The ISCCA flow protocol for the monitoring of anti-CD20 therapies in autoimmune disorders.

BACKGROUND: Anti-CD20 monoclonals (MoAbs) are used in a variety of autoimmune disorders. The aim is to eliminate memory B cells sustaining the tissue damage and the production of pathogenic autoantibodies, while preserving naïve cells. The disappearance of memory B cells and the repopulation by naïve cells correlate with good clinical response, while the reappearance of memory B cells and plasmablasts correlates with relapse or resistance to therapy. Anti-CD20 induce extremely low B cell levels, requiring high-resolution techniques. The immune monitoring protocol developed by ISCCA is described and validated, to provide a standardized method for the clinical decision-making process during anti-CD20 therapies in autoimmune diseases. METHODS: A 10-marker, 8-color staining panel (CD20-V450, CD45-V500c, CD4-FITC + sIgM-FITC, CD38-PE, CD3-PerCP Cy5.5, CD19-PE-Cy7, CD27-APC, CD8-APC H7 + sIgG-APC-H7) is used to identify B cells, plasma cells/blasts, naïve and memory B cells, sIgM+ and sIgG-switched memory B cells, T and NK cells, with high-sensitivity analysis (>106 CD45+ cells). RESULTS: After an anti-CD20 dose, the B cell level is about zero in most patients. If B cells remain virtually absent (<0.1/µl), subsetting is not reliable nor meaningful. If B cells raise >0.3-0.5/µl, subsetting is possible and informative, acquiring >1.0-1.5 × 106 CD45+ events. Further testings can follow the quality of B cell repopulation. If B cells become detectable (>1/µl), the prevalence of memory B cells indicates non-responsiveness or a possible relapse. CONCLUSIONS: The ISCCA Protocol is proposed for a standardized prospective monitoring of patients with autoimmune disorders, to assist the safe and rational usage of anti-CD20 therapies.

Combination therapy with anti-CD20 mAb and IL-10 gene to reverse type 1 diabetes by attenuating pancreatitis and inhibiting apoptosis in NOD mice.

AIMS: To assess the combination therapy of anti-CD20 mabs and adenovirus-mediated interleukin-10 (IL-10) gene delivery on the prevention of type 1 diabetes (T1D) in non-obese diabetes (NOD) mice. MAIN METHODS: In present study, we simultaneously blocked the B cell interactions and recovered the Th cell subset proportion by using through anti-CD20 Mab and adenovirus-mediated gene delivery of IL-10, respectively. After 9 consecutive days of combination therapy, various measurements, including hematoxylin-eosin staining (HE), terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling assay (TUNEL), immunohistochemistry, ELISA, PCR and western blot were applied to further assess the efficacy. KEY FINDINGS: The results suggested that the combination intervention reduced the T1D-associated morbidity of NOD mice, promote insulin secretion, control blood glucose and ease pancreatitis. Moreover, the combination therapy might play a protective role in pancreatic ß cells by suppressing the expression of TNF-α and Fas, blocking the Caspase-8 and Caspase-3 apoptotic pathways and activating the Bcl-2 anti-apoptotic pathway. Finally, the combination intervention may up-regulate the gene expression of CK-19 and PDX-1 and further accelerate the differentiation and proliferation of pancreatic ß cells. SIGNIFICANCE: Therefore, the combination intervention with anti-CD20 mabs and the IL-10 gene plays a role in the prevention of T1D to some extent in NOD mice.

Riesgo de infección asociada a nuevas terapias para el tratamiento de los síndromes linfoproliferativos / Risk of infection associated with new therapies for lymphoproliferative syndromes

En la última década se han experimentado grandes cambios en los tratamientos de los síndromes linfoproliferativos. A la quimioterapia convencional se suma ahora un amplio abanico de terapias dirigidas con diferentes indicaciones. El objetivo de esta revisión es evaluar el riesgo de infección asociado a estas terapias, así como tratar de establecer unas recomendaciones de prevención. En todos los casos, la enfermedad de base del paciente, así como los tratamientos concomitantes o los recibidos previamente, impactan en el riesgo de infección. Los anticuerpos anti-CD20 (rituximab, ofatumumab y obinutuzumab) se asocian a un mayor riesgo de infección bacteriana, vírica y de reactivación de infecciones latentes, así como a infecciones oportunistas. El alemtuzumab se asocia a inmunosupresión grave y mantenida. El ibrutinib y el acalabrutinib se asocian a infecciones bacterianas, especialmente respiratorias, infección fúngica invasiva e infecciones oportunistas. El idelalisib se asocia a un aumento de la incidencia de neumonía por Pneumocystis jirovecii y reactivación de citomegalovirus. El venetoclax se asocia a infecciones respiratorias y neutropenia. Los inhibidores de checkpoint inmune parecen no incrementar, por sí mismos, el riesgo de infección; sin embargo, el uso de glucocorticoides e inmunosupresores para controlar efectos adversos inmunorrelacionados sí conlleva un aumento del número de infecciones, incluyendo infecciones oportunistas. El brentuximab, la lenalidomida y los inhibidores de la histona deacetilasa no parecen asociarse a un mayor riesgo de infección. Aunque existe poca experiencia en el uso de terapias celulares, se ha observado un mayor número de infecciones en pacientes que han recibido más de 3 tratamientos antineoplásicos previamente, o en aquellos que han requerido tocilizumab o glucocorticoides para el manejo del síndrome de liberación de citocinas. En todos los pacientes se recomienda una actualización del calendario vacunal, cribado de infecciones latentes y profilaxis individualizada

