Biomarkers of Efficacy and Safety of the Academic BCMA-CART ARI0002h for the Treatment of Refractory Multiple Myeloma.

PURPOSE: B-cell maturation antigen (BCMA)-chimeric antigen receptor T-cells (CART) improve results obtained with conventional therapy in the treatment of relapsed/refractory multiple myeloma. However, the high demand and expensive costs associated with CART therapy might prove unsustainable for health systems. Academic CARTs could potentially overcome these issues. Moreover, response biomarkers and resistance mechanisms need to be identified and addressed to improve efficacy and patient selection. Here, we present clinical and ancillary results of the 60 patients treated with the academic BCMA-CART, ARI0002h, in the CARTBCMA-HCB-01 trial. PATIENTS AND METHODS: We collected apheresis, final product, peripheral blood and bone marrow samples before and after infusion. We assessed BCMA, T-cell subsets, CART kinetics and antibodies, B-cell aplasia, cytokines, and measurable residual disease by next-generation flow cytometry, and correlated these to clinical outcomes. RESULTS: At cut-off date March 17, 2023, with a median follow-up of 23.1 months (95% CI, 9.2-37.1), overall response rate in the first 3 months was 95% [95% confidence interval (CI), 89.5-100]; cytokine release syndrome (CRS) was observed in 90% of patients (5% grades ≥3) and grade 1 immune effector cell-associated neurotoxicity syndrome was reported in 2 patients (3%). Median progression-free survival was 15.8 months (95% CI, 11.5-22.4). Surface BCMA was not predictive of response or survival, but soluble BCMA correlated with worse clinical outcomes and CRS severity. Activation marker HLA-DR in the apheresis was associated with longer progression-free survival and increased exhaustion markers correlated with poorer outcomes. ARI0002h kinetics and loss of B-cell aplasia were not predictive of relapse. CONCLUSIONS: Despite deep and sustained responses achieved with ARI0002h, we identified several biomarkers that correlate with poor outcomes.

Epstein-Barr virus-acquired immunodeficiency in myalgic encephalomyelitis-Is it present in long COVID?

Both myalgic encephalomyelitis or chronic fatigue syndrome (ME/CFS) and long COVID (LC) are characterized by similar immunological alterations, persistence of chronic viral infection, autoimmunity, chronic inflammatory state, viral reactivation, hypocortisolism, and microclot formation. They also present with similar symptoms such as asthenia, exercise intolerance, sleep disorders, cognitive dysfunction, and neurological and gastrointestinal complaints. In addition, both pathologies present Epstein-Barr virus (EBV) reactivation, indicating the possibility of this virus being the link between both pathologies. Therefore, we propose that latency and recurrent EBV reactivation could generate an acquired immunodeficiency syndrome in three steps: first, an acquired EBV immunodeficiency develops in individuals with "weak" EBV HLA-II haplotypes, which prevents the control of latency I cells. Second, ectopic lymphoid structures with EBV latency form in different tissues (including the CNS), promoting inflammatory responses and further impairment of cell-mediated immunity. Finally, immune exhaustion occurs due to chronic exposure to viral antigens, with consolidation of the disease. In the case of LC, prior to the first step, there is the possibility of previous SARS-CoV-2 infection in individuals with "weak" HLA-II haplotypes against this virus and/or EBV.

Large T cell clones expressing immune checkpoints increase during multiple myeloma evolution and predict treatment resistance.

Tumor recognition by T cells is essential for antitumor immunity. A comprehensive characterization of T cell diversity may be key to understanding the success of immunomodulatory drugs and failure of PD-1 blockade in tumors such as multiple myeloma (MM). Here, we use single-cell RNA and T cell receptor sequencing to characterize bone marrow T cells from healthy adults (n = 4) and patients with precursor (n = 8) and full-blown MM (n = 10). Large T cell clones from patients with MM expressed multiple immune checkpoints, suggesting a potentially dysfunctional phenotype. Dual targeting of PD-1 + LAG3 or PD-1 + TIGIT partially restored their function in mice with MM. We identify phenotypic hallmarks of large intratumoral T cell clones, and demonstrate that the CD27- and CD27+ T cell ratio, measured by flow cytometry, may serve as a surrogate of clonal T cell expansions and an independent prognostic factor in 543 patients with MM treated with lenalidomide-based treatment combinations.

