potential drug candidate for advanced cancer therapy. Although Metformin has antiproliferative and proapoptotic effects on breast cancer, the heterogenous nature of this disease affects the response to metformin leading to the activation of pro-invasive signalling pathways that are mediated by the focal adhesion kinase PYK2 in pure HER2 phenotype breast cancer. METHODS: The effect of metformin on different breast cancer cell lines, representing the molecular heterogenicity of the disease was investigated using in vitro proliferation and apoptosis assays. The activation of PYK2 by metformin in pure HER2 phenotype (HER2+/ER-/PR-) cell lines was investigated by microarrays, quantitative real time PCR and immunoblotting. Cell migration and invasion PYK2-mediated and in response to metformin were determined by wound healing and invasion assays using HER2+/ER-/PR- PYK2 knockdown cell lines. Proteomic analyses were used to determine the role of PYK2 in HER2+/ER-/PR- proliferative, migratory and invasive cellular pathways and in response to metformin. The association between PYK2 expression and HER2+/ER-/PR- patients' cancer-specific survival was investigated using bioinformatic analysis of PYK2 expression from patient gene expression profiles generated by the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) study. The effect of PYK2 and metformin on tumour initiation and invasion of HER2+/ER-/PR- breast cancer stem-like cells was performed using the in vitro stem cell proliferation and invasion assays. RESULTS: Our study showed for the first time that pure HER2 breast cancer cells are more resistant to metformin treatment when compared with the other breast cancer phenotypes. This drug resistance was associated with the activation of PTK2B/PYK2, a well-known mediator of signalling pathways involved in cell proliferation, migration and invasion. The role of PYK2 in promoting invasion of metformin resistant HER2 breast cancer cells was confirmed through investigating the effect of PYK2 knockdown and metformin on cell invasion and by proteomic analysis of associated cellular pathways. We also reveal a correlation between high level of expression of PYK2 and reduced survival in pure HER2 breast cancer patients. Moreover, we also report a role of PYK2 in tumour initiation and invasion-mediated by pure HER2 breast cancer stem-like cells. This was further confirmed by demonstrating a correlation between reduced survival in pure HER2 breast cancer patients and expression of PYK2 and the stem cell marker CD44. CONCLUSIONS: We provide evidence of a PYK2-driven pro-invasive potential of metformin in pure HER2 cancer therapy and propose that metformin-based therapy should consider the molecular heterogeneity of breast cancer to prevent complications associated with cancer chemoresistance, invasion and recurrence in treated patients.

Targeting Multiple Myeloma with AMG 424, a Novel Anti-CD38/CD3 Bispecific T-cell-recruiting Antibody Optimized for Cytotoxicity and Cytokine Release.

PURPOSE: Despite advances in the treatment of multiple myeloma, new therapies are needed to induce more profound clinical responses. T-cell-redirected lysis triggered by bispecific antibodies recruiting T cells to cancer cells is a clinically validated mechanism of action against hematologic malignancies and CD38 is a tumor-associated antigen with near-universal expression in multiple myeloma. Thus, an anti-CD38/CD3 bispecific T-cell-recruiting antibody has the potential to be an effective new therapeutic for multiple myeloma. EXPERIMENTAL DESIGN: Anti-CD38/CD3 XmAb T-cell-recruiting antibodies with different affinities for CD38 and CD3 were assessed in vitro and in vivo for their redirected T-cell lysis activity against cancer cell lines, their lower levels of cytokine release, and their potency in the presence of high levels of soluble CD38. Select candidates were further tested in cynomolgus monkeys for B-cell depletion and cytokine release properties. RESULTS: AMG 424 was selected on the basis of its ability to kill cancer cells expressing high and low levels of CD38 in vitro and trigger T-cell proliferation, but with attenuated cytokine release. In vivo, AMG 424 induces tumor growth inhibition in bone marrow-invasive mouse cancer models and the depletion of peripheral B cells in cynomolgus monkeys, without triggering excessive cytokine release. The activity of AMG 424 against normal immune cells expressing CD38 is also presented. CONCLUSIONS: These findings support the clinical development of AMG 424, an affinity-optimized T-cell-recruiting antibody with the potential to elicit significant clinical activity in patients with multiple myeloma.

Targeting cellular pathways in glioblastoma multiforme.

Glioblastoma multiforme (GBM) is a debilitating disease that is associated with poor prognosis, short median patient survival and a very limited response to therapies. GBM has a very complex pathogenesis that involves mutations and alterations of several key cellular pathways that are involved in cell proliferation, survival, migration and angiogenesis. Therefore, efforts that are directed toward better understanding of GBM pathogenesis are essential to the development of efficient therapies that provide hope and extent patient survival. In this review, we outline the alterations commonly associated with GBM pathogenesis and summarize therapeutic strategies that are aimed at targeting aberrant cellular pathways in GBM.

Interleukin-21 receptor blockade inhibits secondary humoral responses and halts the progression of preestablished disease in the (NZB × NZW)F1 systemic lupus erythematosus model.

OBJECTIVE: Systemic lupus erythematosus (SLE) is a complex autoimmune disease that is driven in part by chronic B and T lymphocyte hyperresponsiveness to self antigens. A deficiency of interleukin-21 (IL-21) or IL-21 receptor (IL-21R) in mice dramatically reduces inflammation and B and T cell activation in models of autoimmunity, including SLE. However, whether IL-21 is essential for the maintenance and amplification of preestablished inflammation has not been widely examined in various animal models. The purpose of this study was to examine the impact of novel mouse IL-21R neutralizing antibodies on recall responses to antigen challenge and on disease progression in the (NZB × NZW)F1 (NZB/NZW) mouse model of SLE. METHODS: Humoral and cellular immune responses to immunization with sheep red blood cells (SRBCs) were measured in mice dosed with IL-21R blocking antibodies. Progression of nephritis and markers of immune activation was monitored in NZB/NZW mice following different anti-IL-21R treatment regimens. RESULTS: IL-21R blockade specifically inhibited secondary IgG responses to SRBC immunization. In NZB/NZW mice, IL-21R blockade completely inhibited the onset of nephritis, which was associated with dramatic reductions in splenomegaly and in B cell and T cell activation. When administered to mice with preexisting disease, anti-IL-21R antibody halted the disease progression and mortality and reversed the nephritis in a subset of mice. Furthermore, treatment cessation was not followed by rapid reemergence of disease. CONCLUSION: Our results highlight the importance of IL-21 in promoting humoral recall responses and in sustaining autoimmune inflammation.

The kinetic mechanism for cytochrome P450 metabolism of type II binding compounds: evidence supporting direct reduction.

The metabolic stability of a drug is an important property that should be optimized during drug design and development. Nitrogen incorporation is hypothesized to increase the stability by coordination of nitrogen to the heme iron of cytochrome P450, a binding mode that is referred to as type II binding. However, we noticed that the type II binding compound 1 has less metabolic stability at sub-saturating conditions than a closely related type I binding compound 3. Three kinetic models will be presented for type II binder metabolism; (1) Dead-end type II binding, (2) a rapid equilibrium between type I and II binding modes before reduction, and (3) a direct reduction of the type II coordinated heme. Data will be presented on reduction rates of iron, the off rates of substrate (using surface plasmon resonance) and the catalytic rate constants. These data argue against the dead-end, and rapid equilibrium models, leaving the direct reduction kinetic mechanism for metabolism of the type II binding compound 1.