Success Of Rituximab Research Articles

P23 Adult onset Still’s disease as a novel autoimmune complication of alemtuzumab therapy: a case report and review of alemtuzumab associated autoimmune disease

Abstract Background Alemtuzumab is a monoclonal antibody targeted to CD52, an antigen of uncertain function on T and B lymphocytes. Alemtuzumab is an established treatment for active relapsing and remitting multiple sclerosis (MS). However, autoimmune disease (AID) is being increasingly recognised as a highly prevalent adverse effect of alemtuzumab treatment. Methods We describe a case of adult onset Still’s disease (AOSD) developing in a patient six months after alemtuzumab therapy, highlighting the need for awareness of the risk of AID in those who develop symptoms after alemtuzumab treatment, some of which can be potentially life-threatening. Results Our patient, a 37 year old female, was admitted to hospital with a three week history of fevers up to 39ºC, a flitting macular rash, sore throat and polyarthritis, neutrophils 35.2x109/L (2-7.5), ferritin 8349 ug/L (normal 15-150), ALP 151 U/L (25-105). She had a history of relapsing and remitting multiple sclerosis, that went into remission after treatment with alemtuzumab six months previously. Despite 72 hours of broad-spectrum intravenous antibiotics, fever and symptoms were unchanged. Immunology and microbiology tests were negative. CT scan of neck, chest abdomen and pelvis were unremarkable. A diagnosis of AOSD was made. She received prednisolone at 40mg daily and symptoms rapidly settled. By day three of prednisolone, ferritin had fallen to 3238, neutrophil count had halved, and ALP normalised. She continues on slow weaning of steroids and remains asymptomatic. Conclusion Alemtuzumab was approved in 2013 as a treatment for active relapsing and remitting MS. Treatment consists of two courses of ‘induction’ treatment 12 months apart, aiming to deplete T and B lymphocytes, with subsequent lymphocyte repopulation. Without requirement for continued ‘maintainance’ treatment, efficacy of alemtuzumab persists for years. The most common alemtuzumab associated AID are thyroid disease, nephropathies and immune thrombocytopenia. However, other AID has been reported, including sarcoidosis. To date, no reports exist describing AOSD post alemtuzumab therapy. Although lymphocyte reconstitution post alemtuzumab is thought to account for its therapeutic effects, this same reconstitution is postulated to contribute to AID. Baker et. al. postulated that rapid re-population of B lymphocytes and slower re-population of regulatory T lymphocytes may trigger B cell mediated AID. Further, rapid repopulation of immature B cells post alemtuzumab is associated with raised serum B cell activating factor (BAFF), which remains elevated for at least a year post treatment. BAFF has been recognised in multiple animal studies to be associated with antibody mediated AID. The hypothesis that B cells play a key role in alemtuzumab related AID may be supported by the success of rituximab in treating 2 cases of alemtuzumab associated AID. The possible critical role of B cells in the pathogenesis of alemtuzumab related AID could indicate a future treatment strategy for such AID. Disclosures N. Narayan None. G. Mazibrada None. N. Amft None.

Rituximab in B-Cell Hematologic Malignancies: A Review of 20 Years of Clinical Experience.

Rituximab is a human/murine, chimeric anti-CD20 monoclonal antibody with established efficacy, and a favorable and well-defined safety profile in patients with various CD20-expressing lymphoid malignancies, including indolent and aggressive forms of B-cell non-Hodgkin lymphoma. Since its first approval 20 years ago, intravenously administered rituximab has revolutionized the treatment of B-cell malignancies and has become a standard component of care for follicular lymphoma, diffuse large B-cell lymphoma, chronic lymphocytic leukemia, and mantle cell lymphoma. For all of these diseases, clinical trials have demonstrated that rituximab not only prolongs the time to disease progression but also extends overall survival. Efficacy benefits have also been shown in patients with marginal zone lymphoma and in more aggressive diseases such as Burkitt lymphoma. Although the proven clinical efficacy and success of rituximab has led to the development of other anti-CD20 monoclonal antibodies in recent years (e.g., obinutuzumab, ofatumumab, veltuzumab, and ocrelizumab), rituximab is likely to maintain a position within the therapeutic armamentarium because it is well established with a long history of successful clinical use. Furthermore, a subcutaneous formulation of the drug has been approved both in the EU and in the USA for the treatment of B-cell malignancies. Using the wealth of data published on rituximab during the last two decades, we review the preclinical development of rituximab and the clinical experience gained in the treatment of hematologic B-cell malignancies, with a focus on the well-established intravenous route of administration. This article is a companion paper to A. Davies, et al., which is also published in this issue.FundingF. Hoffmann-La Roche Ltd., Basel, Switzerland.

