Monoclonal anti-CD20 Antibody Research Articles

Cancer Immunotherapy in Diffuse Large B-Cell Lymphoma.

Remarkable progress has been made in the field of cancer immunotherapy in the past few years. Immunotherapy has become a standard treatment option for patients with various cancers, including melanoma, lymphoma, and carcinomas of the lungs, kidneys, bladder, and head and neck. Promising immunotherapy approaches, such as chimeric antigen receptor (CAR) T cell therapy and therapeutic blockade of immune checkpoints, in particular cytotoxic T lymphocyte-associated protein 4 (CTLA-4) and programmed cell death protein 1 pathway (PD-1/PD-L1), have boosted the development of new therapeutic regimens for patients with cancer. Immunotherapeutic strategies for diffuse large B-cell lymphoma (DLBCL) include monoclonal anti-CD20 antibody (rituximab), monoclonal anti-PD-1 antibodies (nivolumab and pembrolizumab), monoclonal anti-PD-L1 antibodies (avelumab, durvalumab, and atezolizumab) and chimeric antigen receptor (CAR) T cell therapy. In this review, we outline the latest highlights and progress in using immunotherapy to treat patients with DLBCL, with a focus on the therapeutic blockade of PD-1/PD-L1 and CAR T cell therapy in DLBCL. We also discuss current clinical trials of PD-1/PD-L1 and CAR T cell therapy and review the challenges and opportunities of using immunotherapy for the treatment of DLBCL.

Anti-CD52 antibody treatment depletes B cell aggregates in the central nervous system in a mouse model of multiple sclerosis

BackgroundMultiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system (CNS) for which several new treatment options were recently introduced. Among them is the monoclonal anti-CD52 antibody alemtuzumab that depletes mainly B cells and T cells in the immune periphery. Considering the ongoing controversy about the involvement of B cells and in particular the formation of B cell aggregates in the brains of progressive MS patients, an in-depth understanding of the effects of anti-CD52 antibody treatment on the B cell compartment in the CNS itself is desirable.MethodsWe used myelin basic protein (MBP)-proteolipid protein (PLP)-induced experimental autoimmune encephalomyelitis (EAE) in C57BL/6 (B6) mice as B cell-dependent model of MS. Mice were treated intraperitoneally either at the peak of EAE or at 60 days after onset with 200 μg murine anti-CD52 vs. IgG2a isotype control antibody for five consecutive days. Disease was subsequently monitored for 10 days. The antigen-specific B cell/antibody response was measured by ELISPOT and ELISA. Effects on CNS infiltration and B cell aggregation were determined by immunohistochemistry. Neurodegeneration was evaluated by Luxol Fast Blue, SMI-32, and Olig2/APC staining as well as by electron microscopy and phosphorylated heavy neurofilament serum ELISA.ResultsTreatment with anti-CD52 antibody attenuated EAE only when administered at the peak of disease. While there was no effect on the production of MP4-specific IgG, the treatment almost completely depleted CNS infiltrates and B cell aggregates even when given as late as 60 days after onset. On the ultrastructural level, we observed significantly less axonal damage in the spinal cord and cerebellum in chronic EAE after anti-CD52 treatment.ConclusionAnti-CD52 treatment abrogated B cell infiltration and disrupted existing B cell aggregates in the CNS.

Daratumumab induces CD38 internalization and impairs myeloma cell adhesion

ABSTRACTDaratumumab (Dara), a human immunoglobulin G1 kappa (IgG1κ) monoclonal anti-CD38 antibody, has been approved by the U.S. Food and Drug Administration for the treatment of relapsed multiple myeloma (MM) as a single agent as well as in combination with immunomodulatory drugs (IMiDs) and proteasome inhibitors (PI). Although the scientific rationale behind the use of Dara in combination with IMiDs has been extensively explored, the molecular mechanisms underlying Dara-PI regimens have not yet been investigated. Here, we demonstrate that CD38 on the surface of MM cells is rapidly internalized after Dara treatment; we also show that Dara treatment impairs MM cell adhesion, an effect that can be rescued by using the endocytosis inhibitor Dynasore. Finally, we show that Dara potentiates bortezomib (BTZ) killing of MM cells in vitro and in vivo, independent of its function as an immune activator. In conclusion, our data show that Dara impairs MM cell adhesion, which results in an increased sensitivity of MM to proteasome inhibition.

