anti-CD20 Treatment Research Articles

Predictors for insufficient SARS-CoV-2 vaccination response upon treatment in multiple sclerosis.

Disease-modifying therapies (DMT) for multiple sclerosis (MS) influence SARS-CoV-2 vaccination response, which might have implications for vaccination regimens in individual patients. Expanding the knowledge of predictors for an insufficient vaccination response as a surrogate for protection against severe disease courses of infection in people with MS (pwMS) under DMT is of great importance in identifying high-risk populations. Cross-sectional analysis of vaccination titre and its modifiers, in a prospective real-world cohort of 386 individuals (285 pwMS and 101 healthy controls) by two independent immunoassays between October 2021 and June2022. In our cohort, no difference in vaccination antibody level was evident between healthy controls (HC) and untreated pwMS. In pwMS lymphocyte levels, times vaccinated and DMT influence SARS-CoV-2 titre following vaccination. Those treated with selective sphingosine-1-phosphate receptor modulators (S1P) showed comparable vaccination titres to untreated; higher CD8 T cell levels prior to vaccination in B cell-depleted patients resulted in increased anti-spike SARS-CoV2 antibody levels. PwMS under DMT with anti-CD20 treatment, in particular those with decreased CD8 levels before vaccination, as well as non-selective S1P but not selective S1P are at increased risk for insufficient SARS-CoV-2 vaccination response. This argues for a close monitoring of anti-spike antibodies in order to customize individual vaccination regimens within these patients. This work was supported by the German Research Foundation (DFG, CRC-TR-128 to TU, SB, and FZ).

Clinical relevance of molecular aspects in extranodal marginal zone lymphoma: a critical appraisal

Extranodal marginal zone B-cell lymphoma of the mucosa-associated lymphoid tissue (MALT lymphoma) is among the more common types of lymphoma accounting for up to 8% of newly diagnosed lymphoma cases. As opposed to other B-cell lymphomas, however, no predominant genetic hallmark has been defined in MALT lymphoma, but different localizations appear to be affected by different, sometimes distinct changes. Nonetheless, a high proportion of these genetic changes reported in MALT lymphomas dysregulate the pathways leading to NF-kB activation. t(11;18)(q21;q21)/BIRC3::MALT1 appears to be MALT lymphoma specific and is found in 24% of gastric and 40% of pulmonary MALT lymphomas. The translocation is associated with more disseminated disease in gastric MALT lymphoma and is found in a large percentage of patients whose lymphoma is unresponsive to antibiotic eradication of Helicobacter pylori. In addition to t(11;18)(q21;q21), nuclear expression of BCL10 or NF-kB appears to be highly associated with lymphoma cell survival independence of H. pylori-mediated stimulations. Antibiotic eradication, however, is the recommended therapy of choice irrespective of genetic findings, and molecular analysis is not required before initiation of therapy. The influence of genetic translocations including t(11;18)(q21;q21) on systemic therapies, however, is less clearly defined. While small series have shown no influence on the outcome for treatment with the anti-CD20 antibody rituximab (R) or treatment with cladribine (2-CdA), conflicting data have been reported for alkylating agents, especially chlorambucil and the combination of R + chlorambucil. None of other genetic changes seen in MALT lymphoma to date has discernible value in routine clinical applications, but recent data suggest that changes in TNFAIP3(A20), KMTD2 and CARD11 might be associated with response to Bruton kinase inhibitors.

Impact of Treatment with Anti-CD20 Monoclonal Antibody on the Production of Neutralizing Antibody Against Anti-SARS-CoV-2 Vaccination in Mature B-Cell Neoplasms.

Anti-CD20 monoclonal antibodies (MoAbs), rituximab (RIT), and obinutuzumab (OBZ) are the central components of immunochemotherapy for B-cell lymphoma (BCL). However, these agents potentially cause B-cell depletion, resulting in the impairment of antibody (Ab) production. During the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, the optimal prediction of Ab response against anti-SARS-CoV-2 vaccination is critically important in patients with BCL treated by B-cell depletion therapeutics to prevent coronavirus disease 2019 (COVID-19). We investigated the effect of using RIT and/or OBZ on the Ab response in 131 patients with various types of BCL who received the second SARS-CoV-2 mRNA vaccine either after, during, or before immunochemotherapy containing B-cell-depleting moiety between June and November 2021 at seven institutes belonging to the Kyoto Clinical Hematology Study Group. The SARS-Cov-2 neutralizing Ab (nAb) was measured from 14 to 207 days after the second vaccination dose using the iFlash3000 automatic analyzer and the iFlash-2019-nCoV Nab kit. Among 86 patients who received the vaccine within 12 months after B-cell depletion therapy, 8 (9.3%) were seropositive. In 30 patients who received the vaccine after 12 months from B-cell depletion therapy, 22 (73%) were seropositive. In 15 patients who were subjected to B-cell depletion therapy after vaccination, 2 (13%) were seropositive. The multivariate analysis indicated that an interval of 12 months between B-cell depletion therapy and the subsequent vaccination was significantly associated with effective Ab production. Receiver operating characteristic curve analysis identified the optimal threshold period after anti-CD20 MoAb treatment, which determines the seropositivity against SARS-CoV-2, to be 342 days. The use of anti-CD20 MoAb within 12 months before vaccination is a critical risk for poor Ab response against anti-SARS-CoV-2 vaccination in patients with BCL.

