An evaluation of glofitamab, the first fixed-duration bispecific antibody for relapsed or refractory large B-cell lymphomas.

INTRODUCTION: Significant proportion of patients with diffuse large B-cell lymphoma (DLBCL) is refractory or relapse (R/R) after the treatment. The prognosis of this patient cohort remains poor. Novel strategies mainly based on immunotherapy and targeted agents are currently being studied. Glofitamab is novel T-cell-engaging bispecific antibody possessing a 2:1 structure with bivalent CD20 binding. Its safety and efficacy in R/R B-cell non-Hodgkin lymphoma including DLBCL were evaluated in phase I-II NP30179 trial. AREAS COVERED: The article summarizes the milestones and latest reports on glofitamab development in the field of B-cell lymphoma treatment. EXPERT OPINION: Recently, phase II part of the NP30179 study and several other reports were published proving glofitamab potential in R/R DLBCL patients. Based on the published data, glofitamab was approved by regulatory authorities worldwide for the monotherapy of R/R DLBCL in conventional time-limited manner. It is readily accessible in case of rapidly progressing disease, and it compares well with other novel treatment options. Its side effects are similar to those of other T-cell-engaging agents and can be mitigated by pretreatment with obinutuzumab or step-up dosing. Its safety profile with manageable toxicities heads the clinical development toward combination strategies and its use in earlier therapeutic phases.

Characterization of the input material quality for the production of tisagenlecleucel by multiparameter flow cytometry and its relation to the clinical outcome.

Tisagenlecleucel (tisa-cel) is a CD19-specific CAR-T cell product approved for the treatment of relapsed/refractory (r/r) DLBCL or B-ALL. We have followed a group of patients diagnosed with childhood B-ALL (n = 5), adult B-ALL (n = 2), and DLBCL (n = 25) who were treated with tisa-cel under non-clinical trial conditions. The goal was to determine how the intensive pretreatment of patients affects the produced CAR-T cells, their in vivo expansion, and the outcome of the therapy. Multiparametric flow cytometry was used to analyze the material used for manufacturing CAR-T cells (apheresis), the CAR-T cell product itself, and blood samples obtained at three timepoints after administration. We present the analysis of memory phenotype of CD4/CD8 CAR-T lymphocytes (CD45RA, CD62L, CD27, CD28) and the expression of inhibitory receptors (PD-1, TIGIT). In addition, we show its relation to the patients' clinical characteristics, such as tumor burden and sensitivity to prior therapies. Patients who responded to therapy had a higher percentage of CD8+CD45RA+CD27+ T cells in the apheresis, although not in the produced CAR-Ts. Patients with primary refractory aggressive B-cell lymphomas had the poorest outcomes which was characterized by undetectable CAR-T cell expansion in vivo. No clear correlation of the outcome with the immunophenotypes of CAR-Ts was observed. Our results suggest that an important parameter predicting therapy efficacy is CAR-Ts' level of expansion in vivo but not the immunophenotype. After CAR-T cells' administration, measurements at several timepoints accurately detect their proliferation intensity in vivo. The outcome of CAR-T cell therapy largely depends on biological characteristics of the tumors rather than on the immunophenotype of produced CAR-Ts.

Retrospective Analysis of 118 Patients With Cutaneous T-Cell Lymphomas: A Single-Center Experience.