Therapeutic blockade of CD3-mediated signaling ameliorates memory Th2-driven allergic asthma

Abstract Allergic asthma is an inflammatory disease of the lungs characterized by Type 2 helper T cell (Th2)-driven immune responses to inhaled allergens. Memory Th2 cells are a major driving force behind asthma, secreting cytokines like IL-4, IL-5, and IL-13 as well as persisting in the host long after allergen encounter. The selective removal of pre-existing memory Th2 cells could be a key process resulting in enhanced tolerance. One such method could involve targeting CD3 through the use of monoclonal anti-CD3 antibodies, which have been shown to successfully induce tolerance in animal models of autoimmune diseases and have also shown promise in clinical trials. We tested blocking CD3 complex signaling in a HDM model of allergic inflammation, to determine if we could induce airway tolerance. Importantly, pre-sensitized C57BL/6J mice displayed strongly ablated lung inflammation and Th2 responses when treated at the time of memory T cell reactivation with F(ab′)2 anti-CD3 antibody. This treatment was found to specifically inhibit HDM-specific CD4+ T cell proliferation and Th2 cytokine production. Interestingly, mice treated with anti-CD3 also showed an increased frequency of Treg cells, improving the Treg:Th2 cell ratio in favor of airway tolerance. Additionally, a significant reduction in airway inflammation and Th2 responses was maintained with therapeutic anti-CD3 antibody even when given several months later during a further memory T cell recall response to allergen. These data suggest that blocking signaling mediated by the CD3 complex could be employed to inhibit memory recall responses to complex allergens thus promoting airway tolerance.

Deciphering the Role of B Cells in Multiple Sclerosis-Towards Specific Targeting of Pathogenic Function.

B cells, plasma cells and antibodies may play a key role in the pathogenesis of multiple sclerosis (MS). This notion is supported by various immunological changes observed in MS patients, such as activation and pro-inflammatory differentiation of peripheral blood B cells, the persistence of clonally expanded plasma cells producing immunoglobulins in the cerebrospinal fluid, as well as the composition of inflammatory central nervous system lesions frequently containing co-localizing antibody depositions and activated complement. In recent years, the perception of a respective pathophysiological B cell involvement was vividly promoted by the empirical success of anti-CD20-mediated B cell depletion in clinical trials; based on these findings, the first monoclonal anti-CD20 antibody—ocrelizumab—is currently in the process of being approved for treatment of MS. In this review, we summarize the current knowledge on the role of B cells, plasma cells and antibodies in MS and elucidate how approved and future treatments, first and foremost anti-CD20 antibodies, therapeutically modify these B cell components. We will furthermore describe regulatory functions of B cells in MS and discuss how the evolving knowledge of these therapeutically desirable B cell properties can be harnessed to improve future safety and efficacy of B cell-directed therapy in MS.

1036TiP - Phase 2, multicenter, single-arm, open label study evaluating the combination of daratumumab + cyclophosphamide, bortezomib and dexamethasone (Dara-CyBorD) in previously untreated and relapsed patients (pts) with multiple myeloma (MM)

