anti-CD20 Antibody Rituximab Research Articles

Implication of Rituximab Infusion Reactions on Clinical Outcomes in Patients With Diffuse Large B-cell Lymphoma: A Single Institution Experience

BackgroundThe addition of the anti-CD20 monoclonal antibody rituximab to chemotherapy for diffuse large B-cell lymphoma (DLBCL) has led to improvements in progression-free survival and overall survival, although the exact mechanism of rituximab is not known. Rituximab administration often results in transient, non–life-threatening infusion reactions (IRs). We report a retrospective cohort of patients with DLBCL who received rituximab to determine the significance of IRs on clinical outcomes. Patients and MethodsWe identified and analyzed a retrospective cohort of 229 patients with DLBCL. They were stratified into 2 cohorts; those who did and did not have an IR. Univariate and multivariate analyses were performed to evaluate the prognostic significance of rituximab-related IRs relative to DLBCL subtype, International Prognostic Index score, c-Myc translocations or amplifications, chemotherapy regimen, and Ki-67 proliferative index. ResultsBaseline characteristics did not differ significantly between the 2 groups. Rituximab was included as initial treatment in all patients. Patients with an IR had a significantly higher overall survival (hazard ratio, 0.26; 95% confidence interval, 0.07-0.95) at 5 years. In addition, subgroup analysis showed a significantly higher progression-free survival in patients with the germinal center subtype of disease and c-Myc alterations who had a rituximab-related IR (log-rank P < .0001). ConclusionsThe presence of a rituximab-related IR is associated with a better overall survival in patients with DLBCL. Although limited by the small sample size and retrospective nature, these results provide rationale for further investigation into the mechanism of action of rituximab in order to optimize the efficacy of CD20 monoclonal antibodies.

Systemic and Intra-Nodal Activation of NK Cells After Rituximab Monotherapy for Follicular Lymphoma.

Monotherapy with the anti-CD20 monoclonal antibody rituximab can induce complete responses (CR) in patients with follicular lymphoma (FL). Resting FcRγIII+ (CD16+) natural killer (NK) cells respond strongly to rituximab-coated target cells in vitro. Yet, the contribution of NK cells in the therapeutic effect in vivo remains unknown. Here, we followed the NK cell repertoire dynamics in the lymph node and systemically during rituximab monotherapy in patients with FL. At baseline, NK cells in the tumor lymph node had a naïve phenotype albeit they were more differentiated than NK cells derived from control tonsils as determined by the frequency of CD56dim NK cells and the expression of killer cell immunoglobulin-like receptors (KIR), CD57 and CD16. Rituximab therapy induced a rapid drop in NK cell numbers coinciding with a relative increase in the frequency of Ki67+ NK cells both in the lymph node and peripheral blood. The Ki67+ NK cells had slightly increased expression of CD16, CD57 and higher levels of granzyme A and perforin. The in vivo activation of NK cells was paralleled by a temporary loss of in vitro functionality, primarily manifested as decreased IFNγ production in response to rituximab-coated targets. However, patients with pre-existing NKG2C+ adaptive NK cell subsets showed less Ki67 upregulation and were refractory to the loss of functionality. These data reveal variable imprints of rituximab monotherapy on the NK cell repertoire, which may depend on pre-existing repertoire diversity.

Therapy of nodal Follicular Lymphoma (WHO grade 1/2) in clinical stage I/II using response adapted Involved Site Radiotherapy in combination with Obinutuzumab (Gazyvaro) - GAZAI Trial (GAZyvaro and response adapted Involved-site Radiotherapy): a study protocol for a single-arm, non-randomized, open, national, multi-center phase II trial

