Metabolic priming of GD2 TRAC-CAR T cells during manufacturing promotes memory phenotypes while enhancing persistence.

Manufacturing chimeric antigen receptor (CAR) T cell therapies is complex, with limited understanding of how medium composition impacts T cell phenotypes. CRISPR-Cas9 ribonucleoproteins can precisely insert a CAR sequence while disrupting the endogenous T cell receptor alpha constant (TRAC) gene resulting in TRAC-CAR T cells with an enriched stem cell memory T cell population, a process that could be further optimized through modifications to the medium composition. In this study we generated anti-GD2 TRAC-CAR T cells using "metabolic priming" (MP), where the cells were activated in glucose/glutamine-low medium and then expanded in glucose/glutamine-high medium. T cell products were evaluated using spectral flow cytometry, metabolic assays, cytokine production, cytotoxicity assays in vitro, and potency against human GD2+ xenograft neuroblastoma models in vivo. Compared with standard TRAC-CAR T cells, MP TRAC-CAR T cells showed less glycolysis, higher CCR7/CD62L expression, more bound NAD(P)H activity, and reduced IFN-γ, IL-2, IP-10, IL-1ß, IL-17, and TGF-ß production at the end of manufacturing ex vivo, with increased central memory CAR T cells and better persistence observed in vivo. MP with medium during CAR T cell biomanufacturing can minimize glycolysis and enrich memory phenotypes ex vivo, which could lead to better responses against solid tumors in vivo.

Time to resolution of iodine-123 metaiodobenzylguanidine (123 I-MIBG) avidity and local control outcomes for high-risk neuroblastoma following radiation therapy.

INTRODUCTION: 123 I-MIBG scan is used in neuroblastoma (NB) to monitor treatment response. Time to resolution of 123 I-MIBG avidity after radiation therapy (RT) is unknown. We sought to determine time to resolution of 123 I-MIBG avidity after RT and local failure (LF) rate. METHODS: We performed a retrospective review of children with high-risk NB who underwent 123 I-MIBG scans pre- and post-RT from 2003 to 2019. Time from RT to resolution of 123 I-MIBG activity was analysed. LF and cumulative incidence of local progression (CILP) after RT stratified by site, presence of residual disease and use of boost RT were determined. RESULTS: Forty-two patients with median age 3.9 years (1.9-4.7 years) were included, with median follow-up time 3.9 years (1.4-6.9). Eighty-six lesions were treated with RT to median dose of 21.6 Gy. Eighteen of 86 lesions were evaluable for time to resolution of MIBG avidity after RT, with median resolution time of 78 days (36-208). No LF occurred among 26 patients who received RT to primary sites after GTR, versus 4/12 (25%) patients treated with residual primary disease. 2-year CILP was 19% (12% primary disease 25% metastatic disease (P = 0.18)). 2-year CILP for non-residual primary, residual primary, non-residual metastatic and residual metastatic lesions was 0%, 42%, 11% and 30% respectively (P = 0.01) and for boosted and non-boosted residual lesions was 29% and 35% (P = 0.44). CONCLUSION: Median time to MIBG resolution after RT was 78 days. Primary lesions without residual disease had excellent local control. LF rate was higher after RT for residual disease, with no benefit for boost RT.

Comparative Study of the Effect of Radiation Delivered by Lutetium-177 or Actinium-225 on Anti-GD2 Chimeric Antigen Receptor T Cell Viability and Functions.

