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Development of semi-automated devices that can reduce the hands-on time and standardize the production of clinical-grade CAR T-cells, such as CliniMACS Prodigy from Miltenyi, is key to facilitate the development of CAR T-cell therapies, especially in academic institutions. However, the feasibility of manufacturing CAR T-cell products from heavily pre-treated patients with this system has not been demonstrated yet. Here we report and characterize the production of 28 CAR T-cell products in the context of a phase I clinical trial for CD19+ B-cell malignancies (NCT03144583). The system includes CD4-CD8 cell selection, lentiviral transduction and T-cell expansion using IL-7/IL-15. Twenty-seven out of 28 CAR T-cell products manufactured met the full list of specifications and were considered valid products. cell expansion lasted an average of 8.5 days and had a mean transduction rate of 30.6 ± 13.44%. All products obtained presented cytotoxic activity against CD19+ cells and were proficient in the secretion of pro-inflammatory cytokines. Expansion kinetics was slower in patient's cells compared to healthy donor's cells. However, product potency was comparable. CAR T-cell subset phenotype was highly variable among patients and largely determined by the initial product. T and T were the predominant T-cell phenotypes obtained. 38.7% of CAR T-cells obtained presented a T or T phenotype, in average, which are the subsets capable of establishing a long-lasting T-cell memory in patients. An in-depth analysis to identify individual factors contributing to the optimal T-cell phenotype revealed that cell expansion leads to reduced numbers of T , T , and T cells, while T cells increase, both due to cell expansion and CAR-expression. Overall, our results show for the first time that clinical-grade production of CAR T-cells for heavily pre-treated patients using CliniMACS Prodigy system is feasible, and that the obtained products meet the current quality standards of the field. Reduced expansion may yield CAR T-cell products with increased persistence .

Existing immune signatures and tumor mutational burden have only modest predictive capacity for the efficacy of immune check point inhibitors. In this study, we developed an immune-metabolic signature suitable for personalized ICI therapies. A classifier using an immune-metabolic signature (IMMETCOLS) was developed on a training set of 77 metastatic colorectal cancer (mCRC) samples and validated on 4,200 tumors from the TCGA database belonging to 11 types. Here, we reveal that the IMMETCOLS signature classifies tumors into three distinct immune-metabolic clusters. Cluster 1 displays markers of enhanced glycolisis, hexosamine byosinthesis and epithelial-to-mesenchymal transition. On multivariate analysis, cluster 1 tumors were enriched in pro-immune signature but not in immunophenoscore and were associated with the poorest median survival. Its predicted tumor metabolic features suggest an acidic-lactate-rich tumor microenvironment (TME) geared to an immunosuppressive setting, enriched in fibroblasts. Cluster 2 displays features of gluconeogenesis ability, which is needed for glucose-independent survival and preferential use of alternative carbon sources, including glutamine and lipid uptake/β-oxidation. Its metabolic features suggest a hypoxic and hypoglycemic TME, associated with poor tumor-associated antigen presentation. Finally, cluster 3 is highly glycolytic but also has a solid mitochondrial function, with concomitant upregulation of glutamine and essential amino acid transporters and the pentose phosphate pathway leading to glucose exhaustion in the TME and immunosuppression. Together, these findings suggest that the IMMETCOLS signature provides a classifier of tumors from diverse origins, yielding three clusters with distinct immune-metabolic profiles, representing a new predictive tool for patient selection for specific immune-metabolic therapeutic approaches.

Encapsulation of water-soluble bioactive compounds for enabling specific accumulation in tumor locations, while avoiding premature clearance and/or degradation in the bloodstream, is one of the main hallmarks in nanomedicine, especially that of NIR fluorescent probes for cancer theragnosis. The herein reported technology furnishes water-dispersible double-walled polyurethane-polyurea hybrid nanocapsules (NCs) loaded with indocyanine green (ICG-NCs), using a versatile and highly efficient one-pot and industrially scalable synthetic process based on the use of two different prepolymers to set up the NCs walls. Flow cytometry and confocal microscopy confirmed that both ICG-loaded NCs internalized in monocyte-derived dendritic cells (moDCs). The in vivo analysis of xenograft A375 mouse melanoma model revealed that amphoteric functionalization of NCs’ surface promotes the selective accumulation of ICG-NCs in tumor tissues, making them promising agents for a less-invasive theragnosis of cancer.