Background: Dara is a monoclonal anti-CD38 antibody approved for the treatment of relapsed and refractory MM. Addition of dara to bortezomib and dexamethasone increased complete and overall response rates and improved progression-free survival (PFS) in pts with relapsed MM. CyBorD is a bortezomib based regimen which has been shown to be an effective therapy for MM. This study was designed to evaluate the combination of Dara-CyBorD in pts with MM who are previously untreated or have relapsed MM following only one line of prior therapy. Trial design: This is a multicenter, single-arm, open label, Phase 2 study in pts with MM who have received ≤ 1 line of prior therapy. Approximately 100 pts (≥40 with untreated MM and ≥40 with relapsed MM) will receive Dara-CyBorD every 28 days for 4 to 8 cycles. Pts receive oral cyclophosphamide 300 mg/m2 on Days 1, 8, 15, and 22; subcutaneous bortezomib 1.5 mg/m2 on Days 1, 8, and 15; and oral or IV dexamethasone 40 mg weekly. Dara is administered concurrently on 28 day cycles at a dose of 8 mg/kg IV on Days 1 and 2 of Cycle 1, then 16 mg/kg IV weekly from Cycle 1 Day 8 through completion of Cycle 2. For Cycles 3-6, pts receive dara 16 mg/kg IV once every 2 weeks. From Cycle 7 onward, pts receive dara 16 mg/kg IV once every 4 weeks, whether with CyBorD or alone during the maintenance phase. Pts receive 4 to 8 induction cycles of Dara-CyBorD and eligible pts may undergo an autologous stem cell transplant. All eligible pts then receive 12 cycles of maintenance therapy with dara 16 mg/kg IV every 28 days. The primary endpoint is complete response (CR) plus very good partial response (CR+VGPR) following 4 cycles of induction therapy with Dara-CyBorD. Secondary endpoints include overall response rate (CR+VGPR+PR), time to VGPR and partial response, duration of response, PFS, overall survival rate, infusion reaction profile of split-dose infusions and safety. Inclusion criteria include ≥ 18 years of age; documented MM per IMWG 2015 criteria; an Eastern Cooperative Oncology Group performance status score of 0, 1, or 2; no or one prior line of therapy. The estimated primary endpoint analysis date is February 2018. Clinical trial identification: NCT02951819 Legal entity responsible for the study: Janssen Scientific Affairs, LLC Funding: Janssen Scientific Affairs, LLC Disclosure: M. Sharma, L. Ey, H. Parros, S. Murphy, M. Darif, A. Londhe, J. Ukropec, M. Qi, Y. Lutska, T. Lin, S. Gunawardena: Author discloses employment with Janssen and Johnson & Johnson stock ownership.

Immunohistochemical, lectin histochemical and ultrastructural studies of canine transmissible venereal tumor in Brazil

ABSTRACT: Canine transmissible venereal tumor (CTVT) is a naturally occurring contagious round-cell neoplasia, with poorly understood origin and transmission. This study aims to further investigate the tumor nature through immunohistochemistry, lectin histochemistry and transmission electron microscopy (TEM) analysis, and to provide support for diagnostic and differential diagnoses of CTVT. Immunohistochemistry was performed in 10 genital and six exclusively extragenital tumors, which were previously diagnosed by citology and histopathology. CTVT samples were incubated with biotinylated antibodies to specific membrane and cytoplasmic antigens (anti-lysozyme, anti-macrophage, anti-vimentin, anti-CD18, monoclonal anti-CD117, monoclonal anti-CD3, polyclonal anti-CD117, polyclonal CD3 and anti-CD79a), followed by the avidin-biotin-peroxidase complex technique. The lectins Con A, DBA, SBA, PNA, UEA-1, WGA, sWGA, GSL, JSA, PSA, PHA-L, PHA-E and RCA were additionally tested in four genital CTVTs and TEM was performed in eight genital tumors. The anti-vimentin antibody revealed strong immunoreactivity to neoplastic cells in all the assessed samples (16/16). The polyclonal anti-CD3 antibodies showed moderate to strong immunoreactivity in fourteen (14/16) and the polyclonal anti-CD117 in fifteen cases (15/16). There was no immunoreactivity to anti-lysozyme, anti-macrophage, anti-CD18, monoclonal anti-CD117, monoclonal anti-CD3 and anti-CD79a antibodies. At lectin histochemistry, it was observed strong staining of tumor cells to Con-A, PHA-L and RCA. There was no histopathological and immunoreactivity differences between genital and extragenital CTVTs. These findings do not support the hypothesis of histiocytic origin of CTVT. In contrast, the lectin histochemical results were similar to cells from lymphoid/myeloid origin.

CT-P10 (Truxima™): A Rituximab Biosimilar.

CT-P10 (Truxima™) is the first biosimilar of the reference monoclonal anti-CD20 antibody rituximab. It is approved for use in all indications for which reference rituximab is approved, including follicular lymphoma (FL), diffuse large B-cell non-Hodgkin's lymphoma, chronic lymphocytic leukaemia, rheumatoid arthritis (RA), granulomatosis with polyangiitis and microscopic polyangiitis. CT-P10 has similar physicochemical and pharmacodynamic properties to those of reference rituximab, and the pharmacokinetic biosimilarity of the agents has been shown in patients with RA or FL. CT-P10 demonstrated clinical efficacy equivalent to that of reference rituximab in patients with RA, and was generally well tolerated in this population as well as in patients with FL. The tolerability, immunogenicity and safety profiles of CT-P10 were similar to those of reference rituximab, and switching from reference rituximab to CT-P10 had no impact on safety or efficacy. The role of reference rituximab in the management of autoimmune conditions and cancers is well established and CT-P10 provides an effective biosimilar alternative for patients requiring rituximab therapy.