BACKGROUND AND PURPOSE: Chimeric antigen receptor (CAR) T cells have been relatively ineffective against solid tumors. Low-dose radiation which can be delivered to multiple sites of metastases by targeted radionuclide therapy (TRT) can elicit immunostimulatory effects. However, TRT has never been combined with CAR T cells against solid tumors in a clinical setting. This study investigated the effects of radiation delivered by Lutetium-177 (177Lu) and Actinium-225 (225Ac) on the viability and effector function of CAR T cells in vitro to evaluate the feasibility of such therapeutic combinations. After the irradiation of anti-GD2 CAR T cells with various doses of radiation delivered by 177Lu or 225Ac, their viability and cytotoxic activity against GD2-expressing human CHLA-20 neuroblastoma and melanoma M21 cells were determined by flow cytometry. The expression of the exhaustion marker PD-1, activation marker CD69 and the activating receptor NKG2D was measured on the irradiated anti-GD2 CAR T cells. Both 177Lu and 225Ac displayed a dose-dependent toxicity on anti-GD2 CAR T cells. However, radiation enhanced the cytotoxic activity of these CAR T cells against CHLA-20 and M21 irrespective of the dose tested and the type of radionuclide. No significant changes in the expression of PD-1, CD69 and NKG2D was noted on the CAR T cells following irradiation. Given a lower CAR T cell viability at equal doses and an enhancement of cytotoxic activity irrespective of the radionuclide type, 177Lu-based TRT may be preferred over 225Ac-based TRT when evaluating a potential synergism between these therapies in vivo against solid tumors.

The Combination of Radiotherapy and Complement C3a Inhibition Potentiates Natural Killer cell Functions Against Pancreatic Cancer.

Pancreatic cancer is one of the deadliest cancers, against which current immunotherapy strategies are not effective. Herein, we analyzed the immune cell composition of the tumor microenvironment of pancreatic cancer samples in The Cancer Genome Atlas and found that the presence of intratumoral NK cells correlates with survival. Subsequent analysis also indicated that NK cell exclusion from the microenvironment is found in a high percentage of clinical pancreatic cancers and in preclinical models of pancreatic cancer. Mechanistically, NK cell exclusion is regulated in part by complement C3a and its receptor signaling. Inhibition of the C3a receptor enhances NK cell infiltration in syngeneic mouse models of pancreatic cancer resulting in tumor growth delay. However, tumor growth inhibition mediated by NK cells is not sufficient alone for complete tumor regression, but is potentiated when combined with radiation therapy. Our findings indicate that although C3a inhibition is a promising approach to enhance NK cell-based immunotherapy against pancreatic cancer, its combination with radiation therapy hold greater therapeutic benefit.

Syndrome of inappropriate secretion of antidiuretic hormone following high dose rate brachytherapy for prostate cancer: a case report.

BACKGROUND: The syndrome of inappropriate secretion of antidiuretic hormone is a disorder characterized by the excess release of antidiuretic hormone and can result in hyponatremia. If managed inappropriately, severe hyponatremia can cause seizures, cerebral edema, and even death. There are various known causes of this inappropriate release of antidiuretic hormone, including malignancy, CNS disorders, and disturbances in the hypothalamic-pituitary-renal axis. However, reports of syndrome of inappropriate secretion of antidiuretic hormone after brachytherapy for prostate cancer are exceedingly rare. CASE PRESENTATION: We report a case of symptomatic hyponatremia secondary to the inappropriate secretion of antidiuretic hormone after prostate high-dose rate brachytherapy under general anesthesia in a patient with adenocarcinoma of the prostate. CONCLUSIONS: In rare instances, inappropriate secretion of antidiuretic hormone can occur after high-dose rate brachytherapy for prostate cancer. The cause is likely multifactorial, involving pain or discomfort ensuing from the surgical procedure, the general anesthesia or intraoperative drugs administered. However, due to the potential severity of the side effects, timely diagnosis is crucial to ensure prompt, and effective management.

Detection of Recurrence After Thoracic Stereotactic Ablative Radiotherapy Using FDG-PET-CT.

