Transient B-cell Depletion Research Articles

Releasing our model T - chimeric antigen receptor (CAR) T-cells for autoimmune indications.

This review provides an update on the rapidly growing field of engineered cellular therapies for autoimmune disorders, primarily focusing on clinical experience and correlative studies with chimeric antigen receptor (CAR) T-cells. To date, two case series describing treatment with CAR T-cell therapy for systemic lupus erythematosus (SLE) suggest that drug-free remission can be sustained in patients with previously treatment-refractory disease. The outcomes of these studies are similar, despite the use of different CAR constructs and lymphodepletion regimens. Although it is not yet clear whether the patients described have truly been cured, the majority of remissions have remained durable up to last follow-up at 1-2 years from treatment. Meanwhile, mechanistic studies are providing a window into how transient B-cell depletion mediates lasting benefit. With the encouraging data in SLE, CAR T-cells and other novel B-cell-depleting agents (e.g. bispecific T-cell engagers) are now being evaluated as treatment for other autoimmune conditions, with the goal of durable response. Recent reports highlight cellular therapies as a promising strategy for patients with treatment-refractory autoimmune conditions; however, there is still limited experience, and better insight into this therapeutic approach is expected to emerge rapidly.

Delayed B-cell maturation and attenuated vaccine responses in infants exposed to B-cell depleting therapies in utero

In utero exposure to anti-CD20 monoclonal antibodies can result in transient B-cell depletion, delayed B-cell maturation, decreased immunoglobulin production and inadequate vaccine responses in the infant. These children require immunological follow up and personalized vaccine schedules.

Pregnancy in Patents with CLL Systematic Review

Introduction: Chronic Lymphocytic Leukemia (CLL) is a hematologic malignancy characterized by the clonal expansion of non-functional, mature-appearing lymphocytes in the bone marrow, peripheral blood, and lymphoid tissues. While cases of CLL during pregnancy are rare,it is associated with many complications. It was reported that women with CLL are more susceptible to infections due to a weakened immune system; possibly leading to more complications. There is also increased risk of thrombocytopenia, which can increase the risk of bleeding during pregnancy and childbirth. This review aimed at identifying literature available on the complications in CLL and pregnancy as well as the data available on the potential therapeutic strategies. Methods: A scoping review of case reports that discuss pregnant patients who were diagnosed with CLL were isolated. The effects of the disease on pregnancy outcomes regarding both mother and fetus were identified. Animal studies, reviews or non-original articles, and non-English articles were excluded from our study. FDA labels of medications in CLL were reviewed for data on pregnancy and animal outcomes. Information on those medications in pregnant patients were also identified where available. Results: The search strategy across four databases yielded 448 articles. After eligibility assessment, 14 articles were included in the review. Cases were subdivided according to timing of pregnancy relative to CLL diagnosis (diagnosed before pregnancy or during pregnancy at early or late gestational periods). The pre-pregnancy reports were further subclassified based on the presence or absence of infection during pregnancy. None of the cases reported required immediate initiation of CLL therapy and only one case reported mortality post-delivery. This occurred in a patient with Richter transformation and no prior CLL treatments. There were no reported fetal malformations or severe adverse outcomes related to the infants in any of the reported pregnancies ( Figure 2). The included studies provided insights into various aspects of CLL during pregnancy, including patient demographics, diagnosis timing and methods, acquired infections, changes in white blood cell count, treatment approaches, pregnancy outcomes, and complications for both mother and fetus. From the marketed pharmacological alternatives for CLL, most were not studied in pregnant humans with CLL ( Figure 1). However, venetoclax and rituximab have some data in pregnant patients with non-CLL indications. A case reported a pregnant refractory AML patient who received venetoclax with high dose cytarabine and mitoxantrone at 24 weeks of gestation. Bone marrow at 27 weeks showed complete remission and labor was induced at 28 weeks + 5 days after corticosteroid-induced lung maturation. The infant had hyperbilirubinemia, transient B-cell depletion and required ventilation. The baby recovered from all complications, although possible long-term effects are still unclear. The mother underwent allogeneic hematopoietic stem cell transplant. In a 30-year-old female (pregnant at 28 weeks) with primary CNS lymphoma, weekly rituximab was given for 4 weeks with dexamethasone. After delivery through C-section at 31 weeks, the patient started definitive treatment with chemotherapy and autologous transplant. The infant required brief mechanical ventilation and had transient B-cell depletion at delivery until 4 months post-delivery. The baby required a course of post-exposure acyclovir despite not developing an infection. Both mother and baby survived with good outcomes at last follow-up (4 years post-delivery) Conclusion: Despite the rarity of CLL occurrence during pregnancy, there is evident need of further studies and management guidelines in this population. This review provides insights into the possible pregnancy-related concerns in women with CLL at different disease stages. While watchful waiting is generally recommended due to CLL's slow progression, individual evaluation is crucial. If treatment is necessary, delaying therapy until the second or third trimesters may help mitigate the risk of fetal malformations. The increased susceptibility to infections resulting from the combined immunosuppressive effects of CLL and pregnancy should be carefully considered