Anterior ST-elevation myocardial infarction induced by rituximab infusion: A case report and review of the literature

Rituximab is a chimeric monoclonal anti-CD20 antibody approved for the treatment of some lymphoid malignancies as well as for autoimmune diseases including rheumatoid arthritis (RA), idiopathic thrombocytopenic purpura (ITP) and vasculitis. Generally, rituximab is well tolerated; nevertheless, some patients develop adverse effects including infusion reactions. Albeit rare, these reactions may in some cases be life-threatening conditions. Rituximab cardiovascular side effects include more common effects such as hypertension, oedema and rare cases of arrhythmias and myocardial infarction. In this article, we report a case of a 58-year-old man with a history of overlap syndrome including RA and limited scleroderma who was treated with rituximab and developed a dramatic ST-elevation myocardial infarction (STEMI) during the drug administration. This report underlines previous published reports emphasizing the awareness of such an association. This communication also warrants the importance of screening for ischaemic heart disease in selected cases of patients treated with rituximab.

Rituximab for immunologic renal disease: What the nephrologist needs to know.

Rituximab (RTX), a chimeric, monoclonal anti-CD20 antibody, is increasingly used in immune-mediated renal diseases. While licensed in the induction treatment of ANCA-associated vasculitis, it represents one of the most commonly prescribed off-label drugs. Much of the information regarding its safety has been drawn from experience in hematology and rheumatology. Ample evidence illustrates the safety of RTX, however, rare but serious adverse events have emerged that include progressive multifocal leucoencephalopathy and hepatitis B reactivation. Moderate to severe hypogammaglobulinemia and late-onset neutropenia following RTX therapy confer an increased infectious risk and factors predicting these side effects (i.e. a genetic basis) need to be identified. Nephrologists initiating RTX need to bear in mind that long-term risks and optimal dosing for many renal indications remain unclear. Special considerations must be given when RTX is used in women of childbearing age. We summarize practical aspects concerning the use of RTX. This review will provide nephrologists with information to guide their use of RTX alerting them to safety risks and the need for patient counselling.

Granulomatose mit Polyangiitis

Granulomatosis with polyangiitis (GPA) is a potentially life-threatening, rare disease. The etiology is unknown. GPA is histomorphologically characterized by extravascular necrotizing granulomatous inflammation and a systemic necrotizing vasculitis of small to medium-sized vessels. Clinically, a pulmonary-renal syndrome with pulmonary infiltrates, alveolar hemorrhage and a rapidly progressive glomerulonephritis is seen in about 80% of the cases with generalized disease. GPA is associated with proteinase 3-specific anti-neutrophil cytoplasmic autoantibodies (PR3-ANCA). Treatment is guided by severity of organ involvement and disease activity. Cytostatic immunosuppressants or the monoclonal anti-CD20 antibody rituximab are applied.

CD20 role in pathophysiology of Hodgkin's disease.

Hodgkin's lymphoma (HL) is a tumor comprising non-malignant and malignant B-cells. Classical HL expresses CD15+ and CD30+ antigens, and 20 to 40% of patients are CD20+. This antigen is a ligand free protein present in B lymphocyte cells and its function is not well known. Some studies suggest that expression of CD20 may play a major role in Hodgkin's disease pathophysiology and may affect the patients' treatment prognosis, as well as relapse and refractory response. In the past few years, development of monoclonal anti-CD20 antibodies changed drastically the treatment for non-Hodgkin lymphomas in which CD20 is expressed. HL treatment is essentially composed of radiotherapy and chemotherapy; however, monoclonal anti-CD20 antibodies applicability is not well delimitated due to lack of information about clinical outcomes with anti-CD20 monotherapy or combined drug therapy using a classic regimen, as well as about CD20 pathophysiology mechanisms in B-cells tumors. The objective of our review is to discuss CD20 function in Hodgkin's lymphoma development, its influence on disease evolution and outcomes, as well as its effects on therapeutics and patients' prognostic.