Androgen receptor (AR) signaling remains crucial in castration-resistant prostate cancer (CRPC). Since it is also essential in immune cells, we studied whether the expression of AR full-length (ARFL) and its splicing variant ARV7 in peripheral blood mononuclear cells (PBMC) predicts systemic treatment response in mCRPC in comparison with circulating-tumor cells (CTC). We measured ARFL and ARV7 mRNA in PBMC and CTC from patients prior to receiving abiraterone (AA), enzalutamide (E), or taxanes by a pre-amplification plus quantitative reverse-transcription PCR. They were also tested in LNCaP-ARV7-transfected and in 22RV1 docetaxel-resistant (22RV1DR) cells. We studied 171 PBMC from 136 patients and from 24 non-cancer controls, and 47 CTC from 22 patients. High PBMC ARV7 levels correlated with worse AA/E and better taxane response. In taxane-treated patients high PBMC ARFL also correlated with longer progression-free survival (PFS). High ARV7 and ARFL expression were independently associated with better biochemical-PFS. Conversely, high CTC ARV7 and ARFL correlated with shorter radiological-PFS and overall survival, respectively. High ARV7 in 22RV1DR and LNCaP-ARV7 cells correlated with taxane resistance. In conclusion, ARFL and ARV7 at PBMC or CTC have a different predictive role in the taxane response, suggesting a potential influence of the AR pathway from PBMC in such response modulation.

LFA-1 is a leukocyte specific β2 integrin that plays a major role in regulating adhesion and migration of different immune cells. Recent data suggest that LFA-1 on mature dendritic cells (mDCs) may function as a chemokine-inducible anchor during homing of DCs through the afferent lymphatics into the lymph nodes, by transiently switching its molecular conformational state. However, the role of LFA-1 mobility in this process is not yet known, despite that the importance of lateral organization and dynamics for LFA-1-mediated adhesion regulation is broadly recognized. Using single particle tracking approaches we here show that LFA-1 exhibits higher mobility on resting mDCs compared to monocytes. Lymphoid chemokine CCL21 stimulation of the LFA-1 high affinity state on mDCs, led to a significant reduction of mobility and an increase on the fraction of stationary receptors, consistent with re-activation of the receptor. Addition of soluble monomeric ICAM-1 in the presence of CCL21 did not alter the diffusion profile of LFA-1 while soluble ICAM-1 nano-aggregates in the presence of CCL21 further reduced LFA-1 mobility and readily bound to the receptor. Overall, our results emphasize the importance of LFA-1 lateral mobility across the membrane on the regulation of integrin activation and its function as adhesion receptor. Importantly, our data show that chemokines alone are not sufficient to trigger the high affinity state of the integrin based on the strict definition that affinity refers to the adhesion capacity of a single receptor to its ligand in solution. Instead our data indicate that nanoclustering of the receptor, induced by multi-ligand binding, is required to maintain stable cell adhesion once LFA-1 high affinity state is transiently triggered by inside-out signals.

Dendritic cells have been investigated in clinical trials, predominantly with the aim of stimulating immune responses against tumours or infectious diseases. Thus far, however, no clinical studies have taken advantage of their specific immunosuppressive potential. Tolerogenic DCs may represent a new therapeutic strategy for human immune-based diseases, such as Crohn's disease, where the perturbations of the finely tuned balance between the immune system and the microflora result in disease. In the present report, we describe the generation of tolerogenic DCs from healthy donors and Crohn's disease patients using clinical-grade reagents in combination with dexamethasone as immunosuppressive agent and characterize their response to maturation stimuli. Interestingly, we found out that dexamethasone-conditioned DCs keep their tolerogenic properties to Gram-negative bacteria. Other findings included in this study demonstrate that the combination of dexamethasone with a specific cytokine cocktail yielded clinical-grade DCs with the following characteristics: a semi-mature phenotype, a pronounced shift towards anti-inflammatory versus inflammatory cytokine production and low T-cell stimulatory properties. Importantly, in regard to their clinical application, the tolerogenic phenotype of DCs remained stable after the elimination of dexamethasone and after a second stimulation with LPS or bacteria. All these properties make this cell product suitable to be tested in clinical trials of inflammatory conditions including Crohn's disease.

The gut mucosa undergoes continuous antigenic exposure from food antigens, commensal flora derived ligands, and pathogens. This constant stimulation results in controlled inflammatory responses that are effectively suppressed by multiple factors. This tight regulation, necessary to maintain intestinal homeostasis, is affected during inflammatory bowel diseases (IBD) resulting in altered immune responses to harmless microorganisms. Dendritic cells (DCs) are sentinels of immunity, located in peripheral and lymphoid tissues, which are essential for homeostasis of T cell-dependent immune responses. The expression of a particular set of pathogen recognition receptors allows DCs to initiate immune responses. However, in the absence of danger signals, different DC subsets can induce active tolerance by inducing regulatory T cells (Treg), inhibiting inflammatory T helper cell responses, or both. Interestingly, several protocols to generate clinical grade tolerogenic DC (tol-DCs) in vitro have been described, opening the possibility to restore the intestinal homeostasis to bacterial flora by cellular therapy. In this review, we discuss different DC subsets and their role in IBD. Additionally, we will review preclinical studies performed in animal models while describing recent characterization of tol-DCs from Crohn’s disease patients for clinical application.