The role of novel immunotherapies in non-Hodgkin lymphoma.

Immunotherapy is an evolving modality in the treatment of non-Hodgkin lymphoma. Vaccinations with patient-specific tumor-derived antigens have been developed to strengthen immune response to tumor. The success of rituximab, a monoclonal antibody for CD20 on malignant B-cells, fueled further immunotherapy research. The power of the immune system to fight hematologic malignancies is seen in allogeneic stem cell transplant, where donor T cells attack residual malignant cells in the recipient. Now, three innovative therapeutic immunotherapy classes (I) adoptive cellular therapy; (II) immune-checkpoint inhibitors; and (III) novel antibody therapies show promising results in non-Hodgkin lymphoma. Genetically engineered T cells, CAR T cells, obtained remissions in lymphomas refractory to conventional chemotherapy. Immune-checkpoint inhibitors, such as nivolumab and pembrolizumab revolutionized the treatment of many solid tumors, and unprecedented results are now reported in relapsed/refractory lymphoma. Building on the success of rituximab, additional therapeutic monoclonal antibodies were developed for lymphoma treatment. Antibodies have recently been further engineered with multiple binding sites to directly engage both tumor and T cells. There are exciting early clinical trial results for the first bispecific T-cell engager (BiTE), blinatumomab, as well as promising ongoing studies for dual antibody molecules, Dual-Affinity Re-Targeting (DART) proteins. This review highlights these three immunotherapy classes for relapsed/refractory non-Hodgkin lymphomas and discusses the mechanism of action, clinical efficacy, and toxicities of each.

Utilizing CD30 expression as a rational target for therapy of lymphoma

Rituximab, identified through pivotal lymphoma research, was the first monoclonal antibody approved by the US FDA in 1997. Since the success of rituximab, monoclonal antibodies have been a major focus for development of targeted agents for lymphoma treatment. A major hurdle in the development of a new antibody is finding a new target antigen. CD30 is an attractive therapeutic target antigen, because it has been identified as a marker of Reed–Sternberg cells in Hodgkin lymphoma (HL) [1], it is known to be expressed on anaplastic large cell lymphoma (ALCL), some cases of mediastinal large cell lymphoma, primary effusion lymphoma and multiple myeloma. However, its expression on normal tissues is restricted to a small number of activated B- and T-lymphocytes [2]. Thus, based on its expression pattern, CD30 could be an ideal therapeutic target.

Immunotherapy for B-cell lymphoma: current status and prospective advances.

Therapy for non-Hodgkin’s lymphoma has progressed significantly over the last decades. However, the majority of patients remain incurable, and novel therapies are needed. Because immunotherapy ideally offers target selectivity, an ever increasing number of immunotherapies, both passive and active, are undergoing development. The champion of passive immunotherapy to date is the anti-CD20 monoclonal antibody rituximab that revolutionized the standard of care for lymphoma. The great success of rituximab catalyzed the development of new passive immunotherapy strategies that are currently undergoing clinical evaluation. These include improvement of rituximab efficacy, newer generation anti-CD20 antibodies, drug-conjugated and radio labeled anti-CD20 antibodies, monoclonal antibodies targeting non-CD20 lymphoma antigens, and bispecific antibodies. Active immunotherapy aims at inducing long-lasting antitumor immunity, thereby limiting the likelihood of relapse. Current clinical studies of active immunotherapy for lymphoma consist largely of vaccination and immune checkpoint blockade. A variety of protein- and cell-based vaccines are being tested in ongoing clinical studies. Recently completed phase III clinical trials of an idiotype protein vaccine suggest that the vaccine may have clinical activity in a subset of patients. Efforts to enhance the efficacy of active immunotherapy are ongoing with an emphasis on optimization of antigen delivery and presentation of vaccines and modulation of the immune system toward counteracting immunosuppression, using antibodies against immune regulatory checkpoints. This article discusses results of the various immunotherapy approaches applied to date for B-cell lymphoma and the ongoing trials to improve their effect.