Rapid Tumor Debulking of Relapsed/Refractory CLL Patients By PI3Kδ Inhibition and Anti-CD20 Monoclonal Antibody Treatment

Introduction Effective multi-targeted drug combinations could rapidly achieve deep remissions in CLL allowing for defined duration treatment. The combination of a novel highly-specific phosphoinositide 3-kinase delta (PI3Kδ) inhibitor and casein kinase 1 epsilon (CSK1ε) inhibitor, umbralisib (UMB), and a glycoengineered chimeric monoclonal antibody (mAb) targeting a unique epitope on CD20, ublituximab (UBL), with the BCL2 inhibitor venetoclax (VEN) may decrease drug resistance, reduce risk of tumor lysis syndrome (TLS) and achieve higher levels of undetectable minimal residual disease (MRD). This triple agent therapy is being evaluated in relapsed or refractory CLL patients (U2-VEN, ClinicalTrials.gov NCT03379051). Patients received oral 800 mg UMB once-daily. For Cycle 1, UBL was given via IV on Day 1 (150 mg), Day 2 (750 mg), Day 8 (900 mg), Day 15 (900 mg), and then on Day 1 in Cycles 2 and 3 (900 mg); for 3 cycles of 28 days, followed by consolidation with UMB and VEN for cycles 4 to 24. Patient (n = 25) blood samples were collected Pre- and Post-treatment on days of UBL administration during cycles 1-3. Our studies focused on UMB and UBL treatment effects on: 1) The decrease in the circulating CLL tumor burden and thus the subsequent risk of TLS from the addition of VEN to the regimen; and 2) CD20 antigen loss on CLL cells, a major mechanism of anti-CD20 mAb resistance. Methods Blood was analyzed by clinical laboratory automated cell counter and then stained with antibodies against CD3, CD5, CD19, and CD20 for flow cytometry analysis. CLL cell counts were calculated from flow cytometry and clinical laboratory results. To quantitate CD20 surface molecules, peripheral blood mononuclear cells were isolated and stained for flow cytometry analysis with the addition of a Molecules of Equivalent Soluble Fluorochrome standard curve. All human specimen collection and usage was conducted with written informed consent after approval of our Institutional Research Subjects Review Board according to the ethical guidelines of the Declaration of Helsinki. Results Average CLL cell counts (47 x 109/L) decreased rapidly after administration of the first dose of UMB and UBL (C1D1 Post, 26 x 109/L), which rebounded the next day (C1D2 Pre, 43 x 109/L) and then decreased after the second treatment (C1D2 Post, 24 x 109/L). By Day 15, the average CLL cell count had dropped to 9 x 109/L, which decreased further to 5 x 109/L by the start of cycle 3 (Fig. 1). Notably, the variation in CLL cell counts (error bars) decreases over treatment time, suggesting that most CLL patients' tumor burdens are decreasing to a similar low level independent of starting CLL cell count. The change in average CLL cell count between Pre and Post measurements is initially very high (45% decrease C1D1) with decreasing effectiveness over treatment time (8% decrease C3D1). This change could be due to the loss of CLL cell membrane CD20 levels leading to mAb resistance. Pre-treatment (C1D1 Pre) average CLL cell membrane CD20 levels (n=18) were 29,000 CD20 molecules/cell. This decreased to 9,000 molecules/cell after the first treatment (C1D1 Post), stabilized at 9,100 molecules/cell on the next day (C1D2 Pre) and then decreased further after the second treatment (C1D2 Post) to 1600 molecules/cell. After day 8 treatment, CD20 levels remained at 1,000 molecules/cell or less until the end of this study (C3D1). Discussion UMB and UBL rapidly decrease circulating CLL counts over three cycles of treatment, reducing most CLL patient tumor burden to similar low levels. This clinical response could facilitate addition of VEN in the 4thcycle of therapy by decreasing the risk of TLS. The rapid loss of surface CD20 from CLL cells to 1,000 CD20 molecules/cell or less by C1D8 Post has the potential to cause acquired resistance to anti-CD20 mAb therapy. This rapid and sustained loss of CD20 levels is consistent with that reported at standard doses for other anti-CD20 mAbs. However, an effect of concomitant UMB on CD20 levels cannot be ruled out. Previous work suggests that lower anti-CD20 mAb doses can prevent these large decreases in CD20 levels and increase UBL efficacy. In conclusion, our initial data show that treatment with a selective PI3Kδ and CSK1ε inhibitor (UMB) combined with an anti-CD20 mAb (UBL) is effective in decreasing CLL cell counts in all patients assessed in the U2-VEN clinical trial. However, treatment efficacy could be limited by large decreases in the CLL surface CD20 levels. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A Phase Ib/II Study of Polatuzumab Vedotin Plus Obinutuzumab and Lenalidomide in Patients with Relapsed/Refractory Follicular Lymphoma: Final Analysis and Progression-Free Survival Update