Transient B-cell depletion and regulatory T-cells mediation in combination with adenovirus mediated IGF-1 prevents and reverses autoimmune diabetes in NOD mice

Type 1 diabetes (T1D) is one of the T cells mediated autoimmune diseases, although B cells also play an important role in the development. Both T cell and B cell targeted immunotherapies exhibited efficacies in preventing and reversing the T1D. Current study was performed to investigate the protective effects of anti-CD20/CD3 bi-specific antibody (bsAb) in combination with adenovirus mediated mouse insulin-like growth factor 1 (Adv-mIGF-1) gene on T1D in non-obese diabetes (NOD) mice. To simultaneously restore the proportion of Th cells and block the interaction of B cells as well as mediate T cell populations, the NOD model mice were randomly assigned to four groups received the saline, anti-CD20/CD3 bsAb and Adv-mIGF-1 gene alone or combination, respectively. After 16-consecutive weeks intervention, the ELISA, RT-PCR, western blot and histopathological analysis were performed to assess the pancreatic tissues and serum samples to evaluate the treatment effects. Chronic treatment of combination therapy improved T1D morbidity by improving the compartment and function of the CD4+Foxp3+ Tregs, reversing the secretion of insulin, controlling the blood glucose levels (BGLs) and alleviating insulitis as well as cell apoptosis in the NOD model mice. Moreover, current combination therapy also accelerated the proliferation and differentiation of pancreatic β cells via suppressing the apoptosis-related factors, including caspase-3, caspase-8 and Fas, and activating the Bcl-2-related anti-apoptotic pathway. Furthermore, the cytokeratin-19 (CK-19) and pancreatic duodenal homoplasmic box-1 (PDX-1), as two important stem cell markers of pancreas were both significantly improved by treatment of combination therapy. On conclusions, chronic treatment of anti-CD20/CD3 bsAb in combination with Adv-mIGF-1 gene exerts synergistic protection on T1D in the NOD mice.

The CELLO trial: Protocol of a planned phase 4 study to assess the efficacy of Ocrelizumab in patients with radiologically isolated syndrome

BackgroundPatients with radiologically isolated syndrome (RIS) exhibit CNS lesions suggestive of multiple sclerosis (MS) in the absence of overt neurological symptoms characteristic of the disease. They may have concurrent brain atrophy, subtle cognitive impairment, and intrathecal inflammation. At least half ultimately develop MS, cementing RIS as preclinical MS for many. However, high-quality data, including immunologic biomarkers, to guide treatment decisions in this population are lacking. Early intervention with ocrelizumab, a humanized monoclonal antibody approved for relapsing and primary progressive MS that targets CD20+ B-cells, may affect disease course and improve long-term outcomes. The objective of this study is to describe the protocol for CELLO, a clinical trial assessing the effect of ocrelizumab on RIS. MethodsThe CELLO clinical trial, a phase 4, multicenter, randomized, double-blind, placebo-controlled study conducted as an academic-industry collaboration, aims to (1) assess the efficacy of ocrelizumab in patients with RIS and (2) identify biomarkers indicative of emerging autoimmunity as well as immune recovery after transient B-cell depletion. The study will enroll 100 participants across ≥15 sites. Participants will be aged 18 to 40 years, have RIS (defined as meeting 2017 revised McDonald criteria for dissemination in space), and have either been diagnosed with RIS within the last 5 years or have had new brain lesions identified within 5 years of study entry. A screening program of first-degree relatives of patients with MS will be used to boost recruitment. Eligible patients will be randomized 1:1 to receive 3 courses of ocrelizumab or placebo at baseline, week 24, and week 48. Patients will subsequently be followed up for ≥3 years. The primary outcome is time to development of new radiological or clinical evidence of MS. Secondary and exploratory objectives will investigate neuroimaging, serological and immunologic biomarkers, cognitive function, and patient-reported outcomes. A substudy using single-cell RNA sequencing to characterize blood and CSF immune cells will assess markers associated with conversion to clinical MS. ConclusionThe CELLO study will improve the understanding of B-cell biology in early MS disease pathophysiology, characterize the emergence of CNS autoimmunity, and provide evidence to inform treatment decision-making for individuals with RIS. ClinicalTrials.govNCT04877457

KPMW135, a Biosuperior CD3 Bispecific Version of Rituximab Created By a Novel Chemical Conjugation Technology Demonstrates Increased Anti-Tumor Activity By Adding T Cell-Mediated Cytotoxicity Activity to the Existing Mechanisms of Rituximab