Daratumumab (Anti-CD38) interference with serological testing: An emerging challenge for blood banks in developing countries

Daratumumab (DARA), a monoclonal anti-CD38 antibody, belongs to the new generation of immunotherapy in refractory relapsed multiple myeloma. CD38 is weakly expressed on human erythrocytes. By its intrinsic anti-CD38 activity, DARA also interferes in routine pretransfusion compatibility testing such as antibody screening for red blood cells (RBCs) alloantibodies and compatibility testing. Treating RBCs with dithiothreitol eliminates the DARA interference. We report two cases of serological interference of DARA in pretransfusion testing and how timely information before starting the second patient on DARA prevented the delay in pretransfusion compatibility testing and blood availability.

Low-dose rituximab as an adjuvant therapy in pemphigus.

Pemphigus is a chronic autoimmune blistering disease where systemic steroids and immunosuppressants are the mainstay of therapy, but long-term treatment with these agents is associated with many side effects. Rituximab, a chimeric monoclonal anti-CD20 antibody, in low doses has shown efficacy as an adjuvant to reduce the dose of steroids. To study the clinical efficacy and safety of low-dose rituximab as an adjuvant therapy in pemphigus. Fifty patients with extensive pemphigus were selected, who either had recalcitrant pemphigus, were steroid dependent, had relapsed after pulse therapy, had anti-desmoglein levels >20, had contraindications to conventional treatment or wanted to avoid conventional treatment and its side effects. Two doses of rituximab (500 mg) were given 2 weeks apart and patients were regularly followed up every 2 weeks for 3 months and then monthly upto 2 years. Complete blood counts, liver function tests, renal function tests, skin biopsy, direct immunofluorescence and desmoglein levels were checked before and after rituximab administration. Pre-rituximab chest X-ray and electrocardiograph were also obtained. At 3 months, 41 (82%) patients showed complete remission. Nine (18%) patients had partial remission. After 6-12 months, 20 (40% of enrolled patients) continued to be in remission and were off all systemic therapy and the remaining 19 (38%) were continuing to take low doses of steroids with or without other adjuvant immunosuppressants and 2 (4%) had to be given another 2 doses of rituximab and subsequently could be managed with low-dose steroids. Of the 9 patients in partial remission at 3 months, after 6-12 months 5 (10% of the total) were completely off treatment and went into complete remission and 4 (8%) were on additional treatment out of which 2 (4%) had to be given 2 additional doses of rituximab and were in partial remission with low-dose therapy at the end of 12 months. One patient developed urticaria as a side effect. Another developed herpes zoster. Our results show that low-dose rituximab is a well-tolerated and beneficial adjuvant therapy in recalcitrant pemphigus which helps reduce both the severity of disease as well as the dose of steroids and immunosuppressants.

BAFF and CD4 T-Cells Are Major Survival Factors for Splenic Plasma Cells in B Cell Depletion Context: Implications for Autoimmune Diseases