BackgroundLarge field irradiation had been standard for early-stage follicular lymphoma (FL) for a long time. Although involved field radiotherapy (IF-RT) was recently favored because of the toxicity of large field irradiation, smaller irradiation fields have been accompanied with an increased risk of out-of-field recurrence. The MIR (MabThera® and Involved field Radiation) trial has shown that the combination of IF-RT at a dose of 30–40 Gy with the anti-CD20 antibody rituximab has led to similar efficacy compared with large field irradiation but with markedly reduced side effects. Immune modulating radiation therapy alone using low-dose radiotherapy (LDRT) of 2 × 2 Gy has been shown to be effective in FL. The GAZAI (GAZyvaro and response Adapted Involved-site Radiotherapy) trial aims to prove the efficacy of LDRT in combination with a novel anti-CD20 therapy.Methods/designThe GAZAI trial is a non-randomized, open, non-controlled, German, multi-center phase II trial that includes patients with early-stage (I and II) nodular FL (grades 1 and 2) confirmed by central histological review. A maximum of 93 patients will be included in the trial. Patients will receive a combined approach of immunotherapy with the fully humanized anti-CD20 antibody obinutuzumab (Gazyvaro®) and involved site radiotherapy (IS-RT) with 2 × 2 Gy. The primary endpoint of the trial is the rate of metabolic complete response (CR), based on fludeoxyglucose positron emission tomography/computed tomography, after obinutuzumab and 2 × 2 Gy IS-RT in week 18. Secondary endpoints are morphologic CR rate in weeks 7 and 18 and month 6, progression-free survival, toxicity, recurrence patterns, overall survival, and quality of life. Additionally, minimal residual disease response is assessed. The risk for a potentially higher recurrence rate after LDRT will be minimized by additional salvage radiation up to the “full dose” of 40 Gy for patients who have less than a metabolic CR and morphologic partial response/CR, which will be evaluated in week 18, offering a response-adapted approach.DiscussionThe goal of this trial is a further reduction of the radiation dose in patients with nodal early-stage FL showing a good response to a combination of LDRT and anti-CD20 immunotherapy and a comparison with the currently published MIR trial.Trial registrationEudraCT number: 2016-002059-89. ClinicalTrials.gov identifier: NCT03341520.

Expression of CD20 after viral reactivation renders HIV-reservoir cells susceptible to Rituximab

The identification of exclusive markers to target HIV-reservoir cells will represent a significant advance in the search for therapies to cure HIV. Here, we identify the B lymphocyte antigen CD20 as a marker for HIV-infected cells in vitro and in vivo. The CD20 molecule is dimly expressed in a subpopulation of CD4-positive (CD4+) T lymphocytes from blood, with high levels of cell activation and heterogeneous memory phenotypes. In lymph node samples from infected patients, CD20 is present in productively HIV-infected cells, and ex vivo viral infection selectively upregulates the expression of CD20 during early infection. In samples from patients on antiretroviral therapy (ART) this subpopulation is significantly enriched in HIV transcripts, and the anti-CD20 monoclonal antibody Rituximab induces cell killing, which reduces the pool of HIV-expressing cells when combined with latency reversal agents. We provide a tool for targeting this active HIV-reservoir after viral reactivation in patients while on ART.

Flow cytometry-based assessment of direct-targeting anti-cancer antibody immune effector functions.

Monoclonal antibody-based therapies are increasingly being used to treat cancer. Some mediate their therapeutic effects through modifying the function of immune cells globally, while others bind directly to tumor cells and can recruit immune effector cells through their Fc regions. As new direct-binding agents are developed, having the ability to test their Fc-mediated functions in a high-throughput manner is important for selecting antibodies with immune effector properties. Here, using monoclonal anti-CD20 antibody (rituximab) as an example and the CD20+ Raji cell line as tumor target, we describe flow cytometry-based assays for determining an antibody's capacity for mediating antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and complement-dependent cytotoxicity (CDC). These assays are sensitive, reliable, affordable and avoid the use of radioactivity.

Clinical Pharmacokinetic and Pharmacodynamic Considerations in Treating Non-Hodgkin Lymphoma.