Comparative Assessment of Different Clinically Utilized CD20 Directed Antibodies in Chronic Lymphocytic Leukemia (CLL) Cells Reveals Divergent NK-Cell, Monocyte and Macrophage Properties,

Abstract 3717The anti-CD20 antibody rituximab represents the first therapy to contribute to prolongation of survival in CLL yet controversy exists as to how it promotes tumor elimination. Pre-clinical studies in CLL have suggested that innate immune cells, complement, and direct antibody killing might contribute to anti-CD20 antibody efficacy. Murine models of B-cell depletion by CD20 antibodies have shown monocytes to be the most important effector cell, although significant controversy around this point exists. Given the success of rituximab, second generation anti-CD20 antibodies (Ofatumumab and GA101 are now in phase III testing in CLL with reported improved direct (GA101), complement (Ofatumumab), or NK cell effector cell killing (GA101). To date, direct comparison of these three antibodies for direct, complement, and effector cell engagement has not been performed. Additionally, the impact of afucosylation engineering of GA101 on monocyte and macrophage function has not been reported. As a Type II anti-CD20 antibody, GA101 mediated significantly increased cell death (∼25%) without Fc crosslinking when compared to Ofatumumab or rituximab (5–8%). Direct cytotoxicity assessment of 19 pts with cross-linking demonstrated GA101 (25%) to mediate significantly greater (p=0.0003) killing than rituximab (15%) but not Ofatumumab (20%). Complement mediated killing was significantly increased over media with Ofatumumab (∼30%), whereas an average of only 5–10% killing was observed with rituximab or GA101. Immobilized GA101 significantly increased NK cell activation as detected by IFNg production and CD107a induction (p=0.005) more than rituximab or Ofatumumab. In addition, GA101 mediates 2 to 3 fold greater NK cell-mediated Antibody Dependent Cellular Cytotoxicity (ADCC) compared to Ofatumumab or rituximab at higher antibody concentrations, but not at concentrations less than 0.05 mg/ml. This enhanced ADCC with GA101 is seen with normal NK cells as well as NK cells from CLL patients. Ofatumumab mediated significantly greater (p=0.0001) NK cell ADCC than rituximab. Given the importance of macrophages in depletion of anti-CD20 tumors in mouse models of lymphoma, we next focused on this. The three anti-CD20 antibodies show Antibody Dependent Cellular Phagocytosis (ADCP) capability with Monocyte Derived Macrophages (MDM) against CLL B cells, with Ofatumumab exhibiting the greatest ADCP compared to GA101 (60% vs 40%; p=0.0036). In addition, primary monocytes stimulated with immobilized GA101 show less TNFa release, when compared to rituximab or Ofatumumab. To investigate this mechanistically, monocytes were stimulated with plate bound rituximab, Ofatumumab or GA101. Compared to the non Fc engineered antibodies (rituximab and Ofatumumab) Fc engineered GA101 induces reduced pan tyrosine phosphorylation and phosphorylation of ERK. No differences in phosphorylation of FcgRIIa or FcgRIIb was observed between the engineered and non-engineered antibodies. Further mechanistic studies to elucidate these differences in monocytes based upon antibody afucosylation changes, or differential phosphorylation of FcgR are ongoing. Collectively, our data indicate that GA101 and Ofatumumab are both superior to rituximab against CLL cells and have differential properties with respect to apoptosis, CDC, and effector cell-mediated killing. GA-101 mediates the most potent NK cell mediated killing at high concentrations whereas Ofatumumab has the greatest monocyte activation and phagocytosis despite absence of any engineering. These findings have relevance in the choice of the optimal CD20 antibody for treatment of CLL and combination strategies used. Disclosures:No relevant conflicts of interest to declare.

Ongoing Development of Monoclonal Antibodies and Antibody Drug-Conjugates in Lymphoma

Rituximab, a monoclonal antibody (mAb) directed against CD20, has changed practices in the treatment of patients with B-cell lymphoma. The large success of rituximab has contributed to validate immunotherapy with monoclonal antibodies as a valuable strategy in lymphoma. Recently, better-engineered anti-CD20-mAbs have been designed to improve efficacy and safety. Also, new antibodies targeting other lymphoma subtypes including T-cell lymphoma and Hodgkin's disease have been developed. Ongoing pharmacology development is concentrating on immuno-drug-conjugates combining the toxicity of chemotherapy, isotopes, or toxins with the specificity of mAbs. This article offers an overview on the development of new antibodies and immuno conjugates in lymphoma.