Over the last decade, there have been important developments in the treatment of lymphoproliferative disorders. Apart from conventional chemotherapy, a wide array of therapies has been developed, with different indications. The aim of this review is to evaluate the risk of infection associated with these therapies, as well as establishing prevention recommendations. In all cases, the patient's underlying disease as well as concomitant or previous therapies have an impact on the risk of infection. Anti-CD20 antibodies (rituximab, ofatumumab and obinutuzumab) have been associated with a higher risk of bacterial and viral infection, as well as reactivation of latent infections and opportunistic infections. Alemtuzumab is associated with severe, protracted immunosuppression. Ibrutinib and acalabrutinib have been linked to bacterial infections (especially respiratory infections), invasive fungal infections and opportunistic infections. Idelalisib carries a higher risk of Pneumocystis jirovecii and infection and cytomegalovirus reactivation. Venetoclax is associated with respiratory infections and neutropenia. Immune checkpoint inhibitors are not directly associated with a higher risk of infection; nevertheless, the use of corticosteroids and immunosuppressants to control immune-related adverse events results in an increase of the risk of infection. Brentuximab, lenalidomide and histone deacetylase inhibitors do not seem to be associated with a higher risk of infections. Although data are scarce, a higher number of infections have been observed with cellular therapies, mostly in patients with more than 3 previous antineoplastic treatments or those receiving tocilizumab or corticosteroids for managing the cytokine release syndrome. In all patients, we recommend appropriate vaccination, screening for latent infections, and individualized prophylaxis recommendations

Unexpected primary bone lymphoma / Linfoma óseo primario inesperado

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Drug-free macromolecular therapeutics: Impact of structure on induction of apoptosis in Raji B cells.

Recently, we developed a new paradigm in macromolecular therapeutics that avoids the use of low molecular weight drugs. The activity of the "drug-free macromolecular therapeutics" is based on the biorecognition of complementary motifs at cell surface resulting in receptor crosslinking and apoptosis induction. The system is composed of two nanoconjugates: (1) a single-stranded morpholino oligonucleotide (MORF1) attached to an anti-CD20 Fab' fragment (Fab'-MORF1); (2) multiple copies of complementary oligonucleotide MORF2 grafted to a linear polymer of N-(2-hydroxypropyl)methacrylamide (HPMA) - P-(MORF2)x. The two conjugates crosslink CD20 antigens via MORF1-MORF2 hybridization at the surface of CD20+ malignant B-cells and induce apoptosis. Preclinical studies in a murine model of human non-Hodgkin's lymphoma showed cancer cells eradication and long-term survivors. The aim of this study was to determine the relationship between the detailed structure of the nanoconjugates and apoptosis induction in Raji cells to allow system optimization. The factors studied include the length of the MORF sequence, the valence of P-(MORF2)x (varying x), molecular weight of P-(MORF2)x, incorporation of a miniPEG spacer between Fab' and MORF1 and between polymer backbone and pendant MORF2, and comparison of two Fab' fragments, one from 1F5 antibody (Fab'1F5), the other from Rituximab (Fab'RTX). The results of apoptosis induction in human Burkitt's B-cell non-Hodgkin's lymphoma (NHL) Raji cells as determined using three apoptotic assays (Annexin V, Caspase 3, and TUNEL) indicated that: a) An improvement of apoptotic activity was observed for a 28 base pair MORF sequence when compared to MORFs composed of 20 and 25 base pairs. The differences depended on type of assay, concentration and exposure schedule (consecutive vs. premixed). b) The higher the valence of P-(MORF2)x the higher the levels of apoptosis. c) Higher molecular weight of P-(MORF2)x induced higher levels of apoptosis. d) A miniPEG8 spacer was effective in enhancing apoptotic levels in contrast to a miniPEG2 spacer. e) There was not a statistically significant difference when comparing Fab'1F5-MORF1 with Fab'RTX-MORF1.