Non-Hodgkin lymphoma (NHL) includes a variety of closely related malignancies that originate from lymphoid precursors. The majority of NHLs are of B-cell lineage, for which traditional therapy involves chemotherapy in combination with the anti-CD20 monoclonal antibody rituximab. Ongoing research into the pathogenesis of NHL subtypes has given rise to the use of novel agents that target specific molecular pathways. While the incidence of NHL extends over a range of ages from pediatric to elderly settings, the majority of diagnoses occur over age 60years. Increasing the use of concomitant medication coupled with declining organ function among this group of patients creates pharmacokinetic (PK) challenges in administering a number of agents involved in the treatment of NHL. In addition, since many of the new agents are administered orally, there are a number of added PK factors that must be taken into consideration with their prescribing and administration. This article will review the available literature on the PK and pharmacodynamic properties of agents commonly used in the treatment of NHL, and intends to provide information that can assist with properly using these drugs in this setting.

Variable loss of CD30 expression by immunohistochemistry in recurrent cutaneous CD30+ lymphoid neoplasms treated with brentuximab vedotin

Brentuximab vedotin is a monoclonal anti-CD30 antibody-drug conjugate that has been used to treat a variety of CD30+ neoplasms. The phenomenon of antigen loss has been observed in patients treated with the anti-CD20 antibody rituximab. This study seeks to assess for antigen loss in the setting of recurrent CD30+ neoplasms treated with brentuximab vedotin. We report nine cases of persistent/recurrent cutaneous CD30+ lymphoid neoplasms that demonstrated variable CD30 expression after treatment with brentuximab vedotin. Cases include MF (n = 6), cutaneous T-cell lymphoma, not otherwise specified (n = 1), and anaplastic large cell lymphoma (ALCL), both primary (n = 1) and systemic (n = 1). Immunohistochemical staining revealed decreased CD30 expression following brentuximab vedotin therapy in seven of nine cases. In these seven cases, the pre-treatment percent of tumor cells staining for CD30 ranged from 10% to 100% (mean 50.0%, SD 27.8%), compared to 5% to 50% (mean 14.5%, SD 14.8%, P = 0.003) at recurrence. This case series highlights the finding that CD30 positivity can be variable in recurrences after treatment with anti-CD30 antibodies. This serves to raise awareness of the phenomenon of antigen loss after treatment with brentuximab vedotin and underscores the utility of performing multiple biopsies and/or employing molecular diagnostic techniques in patients with recurrent/persistent disease.

Study protocol: multicenter double-blind, randomized, placebo-controlled trial of rituximab for the treatment of childhood-onset early-stage uncomplicated frequently relapsing or steroid-dependent nephrotic syndrome (JSKDC10 trial)

BackgroundEighty percent of children with idiopathic nephrotic syndrome respond well to steroid therapy, but up to 50% of patients with steroid-sensitive nephrotic syndrome exhibit frequently relapsing (FRNS) or steroid-dependent nephrotic syndrome (SDNS). Several studies identified the chimeric anti-CD20 monoclonal antibody rituximab as an effective treatment for patients with complicated FRNS/SDNS. Recent studies suggested that rituximab could also be a first-line treatment for early-stage uncomplicated FRNS/SDNS, although further studies are required to confirm its efficacy and safety.Methods/designWe are conducting a multicenter, double-blind, randomized placebo controlled trial to investigate the efficacy and safety of rituximab for the treatment of childhood-onset early-stage uncomplicated FRNS/SDNS. Patients will be allocated to receive two 375 mg/m2 doses (maximum dose: 500 mg) of either rituximab or placebo. Investigators are permitted to request the disclosure of a subject’s allocation code if he or she exhibits treatment failure. Additionally, if placebo-treated subjects display early relapse (a sign of treatment failure), they have the option to receive rituximab in an unblinded phase. The primary endpoint is relapse-free survival during the observation period.DiscussionThe results will provide important data on the use of rituximab for patients with uncomplicated FRNS/SDNS. In the future, rituximab treatment will enable most patients with uncomplicated FRNS/SDNS to discontinue or reduce steroid therapy without relapse, and it is possible that rituximab could represent an immunosuppressive therapy for these diseases.Trial registrationThis trial was prospectively registered to the JMACCT Clinical Trials Registry on September 6, 2018 (Trial ID: JMA-IIA00380).