The use of monoclonal anti-CD20 antibody (Rituximab) has greatly improved the treatment of B-cell mediated autoimmune diseases, albeit with variable outcomes. Our previous data in humans suggested that Rituximab induced paradoxically the settlement of splenic long-lived plasma cells (LLPC) in the context of 2 autoimmune cytopenia, immune thrombocytopenia and warm autoimmune hemolytic anemia (1) (2). The presence of splenic autoreactive LLPC explained the failure of Rituximab treatment. To investigate whether this mechanism could have a general relevance and decipher the cellular and molecular mechanism of this process, we used both non auto-immune and auto-immune mouse models.We have taken advantage of the knock-in transgenic mouse model AID-CreERT2-EYFP, which allows the irreversible expression of EYFP in B cells engaged in a germinal center-dependent immune response after tamoxifen regimen, to follow plasma cells (PC) at different times of immunization by sheep red blood cells, and upon anti-CD20 regimen (clone 18B12, Biogen Idec), in the spleen and bone marrow (3).By using a set of diagnostic genes that allowed us to distinguish short-lived and long-lived plasma cells, we compared the transcriptional program by multiplex PCR of EYFP+ B220- PC from controls and anti-CD20 treated mice, immunized and analyzed at the same time, corresponding to the nadir of B-cell depletion. While splenic PC of untreated mice displayed an intermediate profile between short-lived and long-lived plasma cells, splenic PC from anti-CD20 treated mice composed a homogeneous population that displayed a more mature program, similar to the one of natural long-lived bone marrow PC. The absolute number of splenic EYFP+ B220- did not change upon anti-CD20 treatment indicating that B-cell depletion promoted PC differentiation rather than a long-lived PC selection. We identified BAFF (B-cell activating factor) as a major player of this process. Indeed, as described in human spleens, we observed that BAFF level was increased in the supernatants of splenocytes after B-cell depletion. Above all, combination of anti-CD20 and anti-BAFF (clone 10F4, GSK) antibodies dramatically reduced the number of splenic EYFP+B220- LLPC (decrease >5 fold compared with anti-CD20 and control groups, P < 0.001). Targeting BAFF had no major impact on protective long-lived bone marrow PC as IgG1 level in the sera remained unchanged after combination therapy. We identified neutrophils as the main source of BAFF production in the spleen. Finally, CD4+ T-cells also appeared to play a key role in context of B-cell depletion for supporting plasma cell survival in the spleen as they appeared to closely interact with EYFP+ plasma cells by confocal microscopy. Moreover, their depletion (clones YTS 191.1 or GK 1.5, Bioxcell) in vivo induced a significant decrease in the number of splenic LLPCs (decrease > 2 fold compared with anti-CD20 group, P < 0.05). To assess whether B-cell depletion could also modify the splenic plasma cell program in an auto-immune context characterized by an ongoing immune response, we used NZB/NZW mice that spontaneously develop a disease closely resembling human systemic lupus. In line with our previous findings, anti-CD20 treatment also promoted the differentiation of LLPC in the spleen of the NZB/NZW model, while a treatment combining anti-CD20 with anti-BAFF induced a marked reduction in total PC numbers(decrease > 3 fold compared with anti-CD20 group, P < 0.05).In conclusion, the process of PC maturation upon anti-CD20 treatment appeared to be a general mechanism, both in non auto-immune and auto-immune models. We identified BAFF and CD4+ T-cells as key factors in the splenic environment responsible for the emergence of such LLPC. Finally, our results suggest that interfering with the plasma cell survival niche with monoclonal anti-BAFF antibody at the time of B-cell depletion might greatly improve the response rate in B-cell mediated auto-immune cytopenia.(1) Mahevas M, et al, Journal of Clinical Investigation , 2013(2) Mahevas M, et al, Journal of Autoimmunity, 2015(3) Dogan I, et al, Nature Immunology, 2009 DisclosuresNo relevant conflicts of interest to declare.

Elimination of FVIII-Specific B Cells By Immunotoxins Comprised of a Single FVIII Domain Fused to Pseudomonas Exotoxin a

The development of inhibitory antibodies (inhibitors) against coagulation factor VIII (FVIII) is the most serious complication for patients with hemophilia A that undergo FVIII replacement therapy. In addition, healthy individuals can spontaneously develop inhibitory anti-FVIII auto-antibodies, which results in acquired hemophilia A. The current standard therapy for patients with hemophilia A and inhibitors, named immune tolerance induction (ITI), is based on frequent and mostly high dose administrations of FVIII. Unfortunately, the eradication of inhibitors can only be achieved in about 70% of patients. Alternative treatment of inhibitor patients with the monoclonal anti-CD20 antibody rituximab results in complete eradication of inhibitors; however, depletion of the entire CD20-positive B cell population is potentially accompanied by severe side effects. Recent studies in hemophilic FVIII knockout mice showed that the application of a FVIII-toxin conjugate resulted in (i) prevention of inhibitor development in naïve mice and (ii) long-term eradication of inhibitors in FVIII-immunized mice.As the use of FVIII for cell targeting of immunotoxins is presumably limited by its high molecular weight (250 kDa) and adhesiveness (off-target reactivity) we explored the potential use of alternative immunotoxins in the current study. The introduced immunotoxins are comprised of a single FVIII domain fused to the Exotoxin A (ETA) from Pseudomonas aeruginosa.The rationale for the use of a single domain instead of full length FVIII as cell-binding component is that immunodominant domains like A2 and C2 might still allow targeting of sufficient amounts of FVIII-specific B-cells by immunotoxins. For proof of concept studies, we generated a histidine-tagged C2 domain-ETA fusion protein (C2-ETA) that was bacterially expressed and purified by affinity chromatography. Purified C2-ETA was recognized by a panel of commercially available monoclonal anti-C2 antibodies in ELISA suggesting proper folding of the C2 domain in the bacterially expressed protein.To test the capacity of C2-ETA to eliminate FVIII-specific B-cells, splenocytes of FVIII-immunized FVIII knockout mice were re-stimulated with FVIII ex vivo in presence and absence of different concentrations of C2-ETA and ETA alone (as control). Re-stimulation of FVIII-specific memory B cells to FVIII- and C2-specific antibody secreting cells (ASC) was analyzed in anEnzyme linked immunospot (ELISPOT) assay using FVIII and C2 as antigens. While differentiation to FVIII-specific ASC was only partially inhibited by C2-ETA, differentiation to C2-specific ASC was completely blocked in a dose-dependent manner. In contrast, the use of ETA alone had no effect. Further analysis of the FVIII domain specificity of antibodies in plasma of FVIII-immunized FVIII knockout mice used for depletion studies revealed a strong contribution of C2-specific antibodies to the overall FVIII-specific immune response.In summary, our results show that the developed C2-ETA immunotoxin is able to specifically eliminate FVIII C2 domain-specific B cells ex vivo. Currently, C2-ETA is tested for its capacity to eliminate FVIII-specific B cells in FVIII knockout mice and additional FVIII domain-ETA immunotoxins are developed. DisclosuresNo relevant conflicts of interest to declare.