Quantitative single-particle digital autoradiography with α-particle emitters for targeted radionuclide therapy using the iQID camera.

PURPOSE: Alpha-emitting radionuclides exhibit a potential advantage for cancer treatments because they release large amounts of ionizing energy over a few cell diameters (50-80 µm), causing localized, irreparable double-strand DNA breaks that lead to cell death. Radioimmunotherapy (RIT) approaches using monoclonal antibodies labeled with α emitters may thus inactivate targeted cells with minimal radiation damage to surrounding tissues. Tools are needed to visualize and quantify the radioactivity distribution and absorbed doses to targeted and nontargeted cells for accurate dosimetry of all treatment regimens utilizing α particles, including RIT and others (e.g., Ra-223), especially for organs and tumors with heterogeneous radionuclide distributions. The aim of this study was to evaluate and characterize a novel single-particle digital autoradiography imager, the ionizing-radiation quantum imaging detector (iQID) camera, for use in α-RIT experiments. METHODS: The iQID camera is a scintillator-based radiation detection system that images and identifies charged-particle and gamma-ray/x-ray emissions spatially and temporally on an event-by-event basis. It employs CCD-CMOS cameras and high-performance computing hardware for real-time imaging and activity quantification of tissue sections, approaching cellular resolutions. In this work, the authors evaluated its characteristics for α-particle imaging, including measurements of intrinsic detector spatial resolutions and background count rates at various detector configurations and quantification of activity distributions. The technique was assessed for quantitative imaging of astatine-211 ((211)At) activity distributions in cryosections of murine and canine tissue samples. RESULTS: The highest spatial resolution was measured at ∼20 µm full width at half maximum and the α-particle background was measured at a rate as low as (2.6 ± 0.5) × 10(-4) cpm/cm(2) (40 mm diameter detector area). Simultaneous imaging of multiple tissue sections was performed using a large-area iQID configuration (ø 11.5 cm). Estimation of the (211)At activity distribution was demonstrated at mBq/µg-levels. CONCLUSIONS: Single-particle digital autoradiography of α emitters has advantages over traditional film-based autoradiographic techniques that use phosphor screens, in terms of spatial resolution, sensitivity, and activity quantification capability. The system features and characterization results presented in this study show that the iQID is a promising technology for microdosimetry, because it provides necessary information for interpreting alpha-RIT outcomes and for predicting the therapeutic efficacy of cell-targeted approaches using α emitters.

Safety and efficacy of ofatumumab in relapsing-remitting multiple sclerosis: a phase 2 study.

OBJECTIVES: We present the first study to explore safety and efficacy of the human CD20 monoclonal antibody ofatumumab in relapsing-remitting multiple sclerosis (RRMS). METHODS: In this randomized, double-blind, placebo-controlled study, patients received 2 ofatumumab infusions (100 mg, 300 mg, or 700 mg) or placebo 2 weeks apart. At week 24, patients received alternate treatment. Safety and efficacy were assessed. RESULTS: Thirty-eight patients were randomized (ofatumumab/placebo, n = 26; placebo/ofatumumab, n = 12) and analyzed; 36 completed the study. Two patients in the 300-mg group withdrew from the study because of adverse events. No unexpected safety signals emerged. Infusion-related reactions were common on the first infusion day but not observed on the second infusion day. None of the patients developed human anti-human antibodies. Ofatumumab was associated with profound selective reduction of B cells as measured by CD19(+) expression. New brain MRI lesion activity was suppressed (>99%) in the first 24 weeks after ofatumumab administration (all doses), with statistically significant reductions (p < 0.001) favoring ofatumumab found in new T1 gadolinium-enhancing lesions, total enhancing T1 lesions, and new and/or enlarging T2 lesions. CONCLUSIONS: Ofatumumab (up to 700 mg) given 2 weeks apart was not associated with any unexpected safety concerns and was well tolerated in patients with RRMS. MRI data suggest a clinically meaningful effect of ofatumumab for all doses studied. Results warrant further exploration of ofatumumab in RRMS. CLASSIFICATION OF EVIDENCE: This study provides Class II evidence that in patients with RRMS, ofatumumab compared with placebo does not increase the number of serious adverse events and decreases the number of new MRI lesions.

Optimization of 90 Y-antiCD20 preparation for radioimmunotherapy.