Abstract 1523: Inhibition of SUMOylation by TAK-981 induces antitumor innate immune responses by modulating macrophage and NK cell function through Type I IFN pathway activation

Abstract TAK-981 is a novel and selective small molecule inhibitor of the small ubiquitin-like modifier (SUMO) ylation enzymatic cascade in Phase 1 clinical trials. SUMOylation is a reversible post-translational modification that regulates protein function by covalent attachment of a SUMO protein to protein substrates. We have found that in vivo inhibition with TAK-981 promotes an antitumor immune response characterized by induction of a Type I interferon (IFN) response in immune cells. Interestingly, others have also reported a role for SUMOylation in restraining Type I IFN responses, upon exposure to pathogenic stimuli, using genetic approaches to inhibit SUMOylation. In this study, we demonstrate that inhibition of SUMOylation with TAK-981 enhances antitumor innate immunity by modulating macrophage and NK cell function. In vitro studies revealed that TAK-981 treatment increased expression of pro-inflammatory markers and prevented expression of anti-inflammatory markers on macrophages. TAK-981 treatment of macrophages further resulted in potent phagocytic activity against cancer cells. TAK-981 also induced expression of activation markers on NK cells and enhanced cell killing activity against cancer cells. Enhancement of macrophage phagocytosis and NK cell cytotoxicity by TAK-981 were shown to be dependent on Type I IFN receptor signaling, and both activities against CD20-positive cancer cells were further enhanced in the presence of the anti-CD20 therapeutic monoclonal antibody rituximab. Evaluation of combination activity in vivo in CD20-positive lymphoma xenograft models demonstrated synergistic antitumor activity in multiple models when TAK-981 was combined with rituximab. Inhibiting SUMOylation with TAK-981 appears to represent a novel and mechanistically unique means to the end of leveraging the potential of Type I IFNs to promote antitumor immune responses. Citation Format: Akito Nakamura, Stephen Grossman, Keli Song, Neeraja Idamakanti, Gary Shapiro, Dennis Huszar. Inhibition of SUMOylation by TAK-981 induces antitumor innate immune responses by modulating macrophage and NK cell function through Type I IFN pathway activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1523.

Recurrent Proliferative Glomerulonephritis With Monoclonal Immunoglobulin Deposits in Kidney Allografts Treated With Anti-CD20 Antibodies.

Proliferative glomerulonephritis with monoclonal immunoglobulin deposits (PGNMID) is a distinct form of glomerulonephritis that often recurs after kidney transplantation causing severe graft injury and often failure. We describe post transplant outcomes and response to therapy in 20 recipients with PGNMID. Evidence of PGNMID recurrence or lack thereof was determined by protocol and clinical biopsies. Histologic recurrence (deposition of monoclonal immunoglobulin) occurred in 18 of 20 recipients (90%), a median of 7 (1 to 65) months post transplant. At diagnosis, recurrence was generally associated with mild or no clinical manifestations and often with mild glomerular morphologic changes by light microcopy. Four of the 18 patients with recurrence did not progress and were not treated. Another 4 patients with recurrences were treated with cyclophosphamide with or without plasmapheresis, and 2 of these grafts were lost from glomerulonephritis. Nine patients with recurrences were treated with anti-CD20 antibodies (rituximab) alone, resulting in improvements in estimated glomerular filtration rate (31.5 ± 16 versus 38.8 ± 13.3 mL/min/1.73 m, P = 0.011) and proteinuria (1280 [117 to 3752] versus 168 [83 to 1613] mg/24 h, P = 0.012) although complete clinical remission was rare. One graft in this later group was lost from recurrence 141 months post transplant. Posttreatment biopsies demonstrated stable or improved glomerular histology in most cases. However, PGNMID did not resolve in any case. Four patients received rituximab 4 months pretransplant to prevent recurrence. However, 3 had mild recurrences. Rituximab treatment of early PGNMID recurrence is effective, resulting in reasonable, long-term graft survival. Whether pretransplant rituximab modifies the course of recurrence requires additional studies.