Fractionated initial infusion and booster dose of ARI0002h, a humanised, BCMA-directed CAR T-cell therapy, for patients with relapsed or refractory multiple myeloma (CARTBCMA-HCB-01): a single-arm, multicentre, academic pilot study.

BACKGROUND: Chimeric antigen receptor (CAR) T-cell therapy is a promising option for patients with heavily treated multiple myeloma. Point-of-care manufacturing can increase the availability of these treatments worldwide. We aimed to assess the safety and activity of ARI0002h, a BCMA-targeted CAR T-cell therapy developed by academia, in patients with relapsed or refractory multiple myeloma. METHODS: CARTBCMA-HCB-01 is a single-arm, multicentre study done in five academic centres in Spain. Eligible patients had relapsed or refractory multiple myeloma and were aged 18-75 years; with an Eastern Cooperative Oncology Group performance status of 0-2; two or more previous lines of therapy including a proteasome inhibitor, an immunomodulatory agent, and an anti-CD38 antibody; refractoriness to the last line of therapy; and measurable disease according to the International Myeloma Working Group criteria. Patients received an initial fractionated infusion of 3 × 106 CAR T cells per kg bodyweight in three aliquots (0·3, 0·9, and 1·8 × 106 CAR-positive cells per kg intravenously on days 0, 3, and 7) and a non-fractionated booster dose of up to 3 × 106 CAR T cells per kg bodyweight, at least 100 days after the first infusion. The primary endpoints were overall response rate 100 days after first infusion and the proportion of patients developing cytokine-release syndrome or neurotoxic events in the first 30 days after receiving treatment. Here, we present an interim analysis of the ongoing trial; enrolment has ended. This study is registered with ClinicalTrials.gov, NCT04309981, and EudraCT, 2019-001472-11. FINDINGS: Between June 2, 2020, and Feb 24, 2021, 44 patients were assessed for eligibility, of whom 35 (80%) were enrolled. 30 (86%) of 35 patients received ARI0002h (median age 61 years [IQR 53-65], 12 [40%] were female, and 18 [60%] were male). At the planned interim analysis (cutoff date Oct 20, 2021), with a median follow-up of 12·1 months (IQR 9·1-13·5), overall response during the first 100 days from infusion was 100%, including 24 (80%) of 30 patients with a very good partial response or better (15 [50%] with complete response, nine [30%] with very good partial response, and six [20%] with partial response). Cytokine-release syndrome was observed in 24 (80%) of 30 patients (all grade 1-2). No cases of neurotoxic events were observed. Persistent grade 3-4 cytopenias were observed in 20 (67%) patients. Infections were reported in 20 (67%) patients. Three patients died: one because of progression, one because of a head injury, and one due to COVID-19. INTERPRETATION: ARI0002h administered in a fractioned manner with a booster dose after 3 months can provide deep and sustained responses in patients with relapsed or refractory multiple myeloma, with a low toxicity, especially in terms of neurological events, and with the possibility of a point-of-care approach. FUNDING: Instituto de Salud Carlos III (co-funded by the EU), Fundación La Caixa, and Fundació Bosch i Aymerich.

FlowCT for the analysis of large immunophenotypic data sets and biomarker discovery in cancer immunology.

Large-scale immune monitoring is becoming routinely used in clinical trials to identify determinants of treatment responsiveness, particularly to immunotherapies. Flow cytometry remains one of the most versatile and high throughput approaches for single-cell analysis; however, manual interpretation of multidimensional data poses a challenge when attempting to capture full cellular diversity and provide reproducible results. We present FlowCT, a semi-automated workspace empowered to analyze large data sets. It includes pre-processing, normalization, multiple dimensionality reduction techniques, automated clustering, and predictive modeling tools. As a proof of concept, we used FlowCT to compare the T-cell compartment in bone marrow (BM) with peripheral blood (PB) from patients with smoldering multiple myeloma (SMM), identify minimally invasive immune biomarkers of progression from smoldering to active MM, define prognostic T-cell subsets in the BM of patients with active MM after treatment intensification, and assess the longitudinal effect of maintenance therapy in BM T cells. A total of 354 samples were analyzed and immune signatures predictive of malignant transformation were identified in 150 patients with SMM (hazard ratio [HR], 1.7; P < .001). We also determined progression-free survival (HR, 4.09; P < .0001) and overall survival (HR, 3.12; P = .047) in 100 patients with active MM. New data also emerged about stem cell memory T cells, the concordance between immune profiles in BM and PB, and the immunomodulatory effect of maintenance therapy. FlowCT is a new open-source computational approach that can be readily implemented by research laboratories to perform quality control, analyze high-dimensional data, unveil cellular diversity, and objectively identify biomarkers in large immune monitoring studies. These trials were registered at www.clinicaltrials.gov as #NCT01916252 and #NCT02406144.