CONTEXT: The advent of monoclonal antibodies such as Rituximab, in recent years, has brought about decisive progress in the treatment of aggressive and indolent non-Hodgkin's lymphoma. AIMS: A further tried and tested improvement to the unmodified antibody has been its coupling to the beta-emitters Y-90. The optimization of 90 Y-antiCD20 radioimmunoconjugate production and quality control methods for future clinical studies in the country was targeted in this work. MATERIALS AND METHODS: The antibody was labeled with 90 Y-yttrium chloride (185 MBq) after conjugation with freshly prepared ccDTPA. Y-90 chloride was obtained by thermal neutron flux (4 × 10 13 n/cm 2 /s) of a natural Y 2 O 3 sample, dissolved in acidic media. Radiolabeling was completed in 24 h by the addition of DTPA-Rituximab conjugate at room temperature. STATISTICAL ANALYSIS USED: All values were expressed as mean ± standard deviation (mean ± SD), and the data were compared using Student's t-test. Statistical significance was defined as P < 0.05. RESULTS: Radiochemical purity of 96% was obtained by using ITLC method for the final radioimmunoconjugate (specific activity = 440-480 MBq/mg). The final isotonic 90 Y-Rituximab complex was checked by gel electrophoresis for protein integrity retention. Biodistribution studies in normal rats were carried out to determine the radioimmunoconjugate distribution up to 72 h. CONCLUSION: The results showed that 90 Y-DTPA-Rituximab could be considered for further evaluation in animals and possibly in humans as a radiopharmaceutical for use in radioimmunotherapy against non-Hodgkin's lymphomas. Because of the importance of developing anti-lymphoma B agents in nuclear medicine for country use, an optimized radiolabeling method has been introduced.

High-dose [131I]tositumomab (anti-CD20) radioimmunotherapy and autologous hematopoietic stem-cell transplantation for adults > or = 60 years old with relapsed or refractory B-cell lymphoma.

PURPOSE: The majority of patients with relapsed or refractory B-cell non-Hodgkin's lymphoma (NHL) are older than 60 years, yet they are often denied potentially curative high-dose therapy and autologous stem-cell transplantations (ASCT) because of the risk of excessive treatment-related morbidity and mortality. Myeloablative anti-CD20 radioimmunotherapy (RIT) can deliver curative radiation doses to tumor sites while limiting exposure to normal organs and may be particularly suited for older adults requiring high-dose therapy. PATIENTS AND METHODS: Patients older than 60 years with relapsed B-cell NHL (B-NHL) received infusions of tositumomab anti-CD20 antibody labeled with 185 to 370 Mbq (5 to 10 mCi) [131I]-tracer for dosimetry purposes followed 10 days later by individualized therapeutic infusions of [131I]tositumomab (median, 19.4 Gbq [525 mCi]; range, 12.1 to 42.7 Gbq [328 to 1,154 mCi]) to deliver 25 to 27 Gy to the critical normal organ receiving the highest radiation dose. ASCT was performed approximately 2 weeks after therapy. RESULTS: Twenty-four patients with a median age of 64 years (range, 60 to 76 years), who had received a median of four prior regimens (range, two to 14 regimens), were treated. Thirteen patients (54%) had chemotherapy-resistant disease. The estimated 3-year overall and progression-free survival rates were 59% and 51%, respectively, with a median follow-up of 2.9 years (range, 1 to 6 years). All patients experienced expected myeloablation with engraftment of platelets (> or = 20 K/microL) and neutrophils ( 500/microL), occurring at a median of 9 and 15 days after ASCT, respectively. There were no treatment-related deaths, and only two patients experienced grade 4 nonhematologic toxicity. CONCLUSION: Myeloablative RIT and ASCT is a safe and effective therapeutic option for older adults with relapsed B-NHL.

Mimotope vaccination for epitope-specific induction of anti-CD20 antibodies.

CD20 is expressed strictly by B-cells and is ubiquitously expressed at high surface densities of malignant human B-cells. This suggests that CD20 may be a tumor target for immunotherapy of B-cell lymphomas. Rituximab, a chimeric monoclonal antibody directed against CD20, has been demonstrated to be an effective treatment for non-Hodgkin's lymphoma (NHL) and some autoimmune diseases. In the current study, we used the phage display technique to generate mimotopes that complemented the screening Ab Rituximab. A total of seven candidate mimotopes were isolated from a 12-mer peptide library from which one mimotope was conjugated to keyhole limpet hemocyanin (KLH) or tetanus toxoid (TT). The immunogenicity of the two vaccines generated was examined in BALB/c mice. Sera from the vaccinated mice demonstrated high-titer specific antibodies to the mimotope conjugates. Antibody binding to native CD20 and Ab-mediated cytotoxicity (CDC, complement-dependent cytotoxicity) were also analyzed. Our data suggest that a Rituximab mimotope may be a useful tool for the construction of a functional vaccine to treat B-cell malignancy as well as some CD20 related autoimmune disorders.