Introduction: Despite recent advances in the treatment of follicular lymphoma (FL), patients frequently require multiple lines of therapy, and an unmet need remains for patients who experience relapsed or refractory (R/R) disease. After a median follow-up of 27.0 months (safety-evaluable population), the novel triplet combination of polatuzumab vedotin (Pola) plus obinutuzumab (G) and lenalidomide (Len) was shown to be efficacious in a heavily pre-treated and refractory FL population and had a safety profile consistent with known profiles of the individual drugs (Diefenbach C, et al. Lancet Haematol 2021). Here, we report the final analysis with updated safety and efficacy data for this combination. Methods: GO29834 (NCT02600897) was an open-label, multicenter study of patients with R/R FL (excluding Grade 3b) who had received ≥1 prior anti-CD20-containing chemoimmunotherapy regimen. The recommended Phase II dose for Pola and Len in combination with G was established in an initial 3+3 dose-escalation phase and expanded into a Phase II study. Patients in the expansion cohort received induction treatment with six 28-day cycles of: intravenous (IV) G 1000mg (Cycle [C]1: Day [D]1, 8, 15; C2-6: D1); IV Pola 1.4mg/kg (D1), and oral Len 20mg (D1-21). Responders and those with stable disease received maintenance treatment for 24 months (G 1000mg on D1 every 2 months and Len 10mg on D1-21 of Months 1-12). The primary endpoint was complete response (CR) at end of induction (EOI), determined by the Independent Review Committee (IRC) based on positron emission tomography-computed tomography scans by modified Lugano 2014 criteria (Cheson BD, et al. J Clin Oncol 2014). Secondary endpoints included progression-free survival (PFS), overall survival (OS), and safety. Results: A total of 56 patients were enrolled across Phase Ib and Phase II; median duration of follow-up was 43.5 months in the safety-evaluable population at the time of final analysis (March 03, 2022). Baseline characteristics were: median age, 62 years; male, 59%; Ann Arbor Stage III-IV, 88%; Follicular Lymphoma International Prognostic Index 1 high-risk (≥3), 55%; bulky disease (≥7cm), 16%; median prior lines of treatment, 3 (range: 1-7); ≥2 prior lines of therapy, 77%; refractory to last line of prior regimen, 59%; refractory to last line of anti-CD20 treatment, 55%; and POD24 (defined as disease progression within 24 months of initiation of first anti-lymphoma treatment with chemoimmunotherapy), 27%. All patients (N=56) had at least one adverse event (AE), 36 (64%) had a serious AE, and 48 (86%) had a Grade 3-4 AE. The most common Grade 3-4 AEs were neutropenia (n=31, 55%), thrombocytopenia (n=14, 25%), infections (n=14, 25%), and anemia (n=8, 14%). AEs leading to dose reduction or interruption of any drug occurred in 18 (32%) and 43 (77%) patients, respectively; the majority were modifications of Len. In addition, 19 (34%) patients had an AE that led to discontinuation of any study drug. Three Grade 5 (fatal) AEs were reported (one case of septic shock and two cases of COVID-19). In the primary efficacy population (N=46), the IRC-assessed objective response rate at EOI was 76%, with a CR rate of 61%. The best overall response rate by investigator assessment (INV) was 91%, with a best CR rate of 72%. After a median follow-up of 43.3 months in the efficacy-evaluable population (N=46), median PFS was not reached (Figure). Landmark PFS by INV was 83% (95% confidence interval [CI]: 72-95) at 12 months, 67% (95% CI: 53-82) at 24 months, and 53% (95% CI: 36-70) at 48 months. Median OS was not reached. Conclusions: In the GO29834 study, we evaluated the safety and efficacy of novel triplet combination Pola-G-Len in patients with R/R FL, a population for whom there is a continued unmet medical need. This final analysis demonstrates a safety profile consistent with the known profiles of the individual drugs. High CR rates at EOI in a heavily pre-treated and refractory population compare favorably with currently available R/R FL therapies. After a median follow-up of 43.3 months in the efficacy-evaluable population, the median PFS and OS were not reached, and more than half of the patients treated with Pola-G-Len had not experienced disease progression after 4 years. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

B-Cell Targeting Anti-CD37 Humanized Antibodies Engineered for Potent Effector Functions and Extended Plasma Half-Life

Background: CD37 is a tetraspanin protein abundant on the surface of B-cells but absent in hematopoietic stem cells and plasma cells. Its expression pattern has attracted interest as a therapeutic target for B-cell malignancies including non-Hodgkin lymphomas (NHL), a heterogeneous disease with substantial unmet clinical need due to relapse and resistance to currently available treatments. During the last years, different approaches have been followed to exploit CD37 in targeting malignant B-cells, such as the generation of CAR-T cells and monoclonal antibodies. Of note, the anti-CD37 antibody-drug conjugates (ADC) naratuximab emtansine and the CD37-targeting radioimmunotherapy (RIT) Betalutin have shown efficacy in NHL patients during clinical testing. Here we report on the design of humanized anti-CD37 IgG1 antibodies (NNV029, NNV031 and NNV032) that are equipped with a unique Fc technology and N-glycan engineering for enhanced effector functions and extended plasma half-life. Methods: The humanized anti-CD37 candidates were characterized in vitro and in vivo and benchmarked against the anti-CD20 antibodies approved for NHL treatment, rituximab and obinutuzumab, and a recombinant version of the bi-paratopic anti-CD37 antibody DuoHexaBody-CD37, currently in Phase I clinical development. We assessed their Fcγ receptor binding properties and their ability to induce antibody-dependent cell cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP) and complement-dependent cytotoxicity (CDC) against CD37-expressing NHL cell lines and patient-derived chronic lymphocytic leukemia (CLL) cells. Moreover, we evaluated their human FcRn binding and transport properties using the human endothelial recycling assay (HERA) followed by studies in human FcRn (Tg32) expressing mice. Lastly, we compared the therapeutic efficacy of the humanized antibodies versus obinutuzumab and recombinant DuoHexaBody-CD37 in a xenograft disseminated Daudi tumor model in Tg32 mice. Results: We revealed that the designed anti-CD37 IgG1 candidates have higher affinity towards FcγRIIa expressed on macrophages and that afucosylation enhanced FcγRIII engagement. Accordingly, using cell-based effector assays, potent ADCP and ADCC were measured. In NHL Burkitt's lymphoma cell lines, ADCP induction by the anti-CD37 humanized antibodies was higher than that displayed by obinutuzumab, rituximab and recombinant DuoHexaBody-CD37, while in DLBCL and MCL cell models it was in a similar range or slightly lower than that shown by the benchmarking antibodies. Moreover, the afucosylated version promoted very strong ADCC, which was superior to that of recombinant DuoHexaBody-CD37 and rituximab, but comparable to that of the afucosylated antibody obinutuzumab. In contrast, all the humanized anti-CD37 IgG1 candidates displayed very modest CDC activation. Fc-engineering for enhanced human FcRn binding was shown to increase human FcRn-mediated rescue from intracellular degradation, which translated into prolonged plasma half-life in Tg32 mice of the anti-CD37 IgG1 antibodies. Finally, all the designed anti-CD37 antibodies performed significantly better than vehicle control treatment in treating Daudi-induced disease in vivo, showing equal therapeutic effect as that of obinutuzumab and recombinant DuoHexaBody-CD37. Conclusions: Taken together, the humanized and Fc-engineered anti-CD37 antibodies show potent effector functions, favorable pharmacokinetic properties and in vivo therapeutic efficacy. As such, the designed CD37 antibodies should be attractive modalities in targeted treatment of B cell malignancies, such as NHL, including lymphomas non-responding to anti-CD20 treatments, but also potentially for B-cell driven autoimmune disorders.