Management of post-transplant lymphoproliferative disorders.

Management of post-transplant lymphoproliferative disorders.

One Size Does Not Fit All: Who Benefits From Maintenance After Frontline Therapy for Follicular Lymphoma?

Follicular lymphoma (FL) is the second most common non-Hodgkin lymphoma and the most common indolent B-cell malignancy. The disease often presents in advanced stage and can often be observed before initiation of therapy. Although the incidence is only approximately 15,000 new cases per year, the prevalence is substantially higher owing to the favorable overall survival (OS) of most patients. The most impactful advance responsible for the improvement of OS in FL was the introduction of the anti-CD20 monoclonal antibody (mAb) rituximab over 20 years ago. Phase III trials demonstrate that rituximab improves the OS in FL when combined with chemotherapy. However, unlike aggressive B-cell lymphomas, advanced stage FL is generally incurable and often displays a pattern of progressively shorter remissions with subsequent lines of therapy. Hence, maintenance strategies have been developed to prolong remissions achieved with frontline therapy. The value of maintenance after frontline therapy has been most extensively studied with extended treatment of anti-CD20 mAb, but recent approaches include chemotherapy-free combinations and targeted therapies given for extended durations. Here, we review relevant data that provide rationale in support of maintenance therapy in FL as well as the risks and limitations of a "one-size-fits-all" approach. Importantly, we note the biologic and clinical heterogeneity across patients with FL that must be considered when making clinical decisions. Finally, we highlight ongoing research that explores response-adapted approaches based on the depth of response as defined by PET scans and assays for minimal residual disease (MRD) that aim to better personalize individual management strategies.

B-Lymphocyte Depletion in Patients With Myalgic Encephalomyelitis/Chronic Fatigue Syndrome: A Randomized, Double-Blind, Placebo-Controlled Trial.

Previous phase 2 trials indicated benefit from B-lymphocyte depletion in myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). To evaluate the effect of the monoclonal anti-CD20 antibody rituximab versus placebo in patients with ME/CFS. Randomized, placebo-controlled, double-blind, multicenter trial. (ClinicalTrials.gov: NCT02229942). 4 university hospitals and 1 general hospital in Norway. 151 patients aged 18 to 65 years who had ME/CFS according to Canadian consensus criteria and had had the disease for 2 to 15 years. Treatment induction with 2 infusions of rituximab, 500 mg/m2 of body surface area, 2 weeks apart, followed by 4 maintenance infusions with a fixed dose of 500 mg at 3, 6, 9, and 12 months (n= 77), or placebo (n= 74). Primary outcomes were overall response rate (fatigue score ≥4.5 for ≥8 consecutive weeks) and repeated measurements of fatigue score over 24 months. Secondary outcomes included repeated measurements of self-reported function over 24 months, components of the Short Form-36 Health Survey and Fatigue Severity Scale over 24 months, and changes from baseline to 18 months in these measures and physical activity level. Between-group differences in outcome measures over time were assessed by general linear models for repeated measures. Overall response rates were 35.1% in the placebo group and 26.0% in the rituximab group (difference, 9.2 percentage points [95% CI, -5.5 to 23.3 percentage points]; P= 0.22). The treatment groups did not differ in fatigue score over 24 months (difference in average score, 0.02 [CI, -0.27 to 0.31]; P= 0.80) or any of the secondary end points. Twenty patients (26.0%) in the rituximab group and 14 (18.9%) in the placebo group had serious adverse events. Self-reported primary outcome measures and possible recall bias. B-cell depletion using several infusions of rituximab over 12 months was not associated with clinical improvement in patients with ME/CFS. The Norwegian Research Council, Norwegian Regional Health Trusts, Kavli Trust, MEandYou Foundation, and Norwegian ME Association.