Background We describe KPMW135, a novel CD20 x CD3 bispecific molecule, in which a CD3-directed ScFv (SP34) is conjugated to rituximab (RTX). RTX is a chimeric monoclonal antibody (mAb) specific for CD20, used to treat B-cell lymphomas and lymphocytic leukemias. Monoclonal Ab therapy enhancer (MATE™) enables efficient site-directed chemical conjugation to "off-the-shelf" therapeutic mAbs and allows the development of bispecific therapeutic candidates. Our MATE platform allows for the chemical engineering of existing Abs without the need to create new DNA vectors or genetic engineering of master cell lines. This approach is superior to previous conjugation methods that 1) lack site-directed conjugation specificity by indiscriminately binding to available amino acid residues or 2) require genetic engineering to create conjugate tags. Methods SP34 scFv was constructed using published CDRs. The expression was in FreeStyle 293 cells, followed by affinity purification and SEC to obtain monomeric scFv. The binding of CD20 to KPMW135 was measured by Bio-Layer Interferometry using Octet (Fortebio). Equilibrium dissociation constant (Kd) was calculated using the Octet HT software. The binding of CD3εδ, CD16a, and FcRn were determined by ELISA using neutravidin-coated plates. Biotinylated Human CD3ε & CD3δ heterodimer protein (Avi tag), CD16a (Avi Tag) and human CD16a were used. Readout was determined with anti-human F(ab) HRP. For in vitro T cell-mediated cytotoxicity assays, unfractionated and NK-cell depleted PBMCs were prepared from freshly-thawed and PHA + IL-2 prestimulated PBMCs. Daudi (CD20+) B lymphoblast cells were engineered to stably express a beta-gal reporter fragment using KILR retroparticles (Eurofins). Target cells were treated with KPMW135, RTX, and controls. PBMCs were introduced at an effector:target ratio of 15:1 and incubated for 18h. Luminescence signal was obtained with luminometer to reflect target cell death. Cynomolgus monkeys were intravenously injected with RTX or KPMW135 (30 μg/kg). Endpoints included clinical observations, cytokine profile and flow cytometric immunophenotyping of T cells, monocytes, granulocytes, NK cells, and B cells (CD45, CD3, CD16, CD14, NKG2A, HLA DR) and cellular activation (CD44 and CD69). Results Binding affinity of CD20 to KPMW135 was 0.75nM. CD20 binding to RTX and KPMW135 was similar, thus conjugation of the CD3-binding ScFv did not negatively affect affinity to CD20. ELISA results show KPMW135 binds to CD3εδ, FcRn, and CD16a, indicating that the conjugation does not interfere with binding to their various moieties. In vitro T cell-mediated cytotoxicity results show the activity of Fc/FcR and CD20 binding by the RTX moiety versus CD3 binding to effector T cells. T cell enrichment by functionally prestimulated PBMCs increased killing of target cells by 2-fold. Mechanically depleted NK cells verified that target cell death was induced by T cells, rather than by NK cells via ADCC. KPMW135 elicited target cell killing with an EC50 of 0.03-0.07nM or 0.09-021nM for prestimulated or freshly thawed PBMCs, respectively. Studies in Cynomolgus monkeys showed that KPMW135 can activate T cells in vivo as shown by increased CD69 and CD44 expression (3- and 2-fold, respectively) with a peak at 4h post-dose compared to equivalent dose RTX. A pronounced depletion of B cells was observed in KPMW135 treated animals as early as half hour post-dose, with a partial recovery by days 7-14, while an equivalent dose of RTX induced a transient B-cell depletion followed by a quicker return to baseline levels. Conclusions KPMW135 is a CD20 x CD3 bispecific created by chemical conjugation of CD3-specific ScFv to "off-the-shelf" RTX using our proprietary MATE™ technology, which maintains RTX native binding to FcRs via the Fc domain and its native binding to CD20 on target cells. KPMW135 retains RTX native ability to target B lymphoid malignancies via Fc effector mechanisms like ADCC and ADCP, but now adds T cell-mediated cytotoxicity. Our in vitro data shows increased target cell killing by KPMW135 compared to RTX, and our in vivo study confirms KPMW135 ability to induce target dependent T cell activation and recruitment, making it superior to unconjugated RTX. CD3 MATEs generated with other Abs show similar increased T cell-mediated cytotoxicity, confirming that this technology can be applied to generate "off-the-shelf" biosuperior antibodies. Disclosures Vidal: Kleo Pharmaceuticals: Current Employment, Current equity holder in private company. Cukan:Kleo Pharmaceuticals: Current Employment. Vaill:Kleo Pharmaceuticals: Current Employment. Bunin:Kleo Pharmaceuticals: Current Employment. Rossi:Kleo Pharmaceuticals: Current Employment. Iben:Kleo Pharmaceuticals: Current Employment. Trinh:Kleo Pharmaceuticals: Current Employment. McGrath:Kleo Pharmaceuticals: Current Employment. Berbasova:Kleo Pharmaceuticals: Current Employment. Alvarez:Kleo Pharmaceuticals: Current Employment. Rastelli:Kleo Pharmaceuticals: Current Employment. Welsch:Kleo Pharmaceuticals: Ended employment in the past 24 months.