Immune biomarkers to predict SARS-CoV-2 vaccine effectiveness in patients with hematological malignancies.

There is evidence of reduced SARS-CoV-2 vaccine effectiveness in patients with hematological malignancies. We hypothesized that tumor and treatment-related immunosuppression can be depicted in peripheral blood, and that immune profiling prior to vaccination can help predict immunogenicity. We performed a comprehensive immunological characterization of 83 hematological patients before vaccination and measured IgM, IgG, and IgA antibody response to four viral antigens at day +7 after second-dose COVID-19 vaccination using multidimensional and computational flow cytometry. Health care practitioners of similar age were the control group (n = 102). Forty-four out of 59 immune cell types were significantly altered in patients; those with monoclonal gammopathies showed greater immunosuppression than patients with B-cell disorders and Hodgkin lymphoma. Immune dysregulation emerged before treatment, peaked while on-therapy, and did not return to normalcy after stopping treatment. We identified an immunotype that was significantly associated with poor antibody response and uncovered that the frequency of neutrophils, classical monocytes, CD4, and CD8 effector memory CD127low T cells, as well as naive CD21+ and IgM+D+ memory B cells, were independently associated with immunogenicity. Thus, we provide novel immune biomarkers to predict COVID-19 vaccine effectiveness in hematological patients, which are complementary to treatment-related factors and may help tailoring possible vaccine boosters.

Epstein-Barr Virus and the Origin of Myalgic Encephalomyelitis or Chronic Fatigue Syndrome.

Myalgic encephalomyelitis or chronic fatigue syndrome (ME/CFS) affects approximately 1% of the general population. It is a chronic, disabling, multi-system disease for which there is no effective treatment. This is probably related to the limited knowledge about its origin. Here, we summarized the current knowledge about the pathogenesis of ME/CFS and revisit the immunopathobiology of Epstein-Barr virus (EBV) infection. Given the similarities between EBV-associated autoimmune diseases and cancer in terms of poor T cell surveillance of cells with EBV latency, expanded EBV-infected cells in peripheral blood and increased antibodies against EBV, we hypothesize that there could be a common etiology generated by cells with EBV latency that escape immune surveillance. Albeit inconclusive, multiple studies in patients with ME/CFS have suggested an altered cellular immunity and augmented Th2 response that could result from mechanisms of evasion to some pathogens such as EBV, which has been identified as a risk factor in a subset of ME/CFS patients. Namely, cells with latency may evade the immune system in individuals with genetic predisposition to develop ME/CFS and in consequence, there could be poor CD4 T cell immunity to mitogens and other specific antigens, as it has been described in some individuals. Ultimately, we hypothesize that within ME/CFS there is a subgroup of patients with DRB1 and DQB1 alleles that could confer greater susceptibility to EBV, where immune evasion mechanisms generated by cells with latency induce immunodeficiency. Accordingly, we propose new endeavors to investigate if anti-EBV therapies could be effective in selected ME/CFS patients.

Immunological Biomarkers of Fatal COVID-19: A Study of 868 Patients.