Therapy of pemphigus

Pemphigus diseases are aheterogeneous group of potentially life-threatening autoimmune bullous disorders. Therefore, rapidly acting and effective therapeutic approaches are essential. In this review, current therapeutic options in line with available guidelines are presented and new therapeutic approaches are discussed. Aliterature search was performed using PubMed. Treatment of pemphigus is based on systemic glucocorticosteroids, frequently combined with potentially corticosteroid-sparing immunosuppressants such as azathioprine and mycophenolate mofetil/mycophenolic acid. Recently, the impressive efficacy of the anti-CD20 antibody rituximab has been shown in aprospective randomized trial. In severe or treatment-refractory cases, immunoadsorption or high-dose intravenous immunoglobulins (IVIG) are recommended. Adjuvant immunoadsorption also seems to be useful within the first 8-12weeks of therapy in patients with very high autoantibody levels. Avariety of new therapeutic approaches is currently evaluated in phase IIa studies. Therapy of pemphigus has been greatly improved by the employment of rituximab. The use of glucocorticosteroids, associated with ahigh number of adverse events and elevated mortality, could be reduced by the additional use of rituximab. After approval of rituximab for the treatment of pemphigus by the US Food and Drug Administration in 2018, licensing in Europe is expected in 2019.

Five-Year Results of an IgA and IgM-Enriched Human Immunoglobulin G-Based Therapy for Early Anti-HLA Donor Specific Antibodies in 158 Lung-Transplanted Patients

Purpose The development of anti-HLA donor specific antibodies early after lung transplantation (eDSA) is associated with antibody-mediated rejection (AMR) and poor graft survival. At our institution, since 2013, we have treated patients with eDSA with successive infusions (first infusion: 2gr/kg, then 0.5gr/kg every 4 weeks for a maximum of 6 months) of IgA and IgM-enriched human intravenous immunoglobulins G (Pentaglobin®, IgGAM), combined in some cases with a single dose of anti-CD20 antibody (Rituximab) and plasmapheresis (PE) or immunoabsorption. Aims of this study were to present the 5-year results of the IgGAM-based therapy and its impact on graft survival. Methods Records of patients transplanted at our institution between 03/2013 and 10/2018 were reviewed. Outcomes of patients with eDSA and treated with IgGAM (IgGAM group) and without eDSA(control group) were compared using the product-limit method of Kaplan-Meier and the log-rank test. Median (IQR) follow-up amounted to 29 (14, 48) months. Results During the study period, among the 705 transplanted patients, 158 (22%) patients formed the IgGAM group and 530 (75%) the control group. Among the remaining 17 (3%) patients, 10 patients developed eDSA but were not treated and 7 were treated only with PE and Rituximab, and thus were not considered in the study. Among the 158 IgGAM patients, 132 (84%) showed only eDSA (possible subclinical AMR) and were pre-emptively treated. The remaining 26 (16%) patients showed graft dysfunction concomitant with eDSA (possible clinical AMR). Median time to eDSA detection was 14 (11, 20) days after transplantation. As of October 2018, treatment was completed in 138 (87%) patients. IgGAM treatment cleared eDSA in 126 (91%) patients (median time 3 months), 15 (12%) patients showing eDSA recurrence at a median of 9 months after treatment end. During treatment time, freedom from biopsy-confirmed rejection (%) was 72 vs. 65 in IgGAM vs. control patients. At 5 years, graft survival (%) was 71 vs. 75 (p=0.74) and freedom from CLAD 84 vs. 70 (p=0.28) in IgGAM vs. control patients, respectively. Conclusion After lung transplantation, a IgGAM-based treatment for eDSA yielded high eDSA clearance. IgGAM patients showed improved 5-year CLAD-free survival in comparison to control patients. Graft survival was similar between groups.