Conditions of tumor-associated antigens as a proper target for therapeutic antibodies against solid cancers

Since the success of rituximab and trastuzumab for treatment of non-Hodgkin's lymphoma and breast cancer, respectively, a huge therapeutic potential of monoclonal antibodies (mAbs) was realized and development of therapeutic mAbs has been widely tried against various cancers. However, the successful examples are still limited and therapeutic mAbs are not yet available for the majority of human cancers. We established a procedure for comprehensive identification of tumor-associated antigens (TAAs) through the extensive isolation of human mAbs that may become therapeutic. Thirty-twoTAAs have been identified and 555 mAbs that bound to one of the TAAs have been isolated to date. Now we are trying to select TAAs as proper targets for therapy and candidate mAbs as drugs from among them. The immunohistochemical analysis using many fresh lung cancer specimens suggested probabilities of proper targets, and moreover, presence of cancer-specific epitopes that could be distinguished from normal epitopes on the same molecules by mAbs. For Abs to efficiently kill the cancer cells they should have the ability to induce immunological cytotoxicity such as ADCC and/or CDC. They should also be able to inhibit the function mediated by the target Ags. For clinical point of view, the continuous presence of the target molecule on the cell surface until cell death might be essential for successful treatment. Therefore, it will be required for targets TAAs to play essential roles in tumorigenesis. Otherwise the cancer cells that do not express them could selectively survive during treatment and finally become dominant. It was also suggested that even the same molecules could play different roles in tumorigenesis quite often in different patients. Therefore when we develop therapeutic Abs, we should obtain information about the conditions of patients including genetic background to whom the treatment will be effective. I will discuss how we can accomplish this purpose.

CD20-Targeted Therapy: The Next Generation of Antibodies

Targeting the CD20 antigen on B lymphocytes with the monoclonal antibody (MoAb) rituximab has greatly improved the outcome of patients with B-cell malignancies. Despite the success of rituximab, resistance occurs in about half of the patients, resulting in non-response to treatment or early relapse with the original disease. A better understanding of the mechanism of rituximab resistance has led to the development of novel, improved anti-CD20 antibodies. This review describes the development of CD20-targeted therapy from its historical background towards the next generation of anti-CD20 MoAbs and explains new strategies to overcome resistance.

Rituximab for the treatment of patients with chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is a lymphoproliferative disorder that originates from antigen-experienced B lymphocytes that do not die and hence accumulate due to external survival signals or undergo apoptosis and are replenished by proliferating precursors. These neoplastic lymphocytes exhibit a characteristic immunophenotype of CD5(+)/CD19(+)/CD20(+)/HLA-DR+/CD23(+)/sIgdim. Thus, the CD20 antigen has been an appealing target for therapy. The introduction of the monoclonal antibody rituximab (anti-CD20) enabled an outstanding advance in CLL treatment. The introduction of this monoclonal antibody into chemotherapy regimens has dramatically improved complete response rates and progression-free survival in patients with both untreated and relapsed CLL. Although only preliminary data from phase III confirmatory trials have been reported, the FCR regimen, which combines fludarabine and cyclophosphamide with rituximab, is currently the most effective treatment regimen for CLL patients, and has also been demonstrated to significantly improve overall survival. The success of rituximab and the identification of other CLL lymphocyte surface antigens have spurred the development of a multitude of monoclonal antibodies targeting distinct proteins and epitopes in an attempt to target CLL cells more effectively.

Rituximab for the treatment of patients with chronic lymphocytic leukemia

Rituximab for the treatment of patients with chronic lymphocytic leukemia M Gentile, E Vigna, C Mazzone, E Lucia, AG Recchia, L Morabito2, MG Bisconte, C Gentile, F Morabito1UOC di Ematologia, Azienda Ospedaliera di Cosenza, Italy; 2Servicio de Hematología y Hemoterapia, Hospital Universitario de Canarias, La Laguna, Tenerife, SpainAbstract: Chronic lymphocytic leukemia (CLL) is a lymphoproliferative disorder that originates from antigen-experienced B lymphocytes that do not die and hence accumulate due to external survival signals or undergo apoptosis and are replenished by proliferating precursors. These neoplastic lymphocytes exhibit a characteristic immunophenotype of CD5+/CD19+/CD20+/HLA-DR+/CD23+/sIgdim. Thus, the CD20 antigen has been an appealing target for therapy. The introduction of the monoclonal antibody rituximab (anti-CD20) enabled an outstanding advance in CLL treatment. The introduction of this monoclonal antibody into chemotherapy regimens has dramatically improved complete response rates and progression-free survival in patients with both untreated and relapsed CLL. Although only preliminary data from phase III confirmatory trials have been reported, the FCR regimen, which combines fludarabine and cyclophosphamide with rituximab, is currently the most effective treatment regimen for CLL patients, and has also been demonstrated to significantly improve overall survival . The success of rituximab and the identification of other CLL lymphocyte surface antigens have spurred the development of a multitude of monoclonal antibodies targeting distinct proteins and epitopes in an attempt to target CLL cells more effectively.Keywords: rituximab, chronic lymphocytic leukemia, chemotherapy

Co-Treatment with Milatuzumab (Anti-CD74 mAb) and Rituximab (Anti-CD20 mAb) Results in the Induction of Mantle Cell Lymphoma Cell Death That Is Dependent On Actin Polymerization and Inhibition of NF-Kb.