Cutaneous T-cell lymphomas (CTCL) represent rare non-Hodgkin lymphomas (NHL) with an incidence less than 1 per 100,000 inhabitants. The most common type of CTCL is mycosis fungoides (MF), which represents approximately 60% of all CTCL, followed by Sézary syndrome (SS), approximately 5%. We retrospectively analyzed the outcome of 118 patients with MF (n=96) and SS (n=22) treated between the years 1998 and 2021 at the Charles University General Hospital in Prague, Czech Republic. The ratio between men and women was 1.2:1 (62 men, and 56 women). The median age at diagnosis was 62 years (23 to 92 years). From the MF cohort 48 patients (50% out of MF cohort) presented with advanced stage disease. Ninety patients (77%) received a systemic treatment at any time from the diagnosis; the median number of therapy lines was two. At the time of database lock, the overall survival (OS) of 96 patients with MF reached 17.7 years with the median follow-up 4.0 years. With the median follow-up 2.6 years, the median OS of 22 patients with SS was 3.5 years. The most common type of systemic therapy for MF included low-dose methotrexate (61%), interferon-alpha (58%), bexarotene (28%), and chlorambucil (25%). The most common type of therapy for SS included bexarotene (64%), extracorporeal photopheresis (50%), and interferon-alpha (45%). Only the minority of patients received innovative targeted agents including brentuximab vedotin, mogamulizumab, or pembrolizumab. Besides the retrospective analysis of the CTCL cohort, current standards and future perspectives of selected innovative agents are summarized and discussed. The analyzed cohort represents the largest cohort of CTCL patients in the Czech Republic. Overall, the survival parameters of our CTCL cohort are comparable to those previously published by other groups. In conclusion, our analysis of 118 real world cohort of consecutive CTCL patients treated at the single center confirmed the efficacy of immune response modifiers and underlines the urgent need for ample implementation of innovative agents and their combinations into earlier lines of therapy.

Chimeric Antigen Receptor Based Cellular Therapy for Treatment Of T-Cell Malignancies.

T-cell malignancies can be divided into precursor (T-acute lymphoblastic leukemia/lymphoblastic lymphoma, T-ALL/LBL) and mature T-cell neoplasms, which are comprised of 28 different entities. Most of these malignancies are aggressive with rather poor prognosis. Prognosis of relapsed/refractory (R/R) disease is especially dismal, with an expected survival only several months after progression. Targeted therapies, such as antiCD30 immunotoxin brentuximab vedotin, antiCD38 antibody daratumumab, and anti-CCR4 antibody mogamulizumab are effective only in subsets of patients with T-cell neoplasms. T-cells equipped with chimeric antigen receptor (CAR-Ts) are routinely used for treatment of R/R B-cell malignancies, however, there are specific obstacles for their use in T-cell leukemias and lymphomas which are fratricide killing, risk of transfection of malignant cells, and T-cell aplasia. The solution for these problems relies on target antigen selection, CRISPR/Cas9 or TALEN gene editing, posttranslational regulation of CAR-T surface antigen expression, and safety switches. Structural chromosomal changes and global changes in gene expression were observed with gene-edited products. We identified 49 studies of CAR-based therapies registered on www.clinicaltrials.gov. Most of them target CD30 or CD7 antigen. Results are available only for a minority of these studies. In general, clinical responses are above 50% but reported follow-up is very short. Specific toxicities of CAR-based therapies, namely cytokine release syndrome (CRS), seem to be connected with the antigen of interest and source of cells for manufacturing. CRS is more frequent in antiCD7 CAR-T cells than in antiCD30 cells, but it is mild in most patients. More severe CRS was observed after gene-edited allogeneic CAR-T cells. Immune effector cell associated neurotoxicity (ICANS) was mild and infrequent. Graft-versus-host disease (GvHD) after allogeneic CAR-T cells from previous hematopoietic stem cell donor was also observed. Most frequent toxicities, similarly to antiCD19 CAR-T cells, are cytopenias. CAR-based cellular therapy seems feasible and effective for T-cell malignancies, however, the optimal design of CAR-based products is still unknown and long-term follow-up is needed for evaluation of their true potential.

Current Immunotherapy Approaches in Non-Hodgkin Lymphomas.

Non-Hodgkin lymphomas (NHLs) are lymphoid malignancies of B- or T-cell origin. Despite great advances in treatment options and significant improvement of survival parameters, a large part of NHL patients either present with a chemotherapy-refractory disease or experience lymphoma relapse. Chemotherapy-based salvage therapy of relapsed/refractory NHL is, however, capable of re-inducing long-term remissions only in a minority of patients. Immunotherapy-based approaches, including bispecific antibodies, immune checkpoint inhibitors and genetically engineered T-cells carrying chimeric antigen receptors, single-agent or in combination with therapeutic monoclonal antibodies, immunomodulatory agents, chemotherapy or targeted agents demonstrated unprecedented clinical activity in heavily-pretreated patients with NHL, including chemotherapy-refractory cases with complex karyotype changes and other adverse prognostic factors. In this review, we recapitulate currently used immunotherapy modalities in NHL and discuss future perspectives of combinatorial immunotherapy strategies, including patient-tailored approaches.

