BackgroundMany cancer immunotherapies rely on cell surface protein engagements in trans. Checkpoint inhibitors block certain interactions between cancer cells and immune cells. Chimeric antigen ...receptor (CAR)-based therapies redirect immune cells towards tumors by binding of surface antigens on cancer cells. This highlights the importance of such cellular interactions and provides a rationale to develop an easy-to-handle, flexible and cost-effective tool to study them. Here we present a novel immunoassay that fulfills these requirements and we show its utility to address research questions arising in the context of immunotherapies.1 Materials and MethodsOur assay makes use of T cell lines equipped with reporter genes, that are translated upon activation of the TCR signaling pathway.2 To create cellular biosensors, the reporter cells are transduced to express chimeric receptors. These receptors consist of an extracellular domain of interest fused to a CD3ζ intracellular signaling domain. The cellular biosensors can then be probed with cells expressing the respective interacting receptor or ligand. This induces a fluorescent signal that is evaluable by flow cytometry.ResultsThe assay provides quantitative information on the inhibitory potency of immune checkpoint inhibitors to the PD-L1/PD1 axis and can be used to characterize small molecule inhibitors blocking the PD1/PD-L1 interaction. While some are highly active, surprisingly others do not interfere with PD1 binding to PD-L1 even at high concentrations. We demonstrate that cellular biosensors can function to probe defined cellular populations for the expression of interaction partners to orphan ligands. In this setting, we do not identify a receptor to the putative immune checkpoint B7-H3 on T cells suggesting alternative mechanisms for B7-H3 mediated immunosuppression. Finally, we apply cellular biosensors to validate binding of CAR antigen recognition domain. Variable chains of three B7-H3 specific antibodies are arranged as single chain fragments (scFv) and evaluated in a cellular biosensor assay. While a mirzotamab derived scFv induces a strong signal, binding of an omburtabmab-scFv is considerably weaker and no signal can be detected with an enoblituzumab derived scFv.ConclusionsIn summary, we present a novel type of immunoassay to study interactions between membrane proteins in trans and highlight potential uses in the context of immunotherapy. It is suitable to study immune checkpoints and can be used to characterize respective inhibitory drugs. It also enables unbiased screening of cellular samples for the expression of interaction partners to orphan ligands. Lastly, it is applicable for the validation of antigen binding domains for CARs. This may accelerate the development of new, functional CAR constructs and could furthermore serve as a method to characterize the role of antigen mutations for immune escape. References Funk MA, Leitner J, Gerner MC, et al. Interrogating ligand-receptor interactions using highly sensitive cellular biosensors. Nat Commun. 2023;14:7804. Jutz S, Hennig A, Paster W, et al. A cellular platform for the evaluation of immune checkpoint molecules. Oncotarget. 2017;8(39):64892-64906. M.A. Funk: None. J. Leitner: None. C. Battin: None. S. Gumpelmair: None. S. Theurich: None. P. Steinberger: None.