Abstract 1694Poster Board I-720Mantle cell lymphoma (MCL) is an incurable B-cell malignancy and patients with this disease have limited therapeutic options. Despite the success of rituximab in treatment of B-cell lymphoma, its use as a single agent or in combination with chemotherapy in MCL has demonstrated modest activity; thus, novel strategies are needed. CD74 is an integral membrane protein expressed on malignant B cells and is implicated in promoting survival and growth, making it an attractive therapeutic target. The humanized anti-CD74 monoclonal antibody (mAb), milatuzumab, (Immunomedics) has shown promising preclinical activity against several human B-cell lymphoma cell lines, but has not been studied in MCL. Since rituximab and milatuzumab target distinct antigens lacking known association, we explored a combination strategy with these mAbs in MCL cell lines, patient samples, and in a preclinical model of MCL. Flow cytometric analysis shows that 6 different MCL cell lines (Mino, JeKo, SP53, Rec-1, Hbl2, Granta-519) and MCL patient primary tumor cells, express variable levels of CD74, with Mino cells showing highest level and Rec-1 the lowest. Incubation of the 6 MCL cell lines and primary cells (7 patients) with immobilized milatuzumab (5 μg/ml) and rituximab (10 μg/ml) resulted in mitochondrial depolarization and in statistically significant enhanced induction of apoptosis determined by Annexin V/PI and flow cytometry. The combination of both agents resulted in additive induction of apoptosis that was caspase independent in 5 MCL cell lines (synergistic in JeKo cells) and in primary cells, at 8, 24 and 48 hours. Importantly, while sensitivity to milatuzumab depends on the level of CD74 expression, the combination of milatuzumab and rituximab was able to induce enhanced cell death in all MCL cell lines and MCL primary cells, regardless of antigen density.We demonstrated that the combination of milatuzumab and rituximab induced enhanced apoptosis in a caspase-independent fashion with no apparent involvement of apoptotic key regulatory proteins such as Bax, Bcl-2, Bcl-Xl and Mcl-1. However, changes in the nuclear level of p65 were observed with either drug alone and with the combination, starting as early as 4 hours after treatment. The association of CD74 with MHC class II led us to explore pro-death mechanisms that become operable during HLA-DR-specific mAb treatment of lymphoma cells (Ivanov A et al., J Clin Invest 2009). We therefore investigated the role of actin polymerization by addition of cytochalasin D and latrunculin B, inhibitors of actin polimerization, prior to treatment with milatuzumab and/or rituximab. These studies showed that milatuzumab-induced MCL cell (Jeko and Mino) death was dependent on actin polymerization.To examine the in vivo activity of rituximab and milatuzumab, a preclinical model of human MCL using the SCID (CB17 scid/scid) mouse depleted of NK cells with TMβ1 mAb (anti-murine IL2Rb) was used. In this model, i.v. injection of 40×106 JeKo cells results in disseminated MCL 3 weeks after engraftment. The primary end-point was survival, defined as the time to develop cachexia/wasting syndrome or hind limb paralysis. Ten mice/group were treated starting at day 15 post-engraftment with intraperitoneal trastuzumab mAb control (300 μg qod), milatuzumab (300 μg qod), rituximab (300 μg qod), or a combination of milatuzumab and rituximab. The mean survival for the combination-treated group was 44.5 days (95%CI:39,51), compared to 28 days for trastuzumab-treated mice (95% CI:24,30), 33.5 days for the milatuzumab-treated mice (95% CI:28,36), and 38 days for the rituximab-treated mice (95%CI:36,42). The combination treatment prolonged survival of this group compared to trastuzumab control (P<0.0001), milatuzumab (P<0.0001) or rituximab (P=0.03). No overt toxicity from milatuzumab or the combination regimen was noted. These preliminary results provide justification for further evaluation of milatuzumab and rituximab in combination in MCL. DisclosuresOff Label Use: Milatuzumab for Mantle Cell Lymphoma Treatment. Goldenberg:Immunomedics, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

Peripheral CD19+ B cells are increased in children with active steroid-sensitive nephrotic syndrome.