Information on the immunopathobiology of coronavirus disease 2019 (COVID-19) is rapidly increasing; however, there remains a need to identify immune features predictive of fatal outcome. This large-scale study characterized immune responses to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection using multidimensional flow cytometry, with the aim of identifying high-risk immune biomarkers. Holistic and unbiased analyses of 17 immune cell-types were conducted on 1,075 peripheral blood samples obtained from 868 COVID-19 patients and on samples from 24 patients presenting with non-SARS-CoV-2 infections and 36 healthy donors. Immune profiles of COVID-19 patients were significantly different from those of age-matched healthy donors but generally similar to those of patients with non-SARS-CoV-2 infections. Unsupervised clustering analysis revealed three immunotypes during SARS-CoV-2 infection; immunotype 1 (14% of patients) was characterized by significantly lower percentages of all immune cell-types except neutrophils and circulating plasma cells, and was significantly associated with severe disease. Reduced B-cell percentage was most strongly associated with risk of death. On multivariate analysis incorporating age and comorbidities, B-cell and non-classical monocyte percentages were independent prognostic factors for survival in training (n=513) and validation (n=355) cohorts. Therefore, reduced percentages of B-cells and non-classical monocytes are high-risk immune biomarkers for risk-stratification of COVID-19 patients.

Immunologic characterization of COVID-19 patients with hematological cancer.

Not available.

Immunogenomic identification and characterization of granulocytic myeloid-derived suppressor cells in multiple myeloma.

Granulocytic myeloid-derived suppressor cells (G-MDSCs) promote tumor growth and immunosuppression in multiple myeloma (MM). However, their phenotype is not well established for accurate monitoring or clinical translation. We aimed to provide the phenotypic profile of G-MDSCs based on their prognostic significance in MM, immunosuppressive potential, and molecular program. The preestablished phenotype of G-MDSCs was evaluated in bone marrow samples from controls and MM patients using multidimensional flow cytometry; surprisingly, we found that CD11b+CD14-CD15+CD33+HLADR- cells overlapped with common eosinophils and neutrophils, which were not expanded in MM patients. Therefore, we relied on automated clustering to unbiasedly identify all granulocytic subsets in the tumor microenvironment: basophils, eosinophils, and immature, intermediate, and mature neutrophils. In a series of 267 newly diagnosed MM patients (GEM2012MENOS65 trial), only the frequency of mature neutrophils at diagnosis was significantly associated with patient outcome, and a high mature neutrophil/T-cell ratio resulted in inferior progression-free survival (P < .001). Upon fluorescence-activated cell sorting of each neutrophil subset, T-cell proliferation decreased in the presence of mature neutrophils (0.5-fold; P = .016), and the cytotoxic potential of T cells engaged by a BCMA×CD3-bispecific antibody increased notably with the depletion of mature neutrophils (fourfold; P = .0007). Most interestingly, RNA sequencing of the 3 subsets revealed that G-MDSC-related genes were specifically upregulated in mature neutrophils from MM patients vs controls because of differential chromatin accessibility. Taken together, our results establish a correlation between the clinical significance, immunosuppressive potential, and transcriptional network of well-defined neutrophil subsets, providing for the first time a set of optimal markers (CD11b/CD13/CD16) for accurate monitoring of G-MDSCs in MM.

Análisis de subpoblaciones monocitarias en relación con los factores de riesgo cardiovascular / Analysis of monocitary subpopulations in relation to cardiovascular risk factors

Introducción: La participación monocitaria en la progresión aterosclerótica y sus efectos pro- o antiinflamatorios dependen de las subpoblaciones circulantes. El objetivo de este estudio es la caracterización de dichas subpoblaciones y su asociación con los factores de riesgo cardiovascular. Métodos: Estudio transversal que incluye 102 pacientes seleccionados; edad media: 65 años (rango 41-86 años), 69% varones. Se utilizó un panel de anticuerpos específicos frente a monocitos clásicos (Mon1, CD14+CD16− CD300e+HLADR+), intermedios (Mon2, CD14+CD16+CD300e+HLADR+) y no clásicos (Mon3, CD14-k-CD16+CD300e+HLADR+). Se establecieron tres grupos de estudio; grupo 1: sujetos asintomáticos con más de un factor de riesgo cardiovascular (n = 17); grupo 2: sujetos asintomáticos, pero con patología vascular por ecografía o microalbuminuria (n = 56); y grupo 3: pacientes con algún evento vascular aterotrombótico previo (n = 19). Asimismo, se calculó el riesgo cardiovascular mediante las escalas Framingham y REGICOR. Resultados: Se observó una asociación entre las subpoblaciones Mon1 y Mon2 y los grupos del estudio (ANOVA, p < 0,05), independiente de la edad y el sexo para los Mon2. Asimismo, las subpoblaciones Mon1 y Mon 2 se asociaron con eventos vasculares adversos (ß = 0,86, p = 0,02 y ß = 0,1 p = 0,002, respectivamente), siendo la asociación de Mon2 independiente de la edad y el sexo. Además, el porcentaje de Mon3 se asoció con la presencia de más de 2 factores de riesgo cardiovascular (ß=0,21, p=0,04) en el análisis univariante. Finalmente, se halló una correlación entre los niveles de Mon1 y Mon2 con el número de leucocitos (r = 0,7, p < 0,001 y r = 0,26 p < 0,01, respectivamente). Conclusiones: El análisis de subpoblaciones monocitarias es de gran interés clínico, ya que permite establecer un diferente perfil inflamatorio según los grupos de riesgo cardiovascular establecidos