The Efficacy and Safety of Anti-CD20 Antibody Treatments in Relapsing Multiple Sclerosis: A Systematic Review and Network Meta-analysis.

Several large randomized controlled trials of anti-CD20 antibodies have been successfully conducted for the treatment of relapsing multiple sclerosis. Despite this, there are few systematic comparisons of different anti-CD20 antibodies and a comprehensive evaluation of their efficacy and safety is yet to be carried out. The objective of this systematic review and network meta-analysis was to evaluate the efficacy and safety of the three approved anti-CD20 antibodies for the treatment of relapsing multiple sclerosis and to aid clinicians in choosing medications. MEDLINE, EMBASE, Cochrane Library, and clinicaltrials.gov were all searched for randomized controlled trials conducted to evaluate anti-CD20 antibodies (rituximab, ocrelizumab, ofatumumab) and corresponding controls up to 31 May, 2022. Review Manager 5.3 and R 3.5.2 software were used to assess the data. The risk ratio and mean difference were analyzed and calculated with a random-effects model. We pooled 4181 patients from ten randomized controlled trials. Without increasing the risk of adverse events and serious adverse events, anti-CD20 antibodies were superior to the active control group in all efficacy outcomes (both p < 0.005, certainty of evidence, very low to high). For the comparison between anti-CD20 groups, rituximab was found to be able to significantly increase the number of patients free of relapse more effectively than the other two interventions; however, the surface under curve ranking area values for serious adverse events were also the highest (84.8%). At the same time, ocrelizumab and ofatumumab exhibited satisfactory efficacy without showing a worse safety than any other interventions. Overall, anti-CD20 antibody treatment is superior to a corresponding control in efficacy and safety measures and ocrelizumab and ofatumumab may be the most suitable anti-CD20 antibodies for treating relapsing multiple sclerosis. Additional large-scale and high-quality studies are still needed to further explore the safety of these therapies.

Effects of anti-CD4 antibody treatment on calcium ions influx in peanut-sensitized C3H/HeJ mice

The precise mechanism underlying the effects of anti-CD4 antibody and calcium ions (Ca2+) in peanut allergy remains unknown. C3H/HeJ mice sensitized with peanut protein extract (PPE) were injected with anti-CD4 antibodies for 4 weeks. Stimulation with PPE increased the specific immunoglobulin (IgE), cytokine, histamine, and mMcp-1 levels, upregulated decorin (Dcn) expression, induced Ca2+ inflow in the spleen, and augmented the expression of the transcription factors GATA-3 and Foxp3, which resulted in Th2 and Treg cell activation. Notably, the Ca2+ levels were positively correlated with the histamine, interleukin (IL)-4, IL-5, and IL-13 levels, and negatively correlated with IL-10 levels. However, administration of anti-CD4 antibodies markedly alleviated allergic symptoms, activated T cells, and reduced Ca2+ inflow, cytokine, histamine, mMcp-1, and the IgHG3, CXCL12, MMP2 and FABP4 gene. Our results indicated that anti-CD4 antibodies can ameliorate PPE-induced allergy, which is probably related to the suppression of Ca2+ inflow, and inhibiting histamine, cytokine and IgHG3, CXCL12, MMP2, and FABP4, thus exerting a protective effect against PPE-sensitized food allergy.

Timing of SARS-CoV-2 Vaccination Matters in People With Multiple Sclerosis on Pulsed Anti-CD20 Treatment.

Background and ObjectivesOur objective was to investigate cellular and humoral immune responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination in a cohort of people with multiple sclerosis (pwMS) on pulsed B-cell–depleting treatment (BCDT). In particular, we intended to evaluate a possible association between immune responses and the timing of vaccination under BCDT.MethodsWe conducted a cross-sectional study among pwMS on pulsed BCDT or without disease-modifying treatment after completed SARS-CoV-2 vaccination. Samples were collected during routine clinical visits at the Multiple Sclerosis Center Dresden, Germany, between June 2021 and September 2021. Blood was analyzed for SARS-CoV-2 spike protein–specific antibodies and interferon-γ release of CD4 and CD8 T cells on stimulation with spike protein peptide pools. Lymphocyte subpopulations and total immunoglobulin levels in the blood were measured as part of clinical routine.ResultsWe included 160 pwMS in our analysis, comprising 133 pwMS on BCDT (n = 132 on ocrelizumab and n = 1 on rituximab) and 27 without disease-modifying treatment. Humoral and cellular anti–SARS-CoV-2 responses were reciprocally regulated by the time between the last BCDT cycle and vaccination. Although antibody responses increased with prolonged intervals between the last BCDT cycle and vaccination, CD4 and CD8 T-cell responses were higher in pwMS vaccinated at early time points after the last BCDT cycle compared with untreated pwMS. T-cellular vaccination responses correlated with total, CD3 CD4, and partly with CD3 CD8 lymphocyte counts. Humoral responses correlated with CD19 lymphocyte counts. Status post coronavirus disease 2019 infection led to significantly increased SARS-CoV-2–specific T-cell and antibody responses.DiscussionDelaying BCDT is currently discussed as a strategy to optimize humoral responses to SARS-CoV-2 vaccination. However, T cells represent an important line of defense against SARS-CoV-2 infection as well, especially in light of emerging variants of concern. We observed enhanced CD4 and CD8 T-cellular responses in pwMS receiving vaccination at early time points after their last BCDT cycle. These data may influence clinical decision making with respect to vaccination strategies in patients receiving BCDT.