Sir, Pathogenesis of steroid-sensitive nephrotic syndrome (SSNS) is thought to be related mainly to T-cell dysfunction [1]. However, the beneficial use of rituximab in cases of frequently relapsing SSNS provided evidence of B cell involvement [2–4]. Our aim was, thus, to investigate prospectively the levels of the circulating CD19+ B cells in children with a first episode of SSNS in sequential stages (presentation, remission on steroids and remission off steroids). Twenty-three children (M/F = 13/10, age = 2.5–14 years, median = 4.32 years) with a first episode of SSNS were studied; 23/23 both at presentation (before steroids initiation) and in remission on steroids (40 mg/m2 on alternate day); 15/23 were tested as well in remission off steroids for at least 6 months. Twenty-five age-matched children who had come to the outpatient haematology clinic in order to be tested for b-thalassaemia trait were found to be negative and served as healthy controls (controls 1). Considering that the presentation of SSNS may be associated with a recent infection, mainly a respiratory tract infection, twenty age-matched children with an upper respiratory tract infection acted as a second control group (controls 2). The percentages of CD3± T cells, as a pan T-cell marker, and the percentages of CD19+ and CD20± B cells were evaluated in all children. The above-mentioned parameters were determined in each sample by flow cytometry using the lysed whole blood method. The duochrome phycoerythrin-cyanin5 (PE-Cy5) conjugated CD3± monoclonal antibody (MoAb) and phycoerythrin (PE) conjugated CD19+ and CD20± MoAbs purchased from Beckman Coulter were used. The samples were analysed with a EPICS-XL flow cytometer. The results were expressed as percentages (%) of fluorescence-positive cells as well as actual numbers (cells/μL) of the circulating CD19+ and CD20+ B cells, based on the white blood cell count. Statistical analysis was performed using the Statistical Package for the Social Sciences for Windows (SPSS version 11.5). The paired t-test and independent-samples t-test were used to compare differences between study groups with and without paired data, respectively. Pearson's coefficient of correlation (r) was used to determine the correlations. A P ≤ 0.05 was considered to be statistically significant. In 5 of 23 children with a first episode of SSNS, there was a recent history of an upper respiratory tract infection. Remission was achieved in all children within 6–15 days after steroid initiation. During remission, all patients presented normoalbuminaemia and were free of proteinuria and albuminuria. Percentages of CD3± T cells were found to be within normal limits in all patients (at presentation of SSNS, in remission still on steroids and in remission off steroids) compared with the two control groups (P ≥ 0.05). As depicted in Figure ​Figure1,1, the circulating CD19+ B cells were significantly higher at presentation of SSNS (mean percentage = 18.13 ± 5.43, mean actual number = 695.34 ± 258.29) compared with remission on steroids (mean percentage = 13.57 ± 4.22 and P 0.05). Moreover, circulating CD19+ B cells were inversely correlated with disease activity (r = −0.465, P 0.05). Fig. 1 Percentage of CD19+ B cells in children with a first episode of SSNS in sequential stages (presentation, remission on steroids, remission off steroids) and in controls 1 and 2. The pathogenesis of SSNS has mainly been focused on T cells dysfunction. The success of rituximab, a chimeric anti-CD20 antibody, in the treatment of cases of frequently relapsing SSNS initiated interest in pathogenic pathways involving B cells [2–4]. There is an interaction between B cells and T cells, and B cells are involved in the presentation of antigens to T cells. However, the effect of B-cell depletion on T-cell function is unknown. Kemper et al. [5] suggested that in children with SSNS, there is a combined T- and B-cell activation. In agreement with our results, recent studies showed that CD19+ B cells may be elevated in relapsing nephrotic children [6,7]. Children with both steroid-sensitive and steroid-resistant nephrotic syndrome were included in this study, and there were no paired data of the circulating CD19+ B cells in remission off steroids. It is worth noting that in our study not all children with SSNS presented elevated CD19+ B cells. We intend to follow up this cohort of 23 nephrotic children in order to identify whether the long-term outcome of the nephrotic syndrome is associated with the up-regulation of CD19+ B cells in these patients taking into account steroid dependency and frequency of relapses. Our findings demonstrated an up-regulation of circulating CD19+ and CD20± B cells in children with a first episode of SSNS. This indicates that B cells may play a role in SSNS and that rituximab may be considered in certain cases. Conflict of interest statement. None declared.

Targeted therapy for chronic lymphocytic leukemia.