Hematologic Responses and Cardiac Organ Improvement in Patients with Heavily Pretreated Cardiac Immunoglobulin Light Chain (AL) Amyloidosis Receiving Daratumumab

▪Introduction: In immunoglobulin light chain (AL) amyloidosis, cardiac involvement is the primary cause of premature death. Light chain suppression, with therapies targeting the underlying plasma cell clone producing amyloidogenic free light chains, has been difficult to achieve in a relapsed/refractory disease setting. Hematologic response is required to obtain a cardiac organ response, which is predictive of survival and is an important, if not primary, therapeutic goal. We have previously reported rapid and favorable hematologic response rates with the monoclonal anti-CD38 antibody daratumumab in a cohort of heavily pretreated relapsed/refractory AL patients. The aim of this study was to evaluate cardiac organ response following light chain suppressive therapy with daratumumab in patients with relapsed/refractory AL.Materials & Methods:Consecutive patients with biopsy-proven AL and cardiac involvement, followed at the Stanford University Amyloid Center, who received daratumumab were retrospectively evaluated for hematologic and cardiac organ response. In accordance with IRB approval, demographic and clinical information was obtained from medical records. Hematologic and cardiac organ response criteria were defined per consensus guidelines in AL (Comenzo et al, Leukemia 2012).Results: Twelve patients with previously treated AL with cardiac involvement received a median of 12 doses (range 5-18) of single agent daratumumab. The antibody was given intravenously at 16 mg/kg weekly for 8 weeks, followed by every other week infusion for 8 doses and then monthly infusions. The median patient age was 67 and 75% of patients were male. The median number of lines of prior therapy was 3; notably, none of the patients had previously achieved a hematologic complete response to prior therapy including high dose melphalan and autologous stem cell transplant in 2 patients. Ten of 12 patients (83%) achieved a partial hematologic response or better with daratumumab (3 complete responses (25%), 3 very good partial responses (25%), and 4 partial responses (33%)). Median NT-pro BNP was 2516 pg/mL prior to daratumumab therapy. Of all 12 treated patients, seven patients were evaluable for cardiac response based on baseline NT-proBNP >650 ng/L. Of these, 3 patients achieved a cardiac organ response by NT-pro BNP criteria (>30% reduction and >300 ng/l decrease). Two patients had cardiac progression by NT-pro BNP criteria (no echocardiographic progression was observed) despite hematologic response with one patient discontinuing therapy to pursue hospice care. Infusion reactions were observed in 8/12 patients with only 1 grade 3 infusion reaction.Conclusions: Daratumumab yielded rapid and significant hematologic responses in our retrospective single institution cohort of heavily pretreated AL patients. At a median daratumumab duration of therapy of only 4 months, evidence of cardiac organ improvement was observed. Daratumumab represents a well tolerated and exceptionally promising new treatment for patients with AL amyloidosis; larger prospective trials to evaluate this agent are warranted. DisclosuresLiedtke:Takeda: Consultancy, Research Funding; Prothena: Consultancy, Research Funding; Celgene: Research Funding; Amgen: Consultancy, Research Funding; Novartis: Research Funding; Gilead: Research Funding; Pfizer: Consultancy, Research Funding.

Oligofructose as an adjunct in treatment of diabetes in NOD mice.