The identification of activated T-lymphocytes restricted to myelin-derived immunogenic peptides in multiple sclerosis (MS) and aquaporin-4 water channel in neuromyelitis optica (NMO) in the blood of patients opened the possibility for developing highly selective and disease-specific therapeutic approaches. Antigen presenting cells and in particular dendritic cells (DCs) represent a strategy to inhibit pro-inflammatory T helper cells. DCs are located in peripheral and lymphoid tissues and are essential for homeostasis of T cell-dependent immune responses. The expression of a particular set of receptors involved in pathogen recognition confers to DCs the property to initiate immune responses. However, in the absence of danger signals different DC subsets have been revealed to induce active tolerance by inducing regulatory T cells, inhibiting pro-inflammatory T helper cells responses or both. Interestingly, several protocols to generate clinical-grade tolerogenic DC (Tol-DC) have been described, offering the possibility to restore the homeostasis to central nervous system-related antigens. In this review, we discuss about different DC subsets and their role in tolerance induction, the different protocols to generate Tol-DCs and preclinical studies in animal models as well as describe recent characterization of Tol-DCs for clinical application in autoimmune diseases and in particular in MS and NMO patients. In addition, we discuss the clinical trials ongoing based on Tol-DCs to treat different autoimmune diseases.

BackgroundImmune check-point blockade (ICB) has shown clinical benefit in mismatch repair-deficient/microsatellite instability high metastatic colorectal cancer (mCRC) but not in mismatch repair-proficient/microsatellite stable patients. Cancer vaccines with autologous dendritic cells (ADC) could be a complementary therapeutic approach to ICB as this combination has the potential to achieve synergistic effects. MethodsThis was a Phase I/II multicentric study with translational sub-studies, to evaluate the safety, pharmacodynamics and anti-tumor effects of Avelumab plus ADC vaccine in heavily pre-treated MSS mCRC patients. Primary objective was to determine the maximum tolerated dose and the efficacy of the combination. The primary end-point was 40% progression-free survival at 6 months with a 2 Simon Stage. ResultsA total of 28 patients were screened and 19 pts were included. Combined therapy was safe and well tolerated. An interim analysis (Simon design first-stage) recommended early termination because only 2/19 (11%) patients were disease free at 6 months. Median PFS was 3.1 months [2.1–5.3 months] and overall survival was 12.2 months [3.2–23.2 months]. Stimulation of immune system was observed in vitro but not clinically. The evaluation of basal RNA-seq noted significant changes between pre and post-therapy liver biopsies related to lipid metabolism and transport, inflammation and oxidative stress pathways. ConclusionsThe combination of Avelumab plus ADC vaccine is safe and well tolerated but exhibited modest clinical activity. Our study describes, for the first-time, a de novo post-therapy metabolic rewiring, that could represent novel immunotherapy-induced tumor vulnerabilities.

During the last 20 years, chemotherapy has improved survival rates of colorectal cancer (CRC). However, the majority of metastatic cases do not respond to or progress after first line conventional chemotherapy and contribute to the fatalities of patients with CRC. Insights into the immune contexture of the tumor microenvironment (TME) have enabled the development of new systemic treatments that boost the host immune system against the tumor—the immune checkpoint inhibitors (ICI). These promising drugs have already shown astonishing efficacies in other cancer types and have raised new hope for the treatment of metastatic CRC (mCRC). In this review, we will summarize the results of the clinical trials that led to their accelerated approval by the U.S. Food and Drug Administration (FDA) in 2017, as well as all relevant recent studies conducted since then—some of which are not published yet. We will focus on therapeutic efficacy, but also discuss the available data for drug safety and security, changes in quality of life indicators and predictive biomarkers for treatment response. The burgeoning evidence for a potential use of ICIs in other settings than mCRC will also be mentioned. For each trial, we have made a preliminary assessment of the quality of clinical trial design and of the “European Society of Medical Oncology (ESMO) magnitude of clinical benefit” (ESMO-MCBS) in order to provide the first evidence-based recommendation to the reader.