The introduction of targeted agents such as the monoclonal antibodies rituximab (anti-CD20) and alemtuzumab (anti-CD52) has brought about a remarkable change in the therapy of chronic lymphocytic leukemia (CLL). Although it is unclear whether overall survival has been improved, the incorporation of these monoclonal antibodies into chemoimmunotherapy regimens has dramatically improved complete response rates and progression-free survival in patients with both newly-diagnosed and relapsed CLL. The success of rituximab and alemtuzumab has spurred the development of other monoclonal antibodies targeting distinct proteins and epitopes on the surface of CLL cells and an exciting array of novel immunotherapeutics. The judicious use of these agents provides the opportunity to develop risk-adapted therapeutic strategies to optimize responses and quality of life in patients with CLL.

Garden of therapeutic delights: new targets in rheumatic diseases

Advances in our understanding of the cellular and molecular mechanisms in rheumatic disease fostered the advent of the targeted therapeutics era. Intense research activity continues to increase the number of potential targets at an accelerated pace. In this review, examples of promising targets and agents that are at various stages of clinical development are described. Cytokine inhibition remains at the forefront with the success of tumor necrosis factor blockers, and biologics that block interleukin-6 (IL-6), IL-17, IL-12, and IL-23 and other cytokines are on the horizon. After the success of rituximab and abatacept, other cell-targeted approaches that inhibit or deplete lymphocytes have moved forward, such as blocking BAFF/BLyS (B-cell activation factor of the tumor necrosis factor family/B-lymphocyte stimulator) and APRIL (a proliferation-inducing ligand) or suppressing T-cell activation with costimulation molecule blockers. Small-molecule inhibitors might eventually challenge the dominance of biologics in the future. In addition to plasma membrane G protein-coupled chemokine receptors, small molecules can be designed to block intracellular enzymes that control signaling pathways. Inhibitors of tyrosine kinases expressed in lymphocytes, such as spleen tyrosine kinase and Janus kinase, are being tested in autoimmune diseases. Inactivation of the more broadly expressed mitogen-activated protein kinases could suppress inflammation driven by macrophages and mesenchymal cells. Targeting tyrosine kinases downstream of growth factor receptors might also reduce fibrosis in conditions like systemic sclerosis. The abundance of potential targets suggests that new and creative ways of evaluating safety and efficacy are needed.

Combination Anti-CD74 (Milatuzumab) and CD20 (Rituximab) Monoclonal Antibody Therapy Has in Vitro and in Vivo Activity in Mantle Cell Lymphoma

Mantle cell lymphoma (MCL) is an incurable B-cell malignancy and patients with this disease have limited therapeutic options. Despite the success of Rituximab in treatment of B-cell malignancies, its use as a single agent or in combination with chemotherapy in MCL has demonstrated modest activity; thus, novel strategies are needed. CD74 is an integral membrane protein expressed on malignant B cells and implicated in promoting survival and growth, making it an attractive therapeutic target. The humanized anti-CD74 monoclonal antibody (mAb), Milatuzumab, (Immunomedics) has shown promising preclinical activity against several human B-cell lymphoma cell lines, but has not been studied in MCL. Since Rituximab and Milatuzumab target distinct antigens lacking known association, we explored a combination strategy with these mAbs in MCL cell lines, patient samples, and in a preclinical model of MCL. Flow cytometric analysis shows that the MCL cell lines Mino and JeKo, and MCL patient tumor cells, express abundant surface CD74 compared to the CD74-negative cell line, Jurkat. Incubation of Mino and JeKo cells with immobilized (goat anti-human IgG) Milatuzumab (5 μg/ml) resulted in mitochondrial depolarization and significant induction of apoptosis determined by Annexin V/PI and flow cytometry (apoptosis at 8hr=38.3±0.85% and 25.4±2.6%; 24hr=73.6±3.47% and 36±3.57%; 48hr=84.9±3.91% and 50.4±4.17%, respectively, compared to Trastuzumab (control). Expression of surviving cells from anti-CD74-treated MCL cells consistently demonstrated marked induction of surface CD74 (MFI 762) compared to control (MFI 6.1). Incubation with immobilized Rituximab (10 μg/ml) resulted in 39.5±2.5% and 37.1±8.35% apoptotic events at 8hr, 58.8±3.14%, 41.2±8.27% at 24hr, and 40.1±1.3% and 45.6±3.25% at 48hr, respectively. Combination treatment of Mino and JeKo cells with Milatuzumab and Rituximab led to significant enhancement in cell death, with 77.6±3.95% and 79.6±2.62% apoptosis at 8hr in Jeko and Mino cells (P=0.0008 and P=0.00004 vs. Milatuzumab alone; P=0.00015 and P=0.001 vs. Rituximab alone); 90.4±3.53% and 76.6±4.3% at 24hr, respectively (P=0.0042 and P=0.0002 vs. Milatuzumab, P=0.0003 and P=0.0027 vs. Rituximab alone); 92.8±0.77% and 85.6±2.62% at 48hr, respectively (P= 0.026 and P=0.0002 vs. Milatuzumab alone, P=0.0000005 and P=0.00008 compared to Rituximab alone, respectively). To examine the in vivo activity of Rituximab and Milatuzumab, a preclinical model of human MCL using the SCID (cb17 scid/scid) mouse depleted of NK cells with TMβ1 mAb (anti-murine IL2Rb) was used. In this model, intravenous injection of 40×106 JeKo cells results in disseminated MCL 3–4 weeks after engraftment. The primary end-point was survival, defined as the time to develop cachexia/wasting syndrome or hind limb paralysis. Mice were treated starting at day 17 postengraftment with intraperitoneal Trastuzumab mAb control (300 μg qod), Milatuzumab (300 μg qod), Rituximab (300 μg qod), or a combination of Milatuzumab and Rituximab. The mean survival for the combination-treated group was 55 days (95%CI:41, upper limit not reached as study was terminated at day 70), compared to 33 days for Trastuzumab-treated mice (95% CI:31,34), 35.5 days for the Milatuzumab-treated mice (95% CI:33,37), and 45 days for the Rituximab-treated mice (95%CI:30,46). The combination treatment prolonged survival of this group compared to Trastuzumab control (P=0.001), Milatuzumab (P=0.0006) and Rituximab (P=0.098). No overt toxicity from Milatuzumab or the combination regimen was noted. A confirmatory study with a larger group of mice and detailed mechanistic studies are now underway. These preliminary results provide justification for further evaluation of Milatuzumab and Rituximab in combination in MCL.