Introduction: Monocytes play an important role in atherosclerotic progression having both pro and anti-inflammatory effects depending on different circulating monocyte subpopulations. The objective of this study is to characterize these subpopulations and their association with cardiovascular risk factors. Methods: Transversal study including 102 selected patients, mean age: 65 years-old (range 41-86), 69% males. A set of specific antibodies against classical monocytes (Mon1, CD14+CD16- CD300e+HLADR+), intermediate (Mon2, CD14+CD16+CD300e+HLADR+) and non-classical (Mon3, CD14-CD16+CD300e+HLADR+) was assayed. Three groups of patients were included: 17 asymptomatic with more than one cardiovascular risk factor (group 1), 56 subjects asymptomatic but with vascular pathology assessed by ultrasound or microalbuminuria (group 2) and 19 patients with a previous atherothrombotic event (group 3). The cardiovascular risk was determined by Framingham and REGICOR scores. Results: An association between study groups and the percentage of Mon1 and Mon2 was observed (ANOVA, p < .05), being independent of age and sex for Mon2. Likewise Mon1 and Mon2 subpopulations were associated with cardiovascular adverse events (ß = 0.86, p =.02 y ß = 0.1 p =.002, respectively), independently of age and sex in the case of Mon2. Moreover the percentage of Mon3 was associated with the presence of several cardiovascular risk factors (ß = 0.21, p = .04) in the univariate analysis. In addition, there was a correlation between the levels of Mon1 and Mon2 and leukocytes (r = 0.7, p < .001 and r = 0.26, p = .01, respectively). Conclusions: The analysis of monocyte subpopulations may be clinically useful to stratify the inflammatory profile related to the different cardiovascular risk groups

Daratumumab in combination with urelumab to potentiate anti-myeloma activity in lymphocyte-deficient mice reconstituted with human NK cells.

Daratumumab is an anti-CD38 fully human IgG1 mAb approved for multiple myeloma treatment. One of the proposed mechanisms of action is the induction of antibody-dependent cellular cytotoxicity (ADCC) mediated by NK cells. NK cells acquire surface CD137 expression in the presence of solid-phase-attached daratumumab and when encountering a daratumumab-coated CD38+ tumor cell line. In this setting, addition of the agonist anti-CD137 mAb urelumab enhances NK-cell activation increasing CD25 expression and IFNÉ£ production. However, in vitro ADCC is not increased by the addition of urelumab both in 4h or 24h lasting experiments. To study urelumab-increased daratumumab-mediated ADCC activity in vivo, we set up a mouse model based on the intravenous administration of a luciferase-transfected multiple myeloma cell line of human origin, human NK cells and daratumumab to immuno-deficient NSG mice. In this model, intravenous administration of urelumab 24h after daratumumab delayed tumor growth and prolonged mice survival.

Analysis of monocitary subpopulations in relation to cardiovascular risk factors. / Análisis de subpoblaciones monocitarias en relación con los factores de riesgo cardiovascular.