Response to SARS-CoV-2 vaccines in patients receiving B-cell modulating antibodies for renal autoimmune disease

BackgroundEffective SARS-CoV-2 vaccination in patients receiving treatment with B-cell depleting agents is challenging. Information on vaccination responses in these patients are a valuable tool to develop efficient vaccination regimens.MethodsIn this single-center retrospective observational study, we report the humoral and cellular response in 34 patients receiving anti-CD20 antibody treatment for renal immune disease.ResultsAfter base immunization with SARS-CoV-2-vaccines, 92.0% developed a cellular, 32.4% a humoral response. Humoral immunity correlated with B-cell counts and the timespan between anti-CD20 antibody treatment and vaccination. All patients with > 21/µl B-cells, or > 197 days after treatment showed humoral response.ConclusionsAdequate timing of SARS-CoV-2-vaccinations after anti-CD20 antibody treatment and CD19 measurements are crucial to generate immunity. Awaiting partial B-cell recovery by postponing regularly scheduled anti-CD20 treatment should be considered in patients with stable immune disease.Trial registration: This study has been retrospectively registered in the German Clinical Trials Register (DRKS00027049) on 29/10/2021.

Broader Epstein-Barr virus-specific T cell receptor repertoire in patients with multiple sclerosis.

Epstein-Barr virus (EBV) infection precedes multiple sclerosis (MS) pathology and cross-reactive antibodies might link EBV infection to CNS autoimmunity. As an altered anti-EBV T cell reaction was suggested in MS, we queried peripheral blood T cell receptor β chain (TCRβ) repertoires of 1,395 MS patients, 887 controls, and 35 monozygotic, MS-discordant twin pairs for multimer-confirmed, viral antigen-specific TCRβ sequences. We detected more MHC-I-restricted EBV-specific TCRβ sequences in MS patients. Differences in genetics or upbringing could be excluded by validation in monozygotic twin pairs discordant for MS. Anti-VLA-4 treatment amplified this observation, while interferon β- or anti-CD20 treatment did not modulate EBV-specific T cell occurrence. In healthy individuals, EBV-specific CD8+ T cells were of an effector-memory phenotype in peripheral blood and cerebrospinal fluid. In MS patients, cerebrospinal fluid also contained EBV-specific central-memory CD8+ T cells, suggesting recent priming. Therefore, MS is not only preceded by EBV infection, but also associated with broader EBV-specific TCR repertoires, consistent with an ongoing anti-EBV immune reaction in MS.

In Vivo Generation of SSA/Ro Antigen-Specific Regulatory T Cells Improves Experimental Sjögren's Syndrome in Mice.

Sjögren's syndrome (SS) is a systemic autoimmune disease, and T cells play an important role in the initiation and perpetuation of the disease. In this study, we developed an immunotherapy for NOD/LtJ mice with SS-like symptoms by combining a transient depletion of CD4+ T cells with the administration of autoantigen-specific peptide Ro480. NOD/LtJ mice were treated with single anti-CD4 monoclonal antibody (mAb) followed 2 days later by a series of 6 intraperitoneal injections of Ro480-494 every other day. Salivary flow rates were determined pre- and posttreatment once a week. Mice were euthanized 6 weeks after the initial anti-CD4 mAb treatment, salivary glands (SGs) were collected for analyses of histologic disease scores and inflammatory cell infiltration, polymerase chain reaction determination of genes was conducted, and flow cytometry analysis including major histocompatibility complex class II tetramer staining of immune cells was performed. In addition, adoptive transfer of Treg cells was administrated to investigate the function of the newly generating Treg cells in vivo. The combination of anti-CD4 mAb with autoantigen-specific peptide Ro480 generated SSA/Ro antigen-specific Treg cells in vivo, which can suppress interferon-γ production of CD4+ T cells and inflammation infiltration in SGs and maintain the function of SGs. Our findings provide a new approach to generating antigen-specific Treg cells in vivo for SS treatment, which may have implications for potential therapy for patients with SS.