INTRODUCTION/BACKGROUND: Differentiating local recurrence (LR) from post-treatment changes following stereotactic ablative radiotherapy (SABR) for thoracic tumors is challenging. We sought to evaluate the performance of FDG-PET-CT in distinguishing recurrence from post-radiation changes in patients with stage I-II non-small cell lung cancer (NSCLC) treated with SABR. MATERIALS AND METHODS: We performed a retrospective review of patients with stage I-II NSCLC treated with SABR and subsequently followed with surveillance FDG-PET-CT scans from 2004 to 2014. The radiology reports were coded as 0 or 1 if minimally or substantially concerning for LR, respectively, and correlated with outcome. Prognostic factors for false-positive FDG-PET-CT were assessed using logistic regression models. RESULTS: We identified 145 patients meeting inclusion criteria for the retrospective analysis. Amongst the 39 (26.9%) patients with FDG-PET-CT scans concerning for LR 3 to 24 months after treatment, 14 were confirmed to have LR. Thus, the positive predictive value (PPV) of FDG-PET-CT in identifying LR was 36% (14/39). Factors associated with a false-positive scan included concerning FDG-PET-CT at the earliest post-treatment time point (3 months) (odds ratio 0.67, P= .04) and older age (odds ratio 2.3, P= .02). CONCLUSION: Our analysis indicates that the PPV of a concerning FDG-PET-CT after SABR for early-stage NSCLC is relatively low, especially at early post-treatment timepoints, but accuracy is improving over time with institutional experience.

Inflammation, Fibrosis and Cancer: Mechanisms, Therapeutic Options and Challenges.

Uncontrolled inflammation is a salient factor in multiple chronic inflammatory diseases and cancers. In this review, we provided an in-depth analysis of the relationships and distinctions between uncontrolled inflammation, fibrosis and cancers, while emphasizing the challenges and opportunities of developing novel therapies for the treatment and/or management of these diseases. We described how drug delivery systems, combination therapy and the integration of tissue-targeted and/or pathways selective strategies could overcome the challenges of current agents for managing and/or treating chronic inflammatory diseases and cancers. We also recognized the value of the re-evaluation of the disease-specific roles of multiple pathways implicated in the pathophysiology of chronic inflammatory diseases and cancers-as well as the application of data from single-cell RNA sequencing in the success of future drug discovery endeavors.

C3aR Signaling Inhibits NK-cell Infiltration into the Tumor Microenvironment in Mouse Models.

Many solid tumors have low levels of cytotoxic CD56dim natural killer (NK) cells, suggesting that CD56dim NK-cell exclusion from the tumor microenvironment (TME) contributes to the decreased response rate of immunotherapy. Complement component 3a (C3a) is known for its tumor-promoting and immunosuppressive roles in solid tumors. Previous reports have implicated the involvement of the C3a receptor (C3aR) in immune cell trafficking into the TME. C3aR is predominantly expressed on the surface of activated cytotoxic NK cells, but a specific role for C3aR in NK-cell biology has not been investigated. Because solid tumors generate elevated C3a and have decreased NK-cell infiltration, we hypothesized that C3aR might play a role in cytotoxic NK-cell recruitment into the TME. Our results indicate that blocking C3aR signaling in NK cells increased NK-cell infiltration into the TME in mouse models and led to tumor regression. Because the critical lymphocyte trafficking integrin LFA-1 orchestrates the migration of activated NK cells, we wanted to gain insight into the interaction between C3aR signaling and LFA-1. Our results demonstrated that direct interaction between C3aR and LFA-1, which led to a high-affinity LFA-1 conformation, decreased NK-cell infiltration into the TME. We propose that approaches to enhance cytotoxic NK-cell infiltration into the TME, through either disrupting C3a and C3aR interaction or inhibiting the formation of high-affinity LFA-1, represent a new strategy to improve the efficiency of immunotherapy for cancer treatment.

Acute and Late Esophageal Toxicity After SABR to Thoracic Tumors Near or Abutting the Esophagus.