INTRODUCTION: Monocytes play an important role in atherosclerotic progression having both pro and anti-inflammatory effects depending on different circulating monocyte subpopulations. The objective of this study is to characterize these subpopulations and their association with cardiovascular risk factors. METHODS: Transversal study including 102 selected patients, mean age: 65 years-old (range 41-86), 69% males. A set of specific antibodies against classical monocytes (Mon1, CD14+CD16- CD300e+HLADR+), intermediate (Mon2, CD14+CD16+CD300e+HLADR+) and non-classical (Mon3, CD14-CD16+CD300e+HLADR+) was assayed. Three groups of patients were included: 17 asymptomatic with more than one cardiovascular risk factor (group 1), 56 subjects asymptomatic but with vascular pathology assessed by ultrasound or microalbuminuria (group 2) and 19 patients with a previous atherothrombotic event (group 3). The cardiovascular risk was determined by Framingham and REGICOR scores. RESULTS: An association between study groups and the percentage of Mon1 and Mon2 was observed (ANOVA, p<.05), being independent of age and sex for Mon2. Likewise Mon1 and Mon2 subpopulations were associated with cardiovascular adverse events (ß=0.86, p=.02 y ß=0.1 p=.002, respectively), independently of age and sex in the case of Mon2. Moreover the percentage of Mon3 was associated with the presence of several cardiovascular risk factors (ß=0.21, p=.04) in the univariate analysis. In addition, there was a correlation between the levels of Mon1 and Mon2 and leukocytes (r=0.7, p<.001 and r=0.26, p=.01, respectively). CONCLUSIONS: The analysis of monocyte subpopulations may be clinically useful to stratify the inflammatory profile related to the different cardiovascular risk groups.

The Mechanism of Action of the Anti-CD38 Monoclonal Antibody Isatuximab in Multiple Myeloma.

PURPOSE: Knowledge about the mechanism of action (MoA) of monoclonal antibodies (mAb) is required to understand which patients with multiple myeloma (MM) benefit the most from a given mAb, alone or in combination therapy. Although there is considerable research about daratumumab, knowledge about other anti-CD38 mAbs remains scarce. EXPERIMENTAL DESIGN: We performed a comprehensive analysis of the MoA of isatuximab. RESULTS: Isatuximab induces internalization of CD38 but not its significant release from MM cell surface. In addition, we uncovered an association between levels of CD38 expression and different MoA: (i) Isatuximab was unable to induce direct apoptosis on MM cells with CD38 levels closer to those in patients with MM, (ii) isatuximab sensitized CD38hi MM cells to bortezomib plus dexamethasone in the presence of stroma, (iii) antibody-dependent cellular cytotoxicity (ADCC) was triggered by CD38lo and CD38hi tumor plasma cells (PC), (iv) antibody-dependent cellular phagocytosis (ADCP) was triggered only by CD38hi MM cells, whereas (v) complement-dependent cytotoxicity could be triggered in less than half of the patient samples (those with elevated levels of CD38). Furthermore, we showed that isatuximab depletes CD38hi B-lymphocyte precursors and natural killer (NK) lymphocytes ex vivo-the latter through activation followed by exhaustion and eventually phagocytosis. CONCLUSIONS: This study provides a framework to understand response determinants in patients treated with isatuximab based on the number of MoA triggered by CD38 levels of expression, and for the design of effective combinations aimed at capitalizing disrupted tumor-stroma cell protection, augmenting NK lymphocyte-mediated ADCC, or facilitating ADCP in CD38lo MM patients.See related commentary by Malavasi and Faini, p. 2946.

Oncolytic adenovirus Delta-24-RGD induces a widespread glioma proteotype remodeling during autophagy.