Dual B-cell targeting therapy ameliorates autoimmune cholangitis

ObjectiveThe ability to regulate B cell development has long been recognized to have therapeutic potential in a variety of autoimmune diseases. However, despite the presence of a classic autoantibody in primary biliary cholangitis (PBC), B cell depleting therapy and indeed therapy with other biologic agents has been disappointing. Unsuccessful treatment using Rituximab is associated with elevation of B-cell activating factor (BAFF) level. Indeed, therapies for PBC remain directed at modulating bile salt biology, rather than targeting effector pathways. With these data in mind, we proposed that targeting two major stages of B cell development, namely long-lived memory B cells and short-lived peripheral autoreactive plasma cells would have therapeutic potential. MethodsTo address this thesis, we administrated anti-BAFF and anti-CD20 monoclonal antibody to ARE-Del mice, a well-characterized murine model of human PBC. We evaluated and compared the therapeutic efficacy of the two agents individually and the combination of anti-BAFF and anti-CD20 in female mice with well-established disease. ResultsOur data demonstrate that there was an increased level of B cell depletion that resulted in a significantly more effective clinical and serologic response using the combination of agents as compared with the use of the individual agents. The combination of anti-BAFF and anti-CD20 treatment was more effective in reducing serum levels of antimitochondrial antibody (AMA), total IgM and IgG compared to mice treated with the 2 individual agents. Combination treatment efficiently depleted B cells in the peripheral blood, peritoneal cavity and spleen. Importantly, we identified a unique IgM+ FCRL5+ B cell subset which was sensitive to dual B-cell targeting therapy and depletion of this unique population was associated with reduced portal infiltration and bile duct damage. Taken together, our data indicate that dual B cell targeting therapy with anti-BAFF and anti-CD20 not only led to the efficient depletion of B cells both in the peripheral blood and tissues, but also led to significant clinical improvement. These findings highlight the potential application of combination of anti-BAFF and anti-CD20 in treating patients with PBC. However, additional studies in other animal models of PBC should be undertaken before considering human trials in those PBC patients who have incomplete responses to conventional therapy.

Effect of a third booster dose of COVID-19 mRNA vaccine in patients with haematological cancer after the initial two-dose vaccination - a single centre report

Novel Coronavirus SARS-CoV-2 causing COVID-19 has been subject to intensive interest since its appearance in 2019, with the risk of severe course being significantly higher for adult patients with hematological malignancy. Results on a two-dose, standard vaccination regimen in patients with hematological cancer have identified risk populations with poor vaccination outcomes (Chronic lymphocytic leukemia, anti-CD20 treatment, etc.). Thus, a booster dose was anticipated with hopes of inducing an immune response in formerly non-respondent individuals. We have vaccinated 394 patients with hematological cancer with the third dose of the mRNA BNT 162b2 COMIRNATY vaccine. Our results show promise, especially for increasing protective antibody levels in patients who retain valid antibody titers. We also identify problematic populations such as chronic lymphocytic leukemia, which still represent a major challenge for prophylaxis and protection against a severe course of COVID-19. Our report brings more insight into vaccination results and behavior. Importantly, we have identified risk groups in which poor outcomes can be anticipated and what extensive preventive measures should be undertaken to avoid COVID-19 infection.

Neutropenia complicating anti-CD20 treatment in patients with multiple sclerosis: A retrospective case series and a systematic review of reported cases

BackgroundNeutropenia is an infrequent complication of treatment with CD20 depleting agents and may require the administration of granulocyte-colony stimulating factors (G-CSF), which have been associated with an increased relapse risk in patients with multiple sclerosis (PwMS). The management of this side effect is still matter of debate. MethodsAim of this study is to evaluate the clinical features and the management of neutropenia occurring in anti-CD20 treated PwMS through a single-center case series and a systematic review of the literature, performed following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. ResultsA total of 19 patients were included (3 from our clinical experience, 16 from the systematic review). Median age was 38 years-old (25-69) and nearly 70% were female, most of these patients had already received a median of 3 (0-4) previous treatments. Neutropenia occurred in 11 patients treated with ocrelizumab and 8 with rituximab, after a median of 2 (1-7) infusions and 9.5 (1-42) months from the first infusion. Most of these patients had late-onset neutropenia, that occurred after a median time of 90 days (2-156). About 70% of patients were symptomatic and most were treated with G-CSF or antibiotics. No relapses after G-CSF were reported. In those who did not suspend anti-CD20 (68.8%), neutropenia reoccurred in 18.2% of cases. Finally, switching between rituximab and ocrelizumab seem not to affect the occurrence of neutropenia. ConclusionOur data provides practical evidence regarding the occurrence and the management of neutropenia during treatment with anti-CD20 in PwMS.

Feeding mode influences dynamic gut microbiota signatures and affects susceptibility to anti-CD3 mAb-induced intestinal injury in neonatal mice.

Feeding modes influence the gut microbiome, immune system, and intestinal barrier homeostasis in neonates; how feeding modes impact susceptibility to neonatal gastrointestinal (GI) diseases is still uncertain. Here, we investigated the impact of dam feeding (DF) and formula feeding (FF) on features of the gut microbiome and physiological inflammation during the first 2 days of postnatal development and on the susceptibility to intestinal injury related to the inflammatory state in neonatal mouse pups. 16S rRNA sequencing data revealed microbiome changes, lower α-diversity, and a distinct pattern of β-diversity including expansion of f_Enterobacteriaceae and f_Enterococcaceae in the ileum of FF pups compared with DF pups by postnatal day (P)2. Together with gut dysbiosis, the FF cohort also had greater ileal mucosa physiological inflammatory activity compared with DF pups by P2 but maintained normal histological features. Interestingly, FF but not DF mouse pups developed necrotizing enterocolitis (NEC)-like intestinal injury within 24 h after anti-CD3 mAb treatment, suggesting that FF influences the susceptibility to intestinal injury in neonates. We further found that NEC-like incidence in anti-CD3 mAb-treated FF neonatal pups was attenuated by antibiotic treatment. Collectively, our data suggest that FF predisposes mouse pups to anti-CD3 mAb-induced intestinal injury due to abnormal f_Enterobacteriaceae and f_Enterococcaceae colonization. These findings advance our understanding of FF-associated microbial colonization and intestinal inflammation, which may help inform the development of new therapeutic approaches to GI diseases like NEC in infants.NEW & NOTEWORTHY This report shows that a feeding mode profoundly affects gut colonization in neonatal mice. Furthermore, our results demonstrate that formula feeding predisposes mouse pups to anti-CD3 mAb-induced necrotizing enterocolitis (NEC)-like intestinal injury upon inadequate microbial colonization. The study suggests the role of the combined presence of formula feeding-associated dysbiosis and mucosal inflammation in the pathogenesis of NEC and provides a new mouse model to study this disease.