Five-Year Experience with Treatment of Early Donor Specific Anti-HLA Antibodies in Pediatric Lung Transplant Recipients

Purpose Experience with treatment of early donor specific anti-HLA antibodies (eDSA) after lung transplantation in children is very limited. At our institution, since 2013, we have treated patients with eDSA with successive infusions (first infusion: 2gr/kg, then 0.5gr/kg every 4 weeks for a maximum of 6 months) of IgA and IgM-enriched human intravenous immunoglobulins (Pentaglobin, IgGAM), combined in some cases with a single dose of anti-CD20 antibody (Rituximab) and plasmapheresis (PE) or immunoabsorption. Aim of this study was to present the 5-year results of the IgGAM-based therapy in pediatric lung recipients. Methods Records of pediatric ( Results During the study period, among the 60 lung-transplanted pediatric patients (median age 13 years), 26 (43%) formed the IgGAM group and 33 (55%) the control group. One (2%) patient was treated only with PE and Rituximab and not considered in the study. Among the 26 IgGAM patients, 14 (54%) showed only eDSA (possible subclinical antibody-mediated rejection, AMR). The remaining 12 (46%) patients showed graft dysfunction concomitant with eDSA (possible clinical AMR). Median time to eDSA detection was 23 (14, 64) days after transplantation. As of October 2018, treatment was completed in 23 (88%) patients. Under IgGAM treatment eDSA cleared in 22 (96%) patients. At follow-up, 5 (20%) IgGAM vs. 5 (15%) control patients developed hypogammaglobulinemia requiring substitution (p=0.73). No IgGAM vs. 3 (10%) control patients developed post-transplant lymphoproliferative disease (p=0.25). At 5 years, graft survival (%) was 76 vs. 84 (p=0.78); freedom (%) from chronic lung allograft rejection (CLAD) was 88 vs. 75 (p=0.54), and from infection requiring hospitalization 53 vs. 23 (p= 0.021), in IgGAM vs. control patients, respectively. Conclusion After lung transplantation, an IgGAM-based treatment for eDSA yielded high eDSA clearance while decreasing the risk of severe infections. IgGAM patients showed 5-year CLAD-free survival at least as good as control patients without eDSA.

Effects of Ex Vivo Perfusion and Il-6 Receptor Blockade on Ischemia Reperfusion Injury in Cardiac Transplantation

Purpose In cardiac transplantation, ischemia reperfusion injury (IRI) remains a major problem associated with poor short and long-term outcomes. Since pro-inflammatory cytokines and endothelial activation have been hallmarks of IRI for decades, they can be addressed by recent therapeutic advancements. Normothermic ex vivo heart perfusion using the Organ Care System, but also pre-emptive treatment strategies for highly immunized recipients using the IL-6 receptor antibody tocilizumab, are in current clinical use. Therefore, we retrospectively studied blood samples from our clinical cardiac transplantation cohort for parameters of IRI. Methods In a novel protocol for heart transplantation (HTx) recipients with preformed donor-specific antibodies (pfDSA), an intraoperative application of tocilizumab (10mg/kg) was given together with 5 sessions of plasmapheresis, followed by one infusion of IgGAM (2g/kg) and a single dose of anti-CD20 antibody (rituximab). This protocol was applied to 6 recipients of OCS-preserved hearts. 10 recipients of OCS-preserved hearts without pfDSA did not receive tocilizumab (group 2). 24 recipients of standard heart transplants preserved on ice represent group 3. Donor or recipient demographics did not differ among the groups. Plasma levels of cytokines and endothelial markers were compared before, immediately after transplant surgery (T0), 24h (T24) and 3 weeks after transplantation. Results IRI could be measured in standard heart recipients at T0 and T24 by significantly increased levels of IL-6, CXCL8/IL-8, IL-18, Ang-2 and IGFBP-1 (all p Conclusion Normothermic ex vivo preservation in the OCS reduces IRI at the cytokine and endothelial level in recipient blood immediately after transplantation. Application of tocilizumab during transplantation blocks binding of IL-6 to its receptor leading to higher plasma levels. These effects may have a clinically relevant impact on ischemia reperfusion injury post cardiac transplantation.

Sustained B cell depletion by CD19-targeted CAR T cells is a highly effective treatment for murine lupus.