BackgroundThe hematopoietic transplant comorbidity index (HTC-CI) has been developed to determine treatment-related morbidity following allogeneic hematopoietic stem cell transplantation (alloHSCT) ...and includes obesity and diabetes as risk factors. On the other hand, chronic low-grade inflammation which is regularly associated with obesity and represents a mechanism of insulin resistance might mediate beneficial immune effects as demonstrated in the context of immune-check point inhibition cancer treatment. Literature on the role of a high body mass index (BMI) prior to alloHSCT remains controversial likely due to the complexity of the involved mechanisms that also comprise of increased catabolic rates and the immunonutritional status. In this study, we evaluated clinical outcomes in a large cohort of consecutive patients who underwent alloHSCT. Specifically, we analyzed pre-transplant BMI and immunonutritional scores as well as their dynamic changes in the early post-transplant phase with regard to survival and toxicities.Materials and MethodsClinical records of 664 consecutive patients undergoing alloHSCT between 2012 and 2017 at the Department of Medicine I, University Hospital of Cologne, Germany, were retrospectively analyzed. Patients were categorized into four BMI classes and three immunometabolic risk groups according to the modified Glasgow Prognostic Score (mGPS) measured pre-transplant and on day 30 post-transplant. Overall survival (OS), non-relapse mortality (NRM) and the development of a clinically relevant acute graft-versus-host disease (GvHD) ≥2 grade were compared using Kaplan-Meier survival analysis. Additional analyses stratified for sex and focused on a disease and transplant setting homogenized cohort.ResultsMedian BMI of the cohort was 24.6 (15.1-50.4) kg/m2. OS and NRM differed significantly between BMI classes (OS p = 0.02; NRM p = 0.05), with a significant survival benefit of overweight (median OS: 21 and 22 months in normal weight and obese, > 50% alive after 60 months in overweight). In contrast to the male cohort, in females also obesity had a favourable impact (p = 0.50; median OS: 16 months in normal weight; 35 months in overweight and >50% alive after 60 months in obese). mGPS classes, both determined pre-transplant and on day 30, experienced significantly different OS and NRM (OS p < 0.001; NRM p < 0.002), in which hypoalbuminemia combined with elevated C-reactive protein (mGPS 2) correlated with worst OS, NRM and a tendency of higher GvHD incidence. The extend of mGPS increase from day 0 to 30 impacted all outcomes significantly (OS p = 0.02; NRM p = 0.05; GvHD p = 0.01). Especially an increase to an mGPS of 2 was associated with significantly worse OS, NRM and higher GvHD incidence (median OS: 13 and 7 months in mGPS 0->2 and 1->2, respectively; 49 months in mGPS 0->1).ConclusionsOur data suggest a more complex role of metabolic pathologies as currently reflected by obesity and diabetes categories within the HTC-CI. Therefore, future prospective studies that include body composition as well as sensitive measures of disturbed glucose tolerance and metabolic rates are warranted to determine immunometabolic risk factors for alloHSCT outcomes. H. Thurisch: None. K. Althoff: None. S. Leitzke: None. U. Holtick: None. C. Scheid: None. S. Oganesian: None. M. Funk: None. D. Cordas dos Santos: None. M. von Bergwelt-Baildon: None. S. Theurich: None.
Abstract Primary cutaneous T-cell lymphomas (CTCL) are non-Hodgkin lymphomas usually running an indolent course. However, some patients progress to tumor stages or leukemic phase for which no ...curative treatment is available. Although initial response rates are high, remissions are often short-lived. Recent reports suggest a potential curative role for allogeneic stem cell transplantation (alloSCT). We searched databases for genetically randomized controlled trials (RCT) comparing alloSCT with conventional therapy. Data extraction and quality assessment were performed following the guidelines of the Cochrane Collaboration. Primary outcome measures were overall survival, secondary criteria included time-to-progression and response rate. A total number of 2077 primary citations were screened for relevant studies. Detailed analysis revealed that no RCTs on this subject have been performed and no systematic meta-analysis could be carried out. Nevertheless, several retrospective analyses and case series addressed the question of alloSCT for patients with advanced CTCL or Sézary syndrome. In this review, we will discuss the currently available data.