COVID-19 infection after two doses of SARS-CoV-2 mRNA vaccine in multiple sclerosis, AQP4-antibody NMOSD and MOGAD

BackgroundIn pre-vaccinated people with multiple sclerosis (MS), certain disease-modifying therapies (DMTs), particularly the anti-CD20 treatments, appear to be associated with an increased risk of COVID-19 infection and indeed with severe infection. It is still not known if such observations extend to vaccinated individuals and there have been considerably fewer studies in aquaporin-4-antibody neuromyelitis optica spectrum disorder (AQP4-NMOSD) and myelin oligodendrocyte glycoprotein-antibody associated disease (MOGAD) patients. In this study, we investigated the rates of symptomatic COVID-19 infection in adult patients with MS, AQP4-NMOSD and MOGAD who had received 2 doses of SARS-CoV-2 mRNA vaccine. MethodsThis was a prospective observational study conducted at the 2 main neuroimmunology referral centres in Singapore. Only patients on active follow-up were recruited to ensure robust data collection. Data on demographics, disease history, DMTs and SARS-CoV-2 mRNA vaccinations were recorded, and for those infected with COVID-19, data on COVID-19 infection was collected. ResultsNineteen (13 MS, 5 AQP4-NMOSD, 1 MOGAD) out of 365 (231 MS, 106 AQP4-NMOSD, 28 MOGAD) patients had COVID-19 infection despite 2 doses of SARS-CoV-2 mRNA vaccine. Amongst the infected patients, 11 patients were on DMTs (3 rituximab, 2 interferons, 1 azathioprine, 1 mycophenolate, 1 prednisolone, 1 cladribine, 1 alemtuzumab, 1 fingolimod), while 8 patients were untreated. The crude infection rate was calculated using time-at-risk analysis, revealing that rituximab had the highest infection rate amongst all the DMTs. A lower crude infection rate was observed in patients who received a third vaccination. The majority of infections were mild and no patients required oxygen supplementation. ConclusionOur findings suggest that patients on rituximab are still at risk of COVID-19 infection after 2 vaccinations and the receipt of a third vaccination may help to prevent infection. Future large scale studies will be required to better delineate the infection risk of different DMTs after the second and subsequent vaccinations.

Response of COVID-19 vaccination in multiple sclerosis patients following disease-modifying therapies: A meta-analysis.

SummaryBackgroundCOVID-19 vaccination is recommended for patients with multiple sclerosis (pwMS), while disease-modifying therapies (DMTs) may influence the efficacy of SARS-CoV-2 vaccines in this population. Thus, we conducted a meta-analysis to evaluate the impact of DMTs on immune response to COVID-19 vaccines in pwMS.MethodsLiterature search from December 1, 2019 to March 31, 2022 was performed in PubMed, MedRxiv, Embase and Cochrane Library. The risk of impaired response to vaccination in pwMS receiving DMTs was estimated in odds ratios (ORs) using random-effects method.FindingsA total of 48 studies comprising 6860 pwMS were included. Overall, pwMS with anti-CD20 (OR=0.02, 95% CI: 0.01-0.03) and sphingosine-1-phosphate receptor modulator (S1PRM) (OR=0.03, 95% CI: 0.01-0.06) treatments had attenuated serologic response after full vaccination compared with those without DMTs. Additionally, pwMS vaccinated within six months since last anti-CD20 therapy were at significantly higher risk of blunted response compared with those receiving anti-CD20 therapy more than six months prior to vaccination (P = 0.001). We found no significant associations between other treatments (including IFN-β, GA, DMF, TERI, NTZ, CLAD, and ALE) and humoral response to SARS-CoV-2 vaccines in pwMS. As for T-cell response, no significant difference was found between pwMS on anti-CD20 and those without DMTs after vaccination, while S1PRM was marginally associated with impaired cellular response (P = 0.03).InterpretationOur findings suggested that routine serological monitoring may be required for pwMS on anti-CD20 and S1PRMs after SARS-CoV-2 vaccination and highlighted the benefits of a booster dose. The effect of cellular response and optimal interval from last anti-CD20 treatment to vaccination should be further addressed.FundingThis study was supported by Natural Science Foundation of Shanghai (21ZR1433000).

P681: EUROFLOW STANDARDIZATION TECHNIQUE AND NORMALIZATION PROCEDURES IN LONGITUDINAL FLOW CYTOMETRIC EXPRESSION ANALYSIS OF CD20 IN CLL PATIENTS RECEIVING ANTI-CD20 DIRECTED THERAPY