1220P - Phase I study evaluating safety, pharmacokinetics (PK), pharmacodynamics, and preliminary efficacy of ABBV-428, first-in-class mesothelin (MSLN)-CD40 bispecific, in patients (pts) with advanced solid tumours

1220P - Phase I study evaluating safety, pharmacokinetics (PK), pharmacodynamics, and preliminary efficacy of ABBV-428, first-in-class mesothelin (MSLN)-CD40 bispecific, in patients (pts) with advanced solid tumours

Phase 1 Study of REGN1979, an Anti-CD20 x Anti-CD3 Bispecific Monoclonal Antibody, in Patients with CD20+ B-Cell Malignancies Previously Treated with CD20-Directed Antibody Therapy

Introduction: REGN1979 is a hinge-stabilized CD20xCD3bispecific full-length antibody (Ab) based on an IgG4isotype modified to reduce Fc binding. It is designed to bind T cells (via CD3) and CD20-expressing cells. Cross-linking results in specific, local T cell activation and engagement ofcytolytic functions, independent of T cell receptor mediated recognition. This mechanism of action (MOA) is distinct from approved anti-CD20 Abs, and may provide additional therapeutic benefit. This report describes safety and clinical outcomes in 25 patients (pts) treated with REGN1979.Methods: The study uses a 3+3 design to assess safety, pharmacokinetics (PK), pharmacodynamics (PD), and activity of REGN1979 inpts with NHL and CLL, in separate dose escalation cohorts. REGN1979 is administered IV over 1-4 hours, weekly (QW) for 4 doses followed by every 4 weeks (Q4W) for 5 doses. Each dose level (DL) consists of an initial starting dose followed by a higher (step-up) dose.Pts with either progressive disease (PD) after initial response or suboptimal initial response could be retreated.Results: As of clinical cut-off, 25pts had been enrolled and treated with REGN1979 at flat doses ranging from 0.03 - 3.0 mg: 20pts with NHLand 5pts with CLL. NHL subtypes included DLBCL (n=12), FL (n=6), and MCL (n=2). Allpts hada medianof3 (range 1-7) prior regimens, and received a median of 5 (range 2-9) doses of REGN1979. Sixpts remained on initial treatment, 3pts completed treatment, and 16pts discontinued treatment (14 for PD; 2 subject decision). Of 4pts who entered retreatment, 2pts remained ongoing.The most common treatment-related treatment-emergent AEs (TR-TEAEs) were pyrexia (56%), infusionrelated reaction (IRR) (40%), chills (36%), cytokine release syndrome (CRS) (28%), fatigue (24%), tachycardia (24%), hypomagnesaemia (20%) and hypotension (20%). Grade >= 3 TR-TEAEs occurred in 8 pts(32%): IRR (12%), CRS (8%), hypotension (8%), and pyrexia, tachycardia, dyspnea, anemia, hypoxia, hypertension, tachypnea, transaminases increased, and tumor pain each in 1 pt(4%). All were grade 3 with the exception of 1 ptwith grade 4 hypotension. There were no protocol-defined DLTs, nor grade 5 TEAE. No ptsdiscontinued REGN1979 due to TR-TEAE. IRR and CRS events were most commonly reported with the first administration of both initial and step-up dose with incidence and severity decreasing with increasing exposure.Tenptsreported at least one TR-SAE: CRS (24%), IRR (20%), and increased AST and tumor pain, each in 1pt(4%). Threeptswith aggressive lymphoma who discontinued REGN1979 due to PD died of their disease within 30 days of their last dose.Notably, modifications to pre-medication(s) and drug administration instructions have improved tolerability (fewer and less severe IRR/CRS). No clinically significant CNS TEAEs have been observed.CT-based (Cheson, 2007) anti-tumor activity was observed in 11 ptswith NHL: PR [n=4,median duration of 50d];SD [n=7, median duration of >196d]. The overall response rate (ORR) in NHL ptsacross DLs was 20%. In 2 highest dose levels, ORR was 27% (Fig 1). Of the 14pts evaluated by PET, 4pts had a partial metabolic response, withmedian duration of 244d perLugano Criteria. Twopts with CLL attained SD as best overall response.PK: REGN1979 concentrations in serum increased with increasing DL and were variable across pts. Variability was less at higher DLs and also decreased over time during therapy. At DLs tested, REGN1979 concentrations did not notably increase over time during treatment.PD/Cytokines: At the lowest dose tested, 0.03 mg, transient B-cell depletion was observed inpts with measurable circulating B cells.Samples from 12pts were available for cytokine analysis. Interim analysis indicates REGN1979 induces cytokine release as expected based on MOA. An increase in IL-6, IL-10 and to lesser extent IFN-γ was observed. The magnitude of cytokine response generally correlated with symptomatic IRR/CRS.Conclusion: REGN1979 demonstrated an acceptable safety profile at flat doses of 0.03 - 3.0 mg (< 1% of rituximab dose) inpts with NHL and CLL. Most TEAEs were associated with IRR/CRS, and were managed with supportive therapy and modification to REGN1979 administration. No clinically significant neurological toxicity was observed. Preliminary antitumor activity demonstrates increased activity at higher doses. Dose escalation and treatment schedule optimization continue. [Display omitted] DisclosuresBrown:Acetylon, Gilead: Research Funding; Celgene, Roche/Genentech, Gilead, Infinity, Janssen, Pharmacyclics, ProNai, Sun BioPharma: Consultancy. Arnason:Gilead: Consultancy. O'Brien:Pharmacyclics, LLC, an AbbVie Company: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria. Chavez:Janssen: Speakers Bureau. Ansell:BMS, Seattle Genetics, Merck, Celldex and Affimed: Research Funding. Topp:Amgen: Consultancy, Honoraria, Other: travel, Speakers Bureau; Roche: Honoraria, Other: travel; Boehringer: Consultancy, Other: Travel, Research Funding; Jazz: Consultancy; Pfizer: Consultancy, Other: Travel. Adriaens:Regeneron Pharmaceuticals Inc.: Employment, Equity Ownership. Ufkin:Regeneron Pharmaceuticals Inc.: Employment, Equity Ownership. Kostic:Regeneron Pharmaceuticals Inc.: Employment, Equity Ownership. Paccaly:Regeneron Pharmaceuticals Inc.: Employment, Equity Ownership. Gao:Regeneron Pharmaceuticals Inc: Employment, Equity Ownership. Trail:Regeneron Pharmaceuticals Inc: Employment, Equity Ownership. Lowy:Regeneron Pharmaceuticals Inc: Employment, Equity Ownership. Brownstein:Regeneron Pharmaceuticals Inc: Employment, Equity Ownership.