BackgroundIn recent years, T cell-based immunotherapies have shown promising results in hematologic malignancies. However, these strategies seem to be limited in solid cancers, posing more complex ...challenges including a hostile TME with nutrient deprivation and tissue hypoxia 1. Additionally, metabolic reprogramming has been identified as a crucial factor for proper cytotoxic T-cell functions upon their activation. Such energy demands are answered by the upregulation of glycolysis, oxidative phosphorylation, and upregulation of nutrient transporters represented by SLCs 2,3. Within the TME, tumor and immune cells compete for nutrients and shape a distinct metabolic milieu, resulting in an ineffective effector function 4. Herein, we aim to metabolically engineer T cells to improve their fitness in the glucose-deprived TME and optimize ACT.Materials and MethodsWe retrovirally overexpressed the glucose transporter Slc2a1/GLUT1 in murine CD8+ T cells (CD8+Slc2a1). To assess T-cell fitness we conducted experiments in physiologic (5mM) and hypoglycemic (0.5mM) media conditions. CellTraceTM-based proliferation experiments and killing assays in the OT1-OVA model are used to examine differences to MOCK in functionality and were analyzed via flow cytometry and microscopy, respectively. Furthermore, Seahorse analyses, bulk RNA-Seq, and metabolomic analyses were performed to examine the mechanical background. Murine in vivo studies are performed to approach the translatability of this system into living organisms.ResultsCD8+Slc2a1 cells possessed a higher proliferative capacity in all conditions tested but most prominently in hypoglycemic (0.5mM) media. This better functional activity of CD8+Slc2a1 was also translated to higher killing rates in coculture assays with tumor cells, especially in low-glucose environments. Metabolic flux analyses and multi-omics suggested greater metabolic activity of CD8+Slc2a1 and revealed higher ROS production and upregulation of correlating anti-oxidative pathways, especially the pentose-phosphate pathway. Preliminary in vivo studies support the in vitro killing in a syngeneic tumor model. Furthermore, signs of altered memory formation were visible, expressed in a higher proportion of effector memory cells.ConclusionsOur data point to the role of GLUT1 overexpression in T cells for improved cytotoxic activity, proliferation, and long-term persistence. Therefore, combinatorial approaches with GLUT1 overexpression could serve as a potential approach to increase efficacy in ACT against solid cancer. We also identified GLUT1-dependent reprogramming in CD8+Slc2a1 cells which is further investigated in ongoing studies. Additionally, we are evaluating the potential risk of this approach to neoplastic formation.References Treating hematological malignancies with cell therapy: where are we now? Landoni E, Savoldo B.; Expert Opin Biol Ther. 2018. Anticancer targets in the glycolytic metabolism of tumors: a comprehensive review; Paolo E. Porporato et al. Frontiers in Pharmacology 2011. Glucose Metabolism on Tumor Plasticity, Diagnosis, and Treatment; Lin Xiaoping et al. Frontiers in Oncology 2020 Fighting in a wasteland: deleterious metabolites and antitumor immunity. Watson MJ, Delgoffe GM. J Clin Invest. 2022. M.E. Kirmaier: None. B.L. Cadilha: None. A. Hadzic: None. W. Schmitz: None. M.R. Benmebarek: None. D. Briukhovetska: None. S. Michaelides: None. V. Buschinger: None. B. Tast: None. C.H. Bönigk: None. S. Oganesian: None. L. Vona: None. A. Tischmacher: None. V. Heissmeyer: None. M. Vaeth: None. V.R. Buchholz: None. M. Eilers: None. M. von Bergwelt-Baildon: None. M. Subklewe: None. S. Kobold: None. S. Theurich: None.
The CXCR4-inhibitor plerixafor mobilizes hematopoietic stem cells amplifying the effects of granulocyte-CSF (G-CSF). Before approval plerixafor was used in a compassionate use program (CUP) for ...patients who failed a previous mobilization. In the German CUP 60 patients from 23 centers (median age 56.5 years (2-75)) were given 240 μg/kg plerixafor SC 9-11 h before apheresis. A total of 78.3% (47/60) received G-CSF for 4 days before plerixafor administration; 76.6% of those (36/47) yielded at least 2.0 × 10(6) CD34(+) cells/μL. The median cell yield was 3.35 × 10(6) CD34+ cells/kg (0-29.53). Nine patients received plerixafor alone or with G-CSF for less than 4 days mobilizing a median of 3.30 × 10(6) CD34+ cells/kg (1.6-5.6). There was no significant difference between G-CSF application for 4 days and for a shorter period of time (P=0.157). A total of 47 patients received plerixafor plus G-CSF combined with chemotherapy yielding a median of 3.28 × 10(6) CD34+ cells/kg (0-24.79). In all, 40 of 60 patients (66.7%) proceeded to transplantation, and achieved a timely and stable engraftment. Side effects were rare and manageable. In conclusion, mobilization with plerixafor in poor mobilizers is safe and results in a sufficient stem cell harvest in the majority of patients.
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