PURPOSE: Our purpose was to evaluate the incidence of acute and late esophageal toxicity in patients with thoracic tumors near or abutting the esophagus treated with SABR. METHODS AND MATERIALS: Among patients with thoracic tumors treated with SABR, we identified those with tumors near or abutting the esophagus. Using the linear-quadratic model with an α/ß ratio of 10, we determined the correlation between dosimetric parameters and esophageal toxicity graded using the Common Terminology Criteria for Adverse Events, version 5.0. RESULTS: Out of 2200 patients treated with thoracic SABR, 767 patients were analyzable for esophageal dosimetry. We identified 55 patients with tumors near the esophagus (52 evaluable for esophagitis grade) and 28 with planning target volume (PTV) overlapping the esophagus. Dose gradients across the esophagus were consistently sharp. Median follow-up and overall survival were 16 and 23 months, respectively. Thirteen patients (25%) developed temporary grade 2 acute esophageal toxicity, 11 (85%) of whom had PTV overlapping the esophagus. Symptoms resolved within 1 to 3 months in 12 patients and 6 months in all patients. No grade 3 to 5 toxicity was observed. Only 3 patients (6%) developed late or persistent grade 2 dysphagia or dyspepsia of uncertain relationship to SABR. The cumulative incidence of acute esophagitis was 15% and 25% at 14 and 60 days, respectively. Acute toxicity correlated on univariate analysis with esophageal Dmax, D1cc, D2cc, Dmax/Dprescription, and whether the PTV was overlapping the esophagus. Esophageal Dmax (BED10) <62 Gy, D1cc (BED10) <48 Gy, D2cc (BED10) <43 Gy, and Dmax/Dprescription <85% were associated with <20% risk of grade 2 acute esophagitis. Only 2 local recurrences occurred. CONCLUSIONS: Although 25% of patients with tumors near the esophagus developed acute esophagitis (39% of those with PTV overlapping the esophagus), these toxicities were all grade 2 and all temporary. This suggests the safety and efficacy of thoracic SABR for tumors near or abutting the esophagus when treating with high conformity and sharp dose gradients.

Durability of response in metastatic melanoma patients after combined treatment with radiation therapy and ipilimumab.

AIM: We previously reported a prospective trial evaluating the safety and efficacy of combining ipilimumab and radiation therapy in patients with metastatic melanoma. Herein, we provide a long-term update on patients with complete response (CR) or partial response (PR). PATIENTS & METHODS: We continued to follow these patients with serial imaging including computed tomography, PET or MRI. RESULTS: Two of the three patients with CR are still alive and without evidence of melanoma but with chronic treatment-induced hypophysitis. The third patient died of hepatocellular carcinoma, but with no evidence of melanoma. Among the three patients with PR, two achieved CR after pembrolizumab monotherapy. CONCLUSION: This long-term follow up reveals the striking durability of the CRs, which appears to correlate with a grade 2-3 hypophysitis.

Design and structure activity relationship of tumor-homing histone deacetylase inhibitors conjugated to folic and pteroic acids.

Histone deacetylase (HDAC) inhibition has recently emerged as a novel therapeutic approach for the treatment of various pathological conditions including cancer. Currently, two HDAC inhibitors (HDACi) ­ Vorinostat and Romidepsin ­ have been approved for the treatment of cutaneous T-cell lymphoma. However, HDACi remain ineffective against solid tumors and are associated with adverse events including cardiotoxicity. Targeted delivery may enhance the therapeutic indices of HDACi and enable them to be efficacious against solid tumors. We showed herein that morphing of folic and pteroic acids into the surface recognition group of HDACi results in hydroxamate and benzamide HDACi which derived tumor homing by targeting folate receptor (FR), a receptor commonly overexpressed in solid tumors. We observed a correlation between the potency of HDAC1 inhibition and cytotoxicity as only the potent pteroate hydroxamates, 11d and 11e, displayed antiproliferative activity against two representative FR-expression cancer cells. Our observation further supports the previous results which suggest that for a drug to be successfully targeted using the FR, it must be extremely potent against its primary target as the FR has a low delivery efficiency.