No Increased Risk of Secondary Neoplasms in Patients Treated with Rituximab for Non-Hodgkin’s Lymphoma : A Meta-Analysis of 9 Trials

Background. Rituximab improved outcomes of all CD20+ non-Hodgkin lymphoma (NHL) subtypes. Rituximab induces a transient B-cell depletion and a dose-dependent T-cell inactivation (Stroopinsky et al., Cancer Immunol Immunother 2012) predisposing to T-cell dependent infections and to a potential impaired T-cell immunosurveillance. Secondary neoplasms (SN) is infrequent in trials including rituximab and the SN risk associated to rituximab across multiple trials has not been reported. We performed a systematic review of published trials comparing chemotherapy with or without rituximab to evaluate SN occurrence.Methods. Our primary endpoint was SN risk in patients with NHL treated with rituximab. We searched PubMed and Embase databases for randomised controlled trials on rituximab and lymphoma where rituximab constituted the only difference between treatment arms and where SN incidence or SN related death were reported. Authors were contacted for SN related rituximab exposure if not detailed. Chronic lymphocytic leukemia and HIV-related lymphomas were excluded due to increased risk of SN. Updated follow-up of eligible trials presented at annual meetings of the American Society of Clinical Oncology and American Society of Hematology were retrieved. Data were extracted independently by two authors. A random effects DerSimonian-Laird meta-analysis was performed to estimate the summary effect of rituximab on the hazard of SN. Statistical heterogeneity was tested using Woolf test.Results. We identified nine trials cumulating 4621 patients with 2312 exposed to rituximab and 2309 not exposed. These nine trials are known with the following names: PRIMA (1), GELA LNH98.5 (2), MINT (3), CORAL (4), IELSG-19 (5), EORTC20981 (6), OSHO#39 (7), SAKK 35/98 (8), RICOVER60 (9). Histology were diffuse large B cell (n=4), follicular (n=4) and marginal zone (n=1) lymphomas. Median age was 58.1 years. Sex distribution was available for seven trials with 1650 (47.6%) women and 1814 (52.4%) men. In all these trials but one (SAKK 35/98), rituximab was used associated with chemotherapy: CHOP, CHOEP, FCM, MCP, DHAP, ICE, or chlorambucil. At a median follow-up of 73 months [interquartile range: 72-84], a total of 334 SN was observed, including 169 SN in patients randomised to rituximab as compared to 165 SN in patients not randomised to rituximab (OR= 0.88; 95%CI: 0.66-1.19) (Figure 1). No evidence of significant heterogeneity was noticed across trials (p = 0.93). Notably, the proportion of females, histology subtypes, use of rituximab in first line, and use of rituximab over prolonged periods in maintenance did not influence SN risk (p = 0.94, p = 0.80, p = 0.87, p = 0.87 respectively). The SN risk was not increased in protocols administrating rituximab over periods of 8 months to 12 months (CORAL , OSHO#39) as opposed to periods of 24 months (PRIMA, EORTC20981) (p=0.86).Conclusions. This meta-analysis of nine trials randomising rituximab in NHL patients suggests no SN predisposition at a median follow-up of 6 years. SN risk associated with the combination of rituximab and new targeted therapies warrants prospective monitoring. [Display omitted] DisclosuresFleury:Lundbeck: Membership on an entity’s Board of Directors or advisory committees, Preceptorship Other. Pfreundschuh:Amgen: Membership on an entity’s Board of Directors or advisory committees, Research Funding; Boehringer Ingelheim: Membership on an entity’s Board of Directors or advisory committees; Celgene: Membership on an entity’s Board of Directors or advisory committees; Onyx: Membership on an entity’s Board of Directors or advisory committees; Pfizer: Membership on an entity’s Board of Directors or advisory committees; Roche: Membership on an entity’s Board of Directors or advisory committees, Research Funding; Spectrum: Research Funding. Salles:Roche: Honoraria, Research Funding. van Oers:Roche: Consultancy. Gisselbrecht:Roche: Research Funding. Zucca:Roche: Consultancy, Membership on an entity’s Board of Directors or advisory committees; Johnson and Johnson: Consultancy, Membership on an entity’s Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity’s Board of Directors or advisory committees. Herold:Roche Pharma AG/Germany: Honoraria, Research Funding. Ghielmini:Roche: Research Funding, Speakers Bureau.