In type 1 diabetes, restoration of normoglycemia can be achieved if the autoimmune attack on beta cells ceases and insulin requirement is met by the residual beta cells. We hypothesize that an adjunctive therapy that reduces insulin demand by increasing insulin sensitivity will improve the efficacy of an immunotherapy in reversing diabetes. We tested the gut microbiota-modulating prebiotic, oligofructose (OFS), as the adjunctive therapy. We treated non-obese diabetic mice with an immunotherapy, monoclonal anti-CD3 antibody (aCD3), with or without concurrent dietary supplement of OFS. After 8 weeks of OFS supplement, the group that received both aCD3 and OFS (aCD3 + OFS) had a higher diabetes remission rate than the group that received aCD3 alone. The aCD3 + OFS group had higher insulin sensitivity accompanied by reduced lymphocytic infiltrate into the pancreatic islets, higher beta-cell proliferation rate, higher pancreatic insulin content, and secreted more insulin in response to glucose. The addition of OFS also caused a change in gut microbiota, with a higher level of Bifidobacterium and lower Clostridium leptum. Hence, our results suggest that OFS can potentially be an effective therapeutic adjunct in the treatment of type 1 diabetes by improving insulin sensitivity and beta-cell function, leading to improved glycemic control.

Efficacy and safety of rituximab therapy in patients with pemphigus vulgaris: first report from Turkey.

Pemphigus vulgaris (PV) is a severe, chronic, and potentially life-threatening autoimmune blistering disease that affects the skin and mucous membranes. Rituximab is a monoclonal anti-CD20 antibody which has been used increasingly in the therapy of PV. The present study sought to test the efficacy and safety of rituximab as an adjuvant therapy by retrospective analysis of clinical and immunological data for 29 patients with PV who were treated with rituximab between 2010 and 2015. Response to therapy, duration of clinical remission, serology of the response, and adverse effects of rituximab were evaluated. The mean ± standard deviation (SD) follow-up time was 17.48 ± 13.18 months. In all patients, findings showed either a decrease in antibody titers or that antibodies were completely undetectable after therapy. Rituximab use resulted in a significant reduction in steroid dosage during follow-up. At the end of the follow-up period, 26 patients (96.2%) had achieved complete remission with or without therapy (one patient had no follow-up and one patient had died, most probably as the result of a thromboembolic event). In 44.4% of patients, a clinical relapse occurred after a mean ± SD period of 13.1 ± 4.7 months after the initiation of rituximab therapy. Relapses were managed with additional infusions of rituximab. Rituximab is a beneficial and relatively safe adjuvant treatment for PV that facilitates prolonged clinical remission and has a significant steroid-sparing effect.

B-Cell Depletion Promotes Aortic Infiltration of Immunosuppressive Cells and Is Protective of Experimental Aortic Aneurysm.

B-cell depletion therapy is widely used for treatment of cancers and autoimmune diseases. B cells are abundant in abdominal aortic aneurysms (AAA); however, it is unknown whether B-cell depletion therapy affects AAA growth. Using experimental models of murine AAA, we aim to examine the effect of B-cell depletion on AAA formation. Wild-type or apolipoprotein E-knockout mice were treated with mouse monoclonal anti-CD20 or control antibodies and subjected to an elastase perfusion or angiotensin II infusion model to induce AAA, respectively. Anti-CD20 antibody treatment significantly depleted B1 and B2 cells, and strikingly suppressed AAA growth in both models. B-cell depletion resulted in lower circulating IgM levels, but did not affect the levels of IgG or cytokine/chemokine levels. Although the total number of leukocyte remained unchanged in elastase-perfused aortas after anti-CD20 antibody treatment, the number of B-cell subtypes was significantly lower. Interestingly, plasmacytoid dendritic cells expressing the immunomodulatory enzyme indole 2,3-dioxygenase were detected in the aortas of B-cell-depleted mice. In accordance with an increase in indole 2,3-dioxygenase+ plasmacytoid dendritic cells, the number of regulatory T cells was higher, whereas the expression of proinflammatory genes was lower in aortas of B-cell-depleted mice. In a coculture model, the presence of B cells significantly lowered the number of indole 2,3-dioxygenase+ plasmacytoid dendritic cells without affecting total plasmacytoid dendritic cell number. The present results demonstrate that B-cell depletion protects mice from experimental AAA formation and promotes emergence of an immunosuppressive environment in aorta.