Background: CD20 expression is still a controversial issue regarding its prognostic value for therapy outcome. While up to 50% of patients relapse within four of years after anti-CD20 directed treatment and constitute a need for a salvage or relapse/refractory treatment, only few publications address the longterm influence on CD20 expression assessed by flow cytometry. The technical complexity of the method and factors affecting the variability of measurements of samples at different time points and from different sources/patients are well recognized by the field and addressed by different approaches as standardization of instruments, automated gating and alignment of population or normalization. Aims: In order to evaluate long term influence of anti-CD20 directed therapy on CD20 expression in patients with chronic lymphocytic leukemia who showed only weak MRD responsiveness to therapy in different clinical trials, we aimed to establish a normalization approach based on the instrument standardization to reduce the proportion of technical variance within data. Methods: Mean fluorescence intensities (MFI) of peak seven of standardized fluorescence beads from daily quality control and instrument standardization of flow cytometers according to the EuroFlow protocol were used to establish a normalization approach. Proof of prinicple was performed using the residual peaks covering the MFI range provided by the beads. To assess different sources of variance ANOVA of a 3x3x3 experiment evaluating marker expressions on different cell populations in the blood of healthy donors using Quantibrite-PE beads was performed addressing technical variation (day, instrument) and biological variation. CD20 and CD19 MFI on CLL cells were retrospectively assessed from MRD measurements of peripheral blood samples of patients enrolled in the phase II GCLLSG trials CLL2-BIG (n=57), -BAG (n=58), -BIO (n=58), -BCG (n=22), combining facultative Bendamustin (B) debulking with either Obinutuzumab (G), Ofatumumab (O), Venetoclax (A), Ibrutinib (I) or Idelalisib (C). Results: Long term shifts of fluorescence intensities, and coefficients of variation were reduced 2 to 10 times across the MFI range covered by the fluorescence beads without distorting the longitudinal course of instrument performance. Most of the variance in assessments of molecules equivalent soluble fluorochrome from MFIs of NK cell and B cell markers in peripheral blood of healthy donors were related to inter-donor variations (p<0.002). Normalized MFIs for CD19 and CD20 on CLL cells do not significantly differ from the measured values in the Mann-Whitney-U test (p>0.85). Higher CD20 expression at therapy start seems to be correlated to strong MRD response solely in the CLL2-BIO trial (p=0.036, Fig. 1). Prognostic factors TP53-, IGHV-, and NOTCH1- mutation did not seem to correlate with the observed CD20 expression differences. Therapy strata showed differences (statistically not significant) in the proportion of firstline and r/r treated patients. Figure 1 Pre-treatment CD20 MFI on CLL cells of patients categorized according to treatment and variable (strong, medium, weak, and no) MRD response. Image:Summary/Conclusion: Following standardized staining and instrument monitoring, it should be possible to robustly assess longitudinal biological variations of marker expression based on MFI values. In a cross trial comparison Obinutzumab showed highest proportion of patients with strong MRD response independent from initial CD20 expression, whereas strong response to Ofatumumab seems to correlate with higher CD20 expression levels.

P1092: NODULAR LYMPHOCYTE PREDOMINANT HODGKIN LYMPHOMA (NLPHL): HISTOLOGYCAL AND CLINICAL REVIEW IN A SINGLE CENTRE.

Background: NLPHL constitutes a subtype of Hodgkin lymphoma with distinct clinical characteristics and evolution. Aims: To review the histopatologycal diagnosis and clinical data in patients with NLPHL in our institution between 1999-2021. Methods: We retrospectively reviewed the histology of lymph node biopsy and clinical data, treatment and outcome of 21 adult patients consecutively diagnosed of a NLPHL between 1999 and 2021. Results: Twenty-one adult patients with diagnosis of NLPHL (3 of which presented transformation areas to diffuse large B-cell lymphoma (t-DLBCL) in the biopsy), were identified. After the histological review of the biopsies 1 patient was reclassified as diffuse large B cell lymphoma (DLBCL) and another patient as NLPLH with t-DLBCL. Six (30%) patients showed a typical histological pattern (A-B) in the biopsy whereas 14 (70%) presented histopathological variants (pattern C-F) including the 4 patients with transformation areas to DLBCL. Thus, the clinical data of a total number of 20 patients with NLPLH (4 of them with transformation areas) were reviewed. Twelve (60%) patients were males and 8 (40%) female with a median age of 46 years (range 17-79). Regarding the clinical stage at diagnosis, 9 (45%), 2 (10%), 5 (25%) and 4 (20%) were in favourable IA stage, favourable IIA stage, unfavourable I-IIA stage (according the German Study Hodgkin Group and European Organisation for Research and Treatment of Cancer criteria) and advanced III-IV stage respectively. Three of the 4 patients in advance stage presented variant histological patterns. Among the 9 patients in favourable clinical IA stage the treatment administered was the following: 2 of them didn’t receive any further treatment (in one by rejection and other patient was in surveillance after lymph node resection), 5 received Radiotherapy and 2 received combined treatment (chemotherapy with ABVD (doxorubicin, bleomycin, vinblastine, dacarbazine) and radiotherapy). The 2 patients in favourable IIA stage received combined treatment. Among the 5 patients in unfavourable I-II clinical stage, 3 received combined treatment (ABVD and radiotherapy), 1 patient was treated with ABVD and 1 with R-CHOP (rituximab, ciclophosphamide, doxorubicin, vincristine and prednisone) due to areas of t-DLBCL in the biopsy. Patients (4) in advanced stage were treated with chemotherapy: 2 with ABVD, 1 with CVP (ciclophosphamide, vincristine and prednisone) and 1 with rituximab and platinum based regimen (transformation areas to DLBCL in the biopsy and previous episode of DLBCL). Regarding to responses, 15 (83.3%) of the 18 treated patients achieved a complete remission and 3 (16.7%) were refractory (2 of them received second line therapy incuding rituximab with a complete remission). One patient relapsed; he was refractory to ABVD and achieved a complete remission with R-CHOP. With a median follow-up of 5.9 years (range 0.8-18) the overall survival is 90%; 2 patients have died (1 of them, a 79 years old patient with comorbidities, due to lymphoma). Summary/Conclusion: We have seen a 95% of concordance with the initial diagnosis after a histological review in our patients with NLPLH. 20% of the biopsies showed transformation areas to DLBCL. Most of the patients (80%) presented in a localized stage. Refractory patients responded well to antiCD20 treatment. The prognosis is good with an overall survival of 90%.