Meta-analysis of the risk of secondary neoplasm in patients treated for non-Hodgkin lymphoma with rituximab.

e19541 Background: Rituximab has improved outcome of patients with CD20+ non-Hodgkin lymphoma (NHL). Rituximab induces a transient B-cell depletion and a dose-dependent T-cell inactivation predisposing to T-cell dependent infections. T-cells contribute to immunosurveillance, but rituximab has not been associated with secondary neoplasm (SN) in NHL. We performed a systematic review of published trials comparing chemotherapy with or without rituximab to evaluate SN risk. Methods: Primary endpoint was SN risk associated with rituximab in induction and/or maintenance for NHL treatment. We searched PubMed and Embase databases for randomized controlled trials on rituximab and lymphoma where treatment arms differed only from rituximab use and where SN incidence or related death were reported. Authors were asked for rituximab associated SN if not detailed. Chronic lymphocytic leukemia and HIV-related lymphomas were excluded due to increased SN risk. Updated follow-up presented at American Society of Clinical Onco...

Hypogammaglobulinaemia after rituximab treatment--incidence and outcomes

Rituximab, a chimeric monoclonal antibody against CD20, is increasingly used in the treatment of B-cell lymphomas and autoimmune conditions. Transient peripheral B-cell depletion is expected following rituximab therapy. Although initial clinical trials did not show significant hypogammaglobulinaemia, reports of this are now appearing in the literature. We performed a retrospective review of patients previously treated with rituximab that were referred to Clinical Immunology with symptomatic or severe hypogammaglobulinaemia. Patient clinical histories, immunological markers, length of rituximab treatment and need for intravenous immunoglobulin replacement therapy (IVIG) were evaluated. An audit of patients receiving rituximab for any condition in a 12-month period and frequency of hypogammaglobulinaemia was also carried out. We identified 19 post-rituximab patients with persistent, symptomatic panhypogammaglobulinaemia. Mean IgG level was 3.42 ± 0.4 g/l (normal range 5.8-16.3 g/l). All patients had reduced or absent B-cells. Haemophilus Influenzae B, tetanus and Pneumococcal serotype-specific antibody levels were all reduced and patients failed to mount an immune response post-vaccination. Nearly all of them ultimately required IVIG. The mean interval from the last rituximab dose and need for IVIG was 36 months (range 7 months-7 years). Of note, 23.7% of 114 patients included in the audit had hypogammaglobulinaemia. With the increasing use of rituximab, it is important for clinicians treating these patients to be aware of hypogammaglobulinaemia and serious infections occurring even years after completion of treatment and should be actively looked for during follow-up. Referral to clinical immunology services and, if indicated, initiation of IVIG should be considered.

Transient B-Cell Depletion Combined With Apoptotic Donor Splenocytes Induces Xeno-Specific T- and B-Cell Tolerance to Islet Xenografts

Peritransplant infusion of apoptotic donor splenocytes cross-linked with ethylene carbodiimide (ECDI-SPs) has been demonstrated to effectively induce allogeneic donor-specific tolerance. The objective of the current study is to determine the effectiveness and additional requirements for tolerance induction for xenogeneic islet transplantation using donor ECDI-SPs. In a rat-to-mouse xenogeneic islet transplant model, we show that rat ECDI-SPs alone significantly prolonged islet xenograft survival but failed to induce tolerance. In contrast to allogeneic donor ECDI-SPs, xenogeneic donor ECDI-SPs induced production of xenodonor-specific antibodies partially responsible for the eventual islet xenograft rejection. Consequently, depletion of B cells prior to infusions of rat ECDI-SPs effectively prevented such antibody production and led to the indefinite survival of rat islet xenografts. In addition to controlling antibody responses, transient B-cell depletion combined with ECDI-SPs synergistically suppressed xenodonor-specific T-cell priming as well as memory T-cell generation. Reciprocally, after initial depletion, the recovered B cells in long-term tolerized mice exhibited xenodonor-specific hyporesponsiveness. We conclude that transient B-cell depletion combined with donor ECDI-SPs is a robust strategy for induction of xenodonor-specific T- and B-cell tolerance. This combinatorial therapy may be a promising strategy for tolerance induction for clinical xenogeneic islet transplantation.