Hemodynamic Response to Gabapentin in Conscious Spontaneously Hypertensive Rats.

Ligands of auxiliary α2δ subunit of voltage-dependent calcium channels (VDCCs) decrease elevated L-type VDCCs surface expression in arterial myocytes and arterial constriction in spontaneously hypertensive rats (SHR). However, their effect on blood pressure (BP) is unclear. In this study, we investigated the hemodynamic response to acute and chronic administration of gabapentin, a ligand of auxiliary α2δ subunit of VDCCs, in adult SHR with established neurogenic hypertension. The acute gabapentin administration lowered BP and heart rate more in conscious SHR than Wistar-Kyoto rats. Both nifedipine (L-type VDCCs blocker) and ω-conotoxin GVIA (N-type VDCCs blocker) also decreased BP more in SHR, but only gabapentin and ω-conotoxin GVIA abolished the nitroprusside-induced reflex tachycardia of baroreceptor-heart rate control. Hypotensive effect of gabapentin was accompanied by a reduction of (1) plasma norepinephrine level, (2) depressor response to ganglionic blocker pentolinium, (3) power of low frequency component of systolic BP variability, and (4) pressor response of mesenteric vascular bed to periarterial nerve stimulation, suggesting the decrease of peripheral sympathetic nerve transmission. Moreover, gabapentin effects on BP and baroreflex were absent in sympathectomized rats. In conclusion, the acute (but not chronic) administration of gabapentin lowered BP more in SHR than in Wistar-Kyoto rats. Besides the known L-type VDCCs involvement in the vascular effect of gabapentin, our data revealed the important role of N-type VDCCs in acute gabapentin effect on sympathetic control of BP. Gabapentin-induced changes of sympathetic nerve transmission indicated major hemodynamic mechanism of the acute response to this drug.

Somatic mutation dynamics in MDS patients treated with azacitidine indicate clonal selection in patients-responders.

Azacitidine (AZA) for higher risk MDS patients is a standard therapy with limited durability. To monitor mutation dynamics during AZA therapy we utilized massive parallel sequencing of 54 genes previously associated with MDS/AML pathogenesis. Serial sampling before and during AZA therapy of 38 patients (reaching median overall survival 24 months (Mo) with 60% clinical responses) identified 116 somatic pathogenic variants with allele frequency (VAF) exceeding 5%. High accuracy of data was achieved via duplicate libraries from myeloid cells and T-cell controls. We observed that nearly half of the variants were stable while other variants were highly dynamic. Patients with marked decrease of allelic burden upon AZA therapy achieved clinical responses. In contrast, early-progressing patients on AZA displayed minimal changes of the mutation pattern. We modeled the VAF dynamics on AZA and utilized a joint model for the overall survival and response duration. While the presence of certain variants associated with clinical outcomes, such as the mutations of CDKN2A were adverse predictors while KDM6A mutations yield lower risk of dying, the data also indicate that allelic burden volatility represents additional important prognostic variable. In addition, preceding 5q- syndrome represents strong positive predictor of longer overall survival and response duration in high risk MDS patients treated with AZA. In conclusion, variants dynamics detected via serial sampling represents another parameter to consider when evaluating AZA efficacy and predicting outcome.

Ontogenetic changes in contribution of calcium sensitization and calcium entry to blood pressure maintenance of Wistar-Kyoto and spontaneously hypertensive rats.