Adenovirus Delta-24-RGD has shown a remarkable efficacy in a phase I clinical trial for glioblastoma. Delta-24-RGD induces autophagy in glioma cells, however, the molecular derangements associated with Delta-24-RGD infection remains poorly understood. Here, proteomics was applied to characterize the glioma metabolic disturbances soon after Delta-24-RGD internalization and late in infection. Minutes post-infection, a rapid survival reprogramming of glioma cells was evidenced by an early c-Jun activation and a time-dependent dephosphorylation of multiple survival kinases. At 48 h post-infection (hpi), a severe intracellular proteostasis impairment was characterized, detecting differentially expressed proteins related to mRNA splicing, cytoskeletal organization, oxidative response, and inflammation. Specific kinase-regulated protein interactomes for Delta-24-RGD-modulated proteome revealed interferences with the activation dynamics of protein kinases C and A (PKC, PKA), tyrosine-protein kinase Src (c-Src), glycogen synthase kinase-3 (GSK-3) as well as serine/threonine-protein phosphatases 1 and 2A (PP1, PP2A) at 48hpi, in parallel with adenoviral protein overproduction. Moreover, the late activation of the nuclear factor kappa B (NF-κB) correlates with the extracellular increment of specific cytokines involved in migration, and activation of different inflammatory cells. Taken together, our integrative analysis provides further insights into the effects triggered by Delta-24-RGD in the modulation of tumor suppression and immune response against glioma. SIGNIFICANCE: The current study provides new insights regarding the molecular mechanisms governing the glioma metabolism during Delta-24-RGD oncolytic adenoviral therapy. The compilation and analysis of intracellular and extracellular proteomics have led us to characterize: i) the cell survival reprogramming during Delta-24-RGD internalization, ii) the proteostatic disarrangement induced by Delta-24-RGD during the autophagic stage, iii) the protein interactomes for Delta-24-RGD-modulated proteome, iv) the regulatory effects on kinase dynamics induced by Delta-24-RGD late in infection, and v) the overproduction of multitasking cytokines upon Delta-24-RGD treatment. We consider that the quantitative molecular maps generated in this study may establish the foundations for the development of complementary adenoviral based-vectors to increase the potency against glioma.

Spatial and temporal proteome dynamics of glioma cells during oncolytic adenovirus Delta-24-RGD infection.

Glioblastoma multiforme (GBM) is the most common and aggressive type of malignant glioma. Oncolytic adenoviruses are being modified to exploit the aberrant expression of proteins in tumor cells to increase the antiglioma efficacy. E1A mutant adenovirus Delta-24-RGD (DNX-2401) has shown a favorable toxicity profile and remarkable efficacy in a first-in-human phase I clinical trial. However, the comprehensive modulation of glioma metabolism in response to Delta-24-RGD infection is poorly understood. Integrating mass spectrometry based-quantitative proteomics, physical and functional interaction data, and biochemical approaches, we conducted a cell-wide study of cytosolic, nuclear, and secreted glioma proteomes throughout the early time course of Delta-24-RGD infection. In addition to the severe proteostasis impairment detected during the first hours post-infection (hpi), Delta-24-RGD induces a transient inhibition of signal transducer and activator of transcription 3 (STAT3), and transcription factor AP-1 (c-JUN) between 3 and 10hpi, increasing the nuclear factor kappa B (NF-κB) activity at 6hpi. Furthermore, Delta-24-RGD specifically modulates the activation dynamics of protein kinase C (PKC), extracellular signal-regulated kinase 1/2 (ERK1/2), and p38 mitogen-activated protein kinase (p38 MAPK) pathways early in infection. At extracellular level, Delta-24-RGD triggers a time -dependent dynamic production of multitasking cytokines, and chemotactic factors, suggesting potential pleiotropic effects on the immune system reactivation. Taken together, these data help us to understand the mechanisms used by Delta-24-RGD to exploit glioma proteome organization. Further mining of this proteomic resource may enable design and engineering complementary adenoviral based-vectors to increase the specificity and potency against glioma.

Effective cancer immunotherapy in mice by polyIC-imiquimod complexes and engineered magnetic nanoparticles.

Encouraging results are emerging from systems that exploit Toll like receptor (TLR) signaling, nanotechnology, checkpoint inhibition and molecular imaging for cancer immunotherapy. A major remaining challenge is developing effective, durable and tumour-specific immune responses without systemic toxicity. Here, we report a simple and versatile system based on synergistic activation of immune responses and direct cancer cell killing by combined TLR ligation using polyIC as TLR3 and imiquimod (R837) as TLR7 agonist, in combination with the model antigen ovalbumin (OVA) and phospholipid micelles loaded with zinc-doped iron oxide magnetic nanoparticles (MNPs). The combination of TLR agonists triggered a strong innate immune response in the lymph nodes (LNs) without systemic release of pro-inflammatory cytokines. The vaccines showed excellent efficacy against aggressive B16-F10 melanoma cells expressing OVA, which was improved with immune checkpoint abrogation of the immunosuppressive programmed death-ligand 1 (PD-L1) at the level of the cancer cells. By magnetic resonance (MR) and nuclear imaging we could track the vaccine migration from the site of injection to LNs and tumour. Overall, we show this synergistic TLR agonists and their combination with MNPs and immune checkpoint blockade to have considerable potential for preclinical and clinical development of vaccines for cancer immunotherapy.