The Future of B Cell-Targeted Therapies in Sjögren‘s Syndrome

Primary Sjögren's syndrome is a systemic autoimmune disease characterized by progressive exocrine gland destruction, resulting clinically in eyes and mouth dryness. To date, no treatment has been proven effective to modify the course of this slow-evolving disease. B cells are now considered to play a central role in the pathogenesis of primary Sjögren's syndrome because their functions are not restrained to antibody production. Thus, several B-cell targeting therapies are under clinical investigation. Rituximab, a monoclonal antibody directed to CD20 and leading to transient blood B-cell depletion, has shown partial improvements in subjective and objective sicca symptoms in small studies. However, the results of two large controlled trials are awaited before considering its use in large populations of patients. Several other therapeutic strategies are being studied, targeting other B-cell surface proteins (epratuzumab and anti-CD22) or major cytokines of B-cell homeostasis (e.g., BAFF, IL-6 and lymphotoxin-β). Although great hope is generated by the trials of these specific therapies, another challenge for clinical researchers is the development of reliable tools to assess the activity of Sjögren's syndrome and its response to treatment.

Transient B-Cell Depletion with Anti-CD20 in Combination with Proinsulin DNA Vaccine or Oral Insulin: Immunologic Effects and Efficacy in NOD Mice

A recent type 1 diabetes (T1D) clinical trial of rituximab (a B cell-depleting anti-CD20 antibody) achieved some therapeutic benefit in preserving C-peptide for a period of approximately nine months in patients with recently diagnosed diabetes. Our previous data in the NOD mouse demonstrated that co-administration of antigen (insulin) with anti-CD3 antibody (a T cell-directed immunomodulator) offers better protection than either entity alone, indicating that novel combination therapies that include a T1D-related autoantigen are possible. To accelerate the identification and development of novel combination therapies that can be advanced into the clinic, we have evaluated the combination of a mouse anti-CD20 antibody with either oral insulin or a proinsulin-expressing DNA vaccine. Anti-CD20 alone, given once or on 4 consecutive days, produced transient B cell depletion but did not prevent or reverse T1D in the NOD mouse. Oral insulin alone (twice weekly for 6 weeks) was also ineffective, while proinsulin DNA (weekly for up to 12 weeks) showed a trend toward modest efficacy. Combination of anti-CD20 with oral insulin was ineffective in reversing diabetes in NOD mice whose glycemia was controlled with SC insulin pellets; these experiments were performed in three independent labs. Combination of anti-CD20 with proinsulin DNA was also ineffective in diabetes reversal, but did show modest efficacy in diabetes prevention (p = 0.04). In the prevention studies, anti-CD20 plus proinsulin resulted in modest increases in Tregs in pancreatic lymph nodes and elevated levels of proinsulin-specific CD4+ T-cells that produced IL-4. Thus, combination therapy with anti-CD20 and either oral insulin or proinsulin does not protect hyperglycemic NOD mice, but the combination with proinsulin offers limited efficacy in T1D prevention, potentially by augmentation of proinsulin-specific IL-4 production.

Transient depletion of B cells in young mice results in activation of regulatory T cells that inhibit development of autoimmune disease in adults.

B-cell depletion therapy can be effective for treating B-cell lymphomas as well as many human and murine autoimmune diseases. B-cell-deficient mice are normally resistant to spontaneous autoimmune thyroiditis (SAT), but they develop SAT if regulatory T cells are transiently depleted during the first 3-6 weeks after birth. This was also a critical time when B-cell depletion effectively inhibited development of SAT in adult mice. The current study was undertaken to test the hypothesis that transient depletion of B cells using anti-CD20 would be sufficient to suppress SAT if B cells were depleted early in life and that inhibition of SAT would be due to the activity of Treg that functioned most effectively when B cells were absent or low. The results presented here support this hypothesis and indicate that development of autoimmune disease in adults is effectively inhibited when anti-CD20 is administered 1-3 weeks after birth. After 3 weeks, transient B-cell depletion is no longer effective, and B-cell depletion must be maintained to effectively suppress autoimmune disease. B-cell depletion in 1- to 3-week-old mice depletes all B-cell subsets, whereas B-cell depletion initiated in adults spares many marginal zone B cells. Following early B-cell depletion, splenic Treg increase in number, and depletion of Treg reverses the inhibitory effect of anti-CD20 on disease development. Early transient depletion of B cells could be useful for preventing autoimmune disease in individuals at high risk for developing autoimmune diseases as adults.

Clonal expansion of B-cells in human systemic lupus erythematosus: Evidence from studies before and after therapeutic B-cell depletion

Anti-B-cell therapy may be beneficial for patients with SLE and active proliferative glomerulonephritis. Using genomic DNA, we examined how rituximab-induced transient B-cell depletion affected the composition of immunoglobulin heavy-chain (Ig-VH/DH/JH) repertoire at the time-point of 33-35% B-cell reconstitution in these patients. Clusters of clonaly-related Ig-VH/DH/JH sequences were evident in all 7 patients with active SLE but in none of 4 healthy subjects studied for comparison. In addition to original somatic mutation(s) shared in the majority of clonaly-related sequences, ongoing mutation suggested expansion of single B-cell precursors and clonal evolution. Otherwise, lupus repertoire was similar to that of healthy subjects with the exception of increased somatic hypermutation. The regenerating repertoire was diverse and comparable to baseline, albeit with fewer somatic mutations but more clonal expansions. Since clonal expansion may lead to preferential survival/growth of potentially autoreactive B-cells further studies are warranted, especially as therapies aiming to reduce B-cell survival emerge.