The failure of anti-CD20 antibody (Rituximab) as therapy for lupus may be attributed to the transient and incomplete B cell depletion achieved in clinical trials. Here, using an alternative approach, we report that complete and sustained CD19+ B cell depletion is a highly effective therapy in lupus models. CD8+ T cells expressing CD19-targeted chimeric antigen receptors (CARs) persistently depleted CD19+ B cells, eliminated autoantibody production, reversed disease manifestations in target organs, and extended life spans well beyond normal in the (NZB × NZW) F1 and MRL fas/fas mouse models of lupus. CAR T cells were active for 1 year in vivo and were enriched in the CD44+CD62L+ T cell subset. Adoptively transferred splenic T cells from CAR T cell-treated mice depleted CD19+ B cells and reduced disease in naive autoimmune mice, indicating that disease control was cell-mediated. Sustained B cell depletion with CD19-targeted CAR T cell immunotherapy is a stable and effective strategy to treat murine lupus, and its effectiveness should be explored in clinical trials for lupus.

Mutations resulting in the formation of hyperactive complement convertases support cytocidal effect of anti-CD20 immunotherapeutics

Anti-CD20 monoclonal antibodies (mAbs) rituximab and ofatumumab are potent activators of the classical complement pathway, and have been approved for the treatment of B-cell malignancies. However, complement exhaustion and overexpression of complement inhibitors by cancer cells diminish their therapeutic potential. The strategies of targeting membrane complement inhibitors by function-blocking antibodies and the supplementation with fresh frozen plasma have been proposed to overcome tumour cell resistance. We present a novel approach, which utilizes gain-of-function variants of complement factor B (FB), a component of alternative C3/C5 convertases, which augment mAb-activated reactions through a positive feedback mechanism called an amplification loop. If complement concentration is limited, an addition of quadruple gain-of-function FB mutant p.D279G p.F286L p.K323E p.Y363A (or selected single mutants) results in significantly increased complement-mediated lysis of ofatumumab-resistant tumour cells, as well as the complete lysis of moderately sensitive cells. Importantly, this effect cannot be achieved by further increasing ofatumumab concentration. Potentiation of cytotoxic effect towards moderately sensitive cells was less apparent at physiological serum concentration. However, an addition of hyperactive FB could compensate the loss of cytotoxic potential of serum collected from the NHL and CLL patients after infusion of rituximab. Residual levels of rituximab in such sera, in combination with added FB, were able to efficiently lyse tumour cells. We suggest that the administration of gain-of-function variants of FB can restore cytotoxic potential of complement-exhausted serum and maximize the therapeutic effect of circulating anti-CD20 mAbs.

Aptamers as quality control tool for production, storage and biosimilarity of the anti-CD20 biopharmaceutical rituximab

Detailed analysis of biopharmaceuticals is crucial for safety, efficacy and stability. Aptamers, which are folded, single-stranded oligonucleotides, can be used as surrogate antibodies to detect subtle conformational changes. We aimed to generate and assess DNA aptamers against the therapeutic anti-CD20 antibody rituximab. Six rituximab-specific aptamers with Kd = 354–887 nM were obtained using the magnetic bead-based systematic evolution of ligands by exponential enrichment (SELEX) technology. Aptamer folds were analysed by online prediction tools and circular dichroism spectroscopy suggesting quadruplex structures for two aptamers while others present B-DNA helices. Aptamer binding and robustness with respect to minor differences in buffer composition or aptamer folding were verified in the enzyme-linked apta-sorbent assay. Five aptamers showed exclusive specificity to the Fab-fragment of rituximab while one aptamer revealed a broader recognition pattern to other monoclonal antibodies. Structural differences upon incubation at 40 °C for 72 h or UV exposure of rituximab were uncovered by four aptamers. High similarity between rituximab originator and biosimilar lots was demonstrated. The most sensitive aptamer (RA2) detected signal changes for all lots of a copy product suggesting conformational differences. For the first time, a panel of rituximab-specific aptamers was generated allowing the assessment of conformational coherence during production, storage, and biosimilarity of different products.