BACKGROUND: Altered calcium sensitization (mediated by RhoA/Rho-kinase pathway) and enhanced calcium entry through L-type voltage-dependent calcium channels (L-VDCCs) participate in blood pressure (BP) maintenance of adult spontaneously hypertensive rats (SHRs). This study aimed to evaluate ontogenetic changes of these two pathways in BP control of SHR and Wistar-Kyoto (WKY) aged 3, 5, 7, 13, 26 and 42 weeks. METHODS: BP response to acute administration of Rho-kinase inhibitor fasudil or L-VDCC blocker nifedipine and the expression of particular components of RhoA/Rho-kinase pathway were determined in young and adult animals. RESULTS: Fasudil-induced BP reduction was attenuated in young SHR compared with WKY, but was enhanced in adult SHR. In contrast, BP response to nifedipine was similar in 3-week-old SHR and WKY and it was augmented with age in SHR but not in WKY. Consequently, the ratio between fasudil-induced and nifedipine-induced BP changes was lower in all age groups of SHR compared with WKY. Fasudil effects on contractility of isolated arteries were attenuated in young but not in adult SHR. mRNA expression of selected Rho-GEFs (Arhgef1, Arhgef11 and Arhgef12) was decreased only in adult SHR, whereas p63RhoGEF and CPI-17 expression was reduced in both age groups of SHR. Active RhoA and phosphorylated CPI-17 were increased in adult but not in young SHR. CONCLUSION: The importance of RhoA/Rho-kinase pathway for BP/vascular tone control is attenuated in SHR from prehypertensive stages. Enhanced RhoA activation and/or CPI-17 phosphorylation might be counteracted by reduced expression of upstream activators of Rho-kinase (Rho-GEFs) together with lower expression of CPI-17 (in downstream cascade of Rho-kinase).

The erythropoietin analogue ARA290 modulates the innate immune response and reduces Escherichia coli invasion into urothelial cells.

Urinary tract infections (UTI) are one of the most common infectious diseases worldwide. The majority is caused by uropathogenic Escherichia coli. Emerging resistances against conventional antimicrobial therapy requires novel treatment strategies. Beside its role in erythropoiesis, erythropoietin has been recognized to exert tissue-protective and immunomodulatory properties. Here, we investigated the nonerythropoietic erythropoietin analogue ARA290 for potential properties to modulate uroepithelial infection by E. coli in a cell culture model. Expression of the erythropoietin receptor was increased by bacterial stimuli and further enhanced by ARA290 in bladder epithelial cell lines and primary cells as well as in the monocytic cell line THP-1. Stimulation with ARA290 promoted an immune response, inducing a strong initial, but temporarily limited interleukin-8 induction. Moreover, the invasion of bladder epithelial cells by E. coli was significantly reduced in cells costimulated with ARA290. Our results indicate that the erythropoietin analogue ARA290 might be a candidate for the development of novel treatment strategies against UTI, by boosting an early immune response and reducing bacterial invasion as a putative source for recurrent infections.

Effect of saliva processing on bacterial DNA extraction.

More than 700 bacterial species inhabit oral cavity of humans. Various oral diseases are related to changes in the structure of this complex community. Their pathogenesis can, thus, be better understood by study of oral microbial flora. As many bacteria are refractory to cultivation, molecular approaches based on PCR followed by downstream analysis are more suitable for community analysis than culture dependent methods. Effective DNA extraction from the sample matrix is a fundamental part of the pre-analytical phase but it can be influenced by processing of the starting material. The aim of this study was to analyze the effects of saliva processing on DNA extraction using several non-commercial isolation procedures. Bacterial chromosomal DNA was extracted from three different sample matrices: fresh saliva, diluted saliva and pelleted saliva using four different extraction methods: phenol chloroform protocol, benzyl-chloride protocol, extraction with Chelex-100 and extraction with Triton X. Extraction from different saliva samples and the use of different extraction methods significantly affected the effectiveness of DNA extraction. The most suitable material for bacterial DNA extraction for molecular analysis is a fresh saliva sample. The most effective methods for isolating salivary DNA are the benzyl-chloride protocol and Chelex-100 extraction. Our results have implications for studies concentrating on salivary microbiome and its role in the pathogenesis of oral diseases.