Chemotherapy induced transient B-cell depletion boosts antibody-forming cells expansion driven by an epidermal growth factor-based cancer vaccine

Cancer vaccines efficacy may improve inducing a rapid and persistent immune response, early at diagnosis along with standard therapies. EGF chemically conjugated to the carrier protein P64k from Neisseria meningitidis in montanide ISA 51 adjuvant is under evaluation, aiming to stimulate a B-cell response. High-dose cyclophosphamide and doxorubicin after priming enhanced the long-term frequency of EGF-specific antibody-forming cells (AFC) of IgM and IgG isotypes, but not the P64k response. Resulting combination, limitedly operational in Btk deficient xid mice, suggests that preferential B-cell lymphocyte space promoted by cyclophosphamide facilitates remaining EGF-specific AFC undergo homeostatic proliferation driven by boosting, amplifying the response.

Les nouveaux immunomodulateurs dans le traitement de l’ophtalmopathie basedowienne

Treatment of severe Graves’ophtalmopathy (GO) is a complex therapeutic challenge and spite any efforts, about one third of patients are disappointed with the outcome of treatment. Intravenous glucocorticoids (GCIV), orbital radiotherapy, or the combination of both are most frequently used for their immunosuppressive effects. The anti-CD 20 monoclonal antibody rituximab (RTX) induces transient B-cell depletion that may potentially modify the active inflammatory phase of thyroid-associated ophtalmopathy (TAO). A preliminary study in nine patients with TAO treated with RTX and twenty patients treated with GCIV was reported by Salvi and al. All patients attained peripheral B-cell depletion after the first RTX infusion. At the end of follow-up, clinical activity score values decreased more significantly compared with GCIV. Proptosis and the degree of inflammation decrease significantly in response to RTX. Relapse of active TAO are not observed in patients treated with RTX but occurred in 10 % of those treated with GCIV. If these results are confirmed in large controlled studies, RTX may represent a useful therapeutic tool in patients with active TAO.

Rituximab Induces Distinct Intraorbital and Intrathyroidal Effects in One Patient Satisfactorily Treated for Graves’ Ophthalmopathy

Hyperthyroid Graves' disease (GD) is a B-cell-mediated disease caused by antibodies stimulating the thyroid stimulating hormone (TSH) receptor (TRAb). A proportion of patients (40-60%) present with an associated ophthalmopathy (TAO), a progressive inflammatory autoimmune disease of the retroorbital tissue. We thought that the anti-CD20 monoclonal antibody rituximab (RTX), by inducing transient B-cell depletion, may potentially modify the active inflammatory phase of TAO. One patient with GD and TAO in its active phase and unresponsive to steroid, was treated with RTX. Whereas the ophthalmopathy responded to RTX therapy and a decrease in the clinical activity score from 5 to 2 was observed during the B-cell depletion, serum antithyroid antibodies, and in particular serum TRAb, were not affected by therapy. When the patient underwent total thyroidectomy, we found B-cells in the thyroid tissue specimens. The eye disease remained stable (clinical activity score=2) and the patient subsequently underwent orbital decompression to correct proptosis of the eye. At that time we did not find lymphocytes in any of the orbital tissue specimens. We believe that RTX therapy in GD may cause amelioration of ophthalmopathy by depleting total lymphocyte population in the orbit, but not lymphocyte depletion in thyroid tissue with consequent unchanged serum TRAb levels.

Delayed redistribution of CD27, CD40 and CD80 positive B cells and the impaired in vitro immunoglobulin production in patients with non‐Hodgkin lymphoma after rituximab treatment as an adjuvant to autologous stem cell transplantation

Recent studies have indicated that patients who received rituximab as an adjuvant to stem cell transplantation (SCT) demonstrated an increased risk of developing severe hypogammaglobulinaemia, which was found to be a result of delayed recovery of CD27 positive memory B cells and impaired isotype expression. It appears that rituximab influences both the quantity and quality of B-cell redistribution. Precisely how the B-cell repertoire regenerates after anti-CD20-mediated transient B-cell depletion in patients with non-Hodgkin lymphoma (NHL) remains to be elucidated. This study performed a phenotypical analysis of B cells in 17 NHL patients who received rituximab as an adjuvant to autologous SCT. The median period after final administration of rituximab was 36 months (range, 12-43 months). Surface antigen expression of CD27, CD40 and CD80 in NHL patients was statistically significantly different from healthy controls (n = 14). Moreover, B cells from NHL patients showed significantly impaired IgG and IgA production upon engagement of surface immunoglobulin receptors in the presence of interleukin (IL)-2, IL-10 and CD40 ligand in comparison with samples from healthy controls. The delayed recovery of memory B cells with an abnormal cell marker expression and function demonstrates that naive B cells may fail to differentiate into plasma cells, resulting in hypogammaglobulinaemia after autologous SCT and rituximab therapy.