Target Expression, Generation, Preclinical Activity, and Pharmacokinetics of the BCMA-T Cell Bispecific Antibody EM801 for Multiple Myeloma Treatment.

We identified B cell maturation antigen (BCMA) as a potential therapeutic target in 778 newly diagnosed and relapsed myeloma patients. We constructed an IgG-based BCMA-T cell bispecific antibody (EM801) and showed that it increased CD3+ T cell/myeloma cell crosslinking, followed by CD4+/CD8+ T cell activation, and secretion of interferon-γ, granzyme B, and perforin. This effect is CD4 and CD8 T cell mediated. EM801 induced, at nanomolar concentrations, myeloma cell death by autologous T cells in 34 of 43 bone marrow aspirates, including those from high-risk patients and patients after multiple lines of treatment, tumor regression in six of nine mice in a myeloma xenograft model, and depletion of BCMA+ cells in cynomolgus monkeys. Pharmacokinetics and pharmacodynamics indicate weekly intravenous/subcutaneous administration.

Microdosed Lipid-Coated (67)Ga-Magnetite Enhances Antigen-Specific Immunity by Image Tracked Delivery of Antigen and CpG to Lymph Nodes.

Development of vaccines to prevent and treat emerging new pathogens and re-emerging infections and cancer remains a major challenge. An attractive approach is to build the vaccine upon a biocompatible NP that simultaneously acts as accurate delivery vehicle and radiotracer for PET/SPECT imaging for ultrasensitive and quantitative in vivo imaging of NP delivery to target tissues/organs. Success in developing these nanovaccines will depend in part on having a "correct" NP size and accommodating and suitably displaying antigen and/or adjuvants (e.g., TLR agonists). Here we develop and evaluate a NP vaccine based on iron oxide-selective radio-gallium labeling suitable for SPECT((67)Ga)/PET((68)Ga) imaging and efficient delivery of antigen (OVA) and TLR 9 agonists (CpGs) using lipid-coated magnetite micelles. OVA, CpGs and rhodamine are easily accommodated in the hybrid micelles, and the average size of the construct can be controlled to be ca. 40 nm in diameter to target direct lymphatic delivery of the vaccine cargo to antigen presenting cells (APCs) in the lymph nodes (LNs). While the OVA/CpG-loaded construct showed effective delivery to endosomal TLR 9 in APCs, SPECT imaging demonstrated migration from the injection site to regional and nonregional LNs. In correlation with the imaging results, a range of in vitro and in vivo studies demonstrate that by using this microdosed nanosystem the cellular and humoral immune responses are greatly enhanced and provide protection against tumor challenge. These results suggest that these nanosystems have considerable potential for image-guided development of targeted vaccines that are more effective and limit toxicity.

Drafting the proteome landscape of myeloid-derived suppressor cells.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of cells that are defined by their myeloid origin, immature state, and ability to potently suppress T-cell responses. They regulate immune responses and the population significantly increases in the tumor microenvironment of patients with glioma and other malignant tumors. For their study, MDSCs are usually isolated from the spleen or directly of tumors from a large number of tumor-bearing mice although promising ex vivo differentiated MDSC production systems have been recently developed. During the last years, proteomics has emerged as a powerful approach to analyze MDSCs proteomes using shotgun-based mass spectrometry (MS), providing functional information about cellular homeostasis and metabolic state at a global level. Here, we will revise recent proteome profiling studies performed in MDSCs from different origins. Moreover, we will perform an integrative functional analysis of the protein compilation derived from these large-scale proteomic studies in order to obtain a comprehensive view of MDSCs biology. Finally, we will also discuss the potential application of high-throughput proteomic approaches to study global proteome dynamics and post-translational modifications (PTMs) during the differentiation process of MDSCs that will greatly boost the identification of novel MDSC-specific therapeutic targets to apply in cancer immunotherapy.