Synthesis and structure-activity relationship of 3-hydroxypyridine-2-thione-based histone deacetylase inhibitors.

We previously identified 3-hydroxypyridine-2-thione (3HPT) as a novel zinc binding group for histone deacetylase (HDAC) inhibition. Early structure-activity relationship (SAR) studies led to various small molecules possessing selective inhibitory activity against HDAC6 or HDAC8 but devoid of HDAC1 inhibition. To delineate further the depth of the SAR of 3HPT-derived HDAC inhibitors (HDACi), we have extended the SAR studies to include the linker region and the surface recognition group to optimize the HDAC inhibition. The current efforts resulted in the identification of two lead compounds, 10d and 14e, with potent HDAC6 and HDAC8 activities that are inactive against HDAC1. These new HDACi possess anticancer activities against various cancer cell lines including Jurkat J.γ1 for which SAHA and the previously disclosed 3HPT-derived HDACi were inactive.

3-Hydroxypyridin-2-thione as novel zinc binding group for selective histone deacetylase inhibition.

Small molecules bearing hydroxamic acid as the zinc binding group (ZBG) have been the most effective histone deacetylase inhibitors (HDACi) to date. However, concerns about the pharmacokinetic liabilities of the hydroxamic acid moiety have stimulated research efforts aimed at finding alternative nonhydroxamate ZBGs. We have identified 3-hydroxypyridin-2-thione (3-HPT) as a novel ZBG that is compatible with HDAC inhibition. 3-HPT inhibits HDAC 6 and HDAC 8 with an IC50 of 681 and 3675 nM, respectively. Remarkably, 3-HPT gives no inhibition of HDAC 1. Subsequent optimization led to several novel 3HPT-based HDACi that are selective for HDAC 6 and HDAC 8. Furthermore, a subset of these inhibitors induces apoptosis in various cancer cell lines.

Targeted cancer therapy: giving histone deacetylase inhibitors all they need to succeed.

Histone deacetylase inhibitors (HDACis) have now emerged as a powerful new class of small-molecule therapeutics acting through the regulation of the acetylation states of histone proteins (a form of epigenetic modulation) and other non-histone protein targets. Over 490 clinical trials have been initiated in the last 10 years, culminating in the approval of two structurally distinct HDACis - SAHA (vorinostat, Zolinza™) and FK228 (romidepsin, Istodax™). However, the current HDACis have serious limitations, including ineffectively low concentrations in solid tumors and cardiac toxicity, which is hindering their progress in the clinic. Herein, we review the primary paradigms being pursued to overcome these hindrances, including HDAC isoform selectivity, localized administration, and targeting cap groups to achieve selective tissue and cell type distribution.

Tamoxifen-poly(ethylene glycol)-thiol gold nanoparticle conjugates: enhanced potency and selective delivery for breast cancer treatment.

The breast cancer treatment drug tamoxifen has been widely administered for more than three decades. This small molecule competes with 17beta-estradiol for binding to estrogen receptor, a hormone receptor upregulated in a majority of breast cancers, subsequently initiating programmed cell death. We have synthesized a thiol-PEGylated tamoxifen derivative that can be used to selectively target and deliver plasmonic gold nanoparticles to estrogen receptor positive breast cancer cells with up to 2.7-fold enhanced drug potency in vitro. Optical microscopy/spectroscopy, time-dependent dose-response data, and estrogen competition studies indicate that augmented activity is due to increased rates of intracellular tamoxifen transport by nanoparticle endocytosis, rather than by passive diffusion of the free drug. Both ligand- and receptor-dependent intracellular delivery of gold nanoparticles suggest that plasma membrane localized estrogen receptor alpha may facilitate selective uptake and retention of this and other therapeutic nanoparticle conjugates. Combined targeting selectivity and enhanced potency provides opportunities for both multimodal endocrine treatment strategies and adjunctive laser photothermal therapy.