Therapie bösartiger Hämoblastosen mit monoklonalen Antikörpern

The success of rituximab, the first monoclonal antibody ever licensed for the treatment of a human malignancy, has not only increased survival and cure rates in many non-Hodgkin lymphomas of the B-cell type, but has prompted an explosion in the development of novel antibodies and biologically active substances with specific cellular targets ("targeted therapy") both in the field of hematological malignancies and solid tumors. The chimeric anti-CD20 monoclonal antibody rituximab is the first drug that was shown to increase overall survival in follicular lymphomas and to cut lymphoma-associated deaths in patients with CD20+ aggressive lymphomas into half. In this review the current role of monoclonal antibodies in the treatment of different hematological neoplasms will be discussed and perspectives for their future use in leukemia and lymphomas will be shown.

Rituximab in Warfarin Resistance Treatment in Patients with Thrombophilia Due to Primary Antiphospholipid Syndrome: A Pilot Study.

Introduction: The inability to obtain a therapeutic value of the INR regardless of the warfarin high doses administration represents a significant problem to resolve in the oral anticoagulation therapy, this phenomena is called “Warfarin Resistance”. Based on the experience and success of Rituximab in the treatment of autoimmune diseases like Immune Thrombocytopenic Purpura, we treated 7 patients with Primary Antiphospholipid Syndrome (PAS) and Warfarin Resistance.Objective: To assess if Rituximab can revert the warfarin resistance in patients with thrombophilia associated to PASMaterial and Methods: We evaluated 7 patients with PAS who were receiving 15 mg of warfarin and had persistent low INR values. Rituximab was given weekly at dose of 375 mg/m2 for 4 doses. Effectiveness and safety were registered weekly during 2 months since the start of treatment with clinical evaluation and INR, aPTT, warfarin serum levels and CD20 blood lymphocytes count determinations.Results: All patients were women with a mean of 35 years old (37– 48) and INR of 1.11 (0.79–1.50). All the patients (100%) reached the therapeutic level of INR between days 8 to 15, but the response lasted only a mean of 24 days (8 – 40). The maximum INR values were observed through the 2nd to the 3rd doses of Rituximab. The warfarin serum levels did not change during the assessment period.Conclusion: Rituximab was effective in reverting resistance to Warfarin therapy in patients with thrombosis for primary Antiphospholipid Syndrome, however it lasted a very short time and eventually all patients relapsed.

Complement-Induced Cell Death by Rituximab Depends on CD20 Expression Level and Acts Complementary to Antibody-Dependent Cellular Cytotoxicity

The use of the CD20-specific antibody rituximab has greatly improved the response to treatment of CD20+ follicular lymphoma. Despite the success of rituximab, resistance has been reported and prognostic markers to predict individual response are lacking. The level of CD20 expression on tumors has been related to response, but results of several studies are contradictory and no clear relationship could be established. Complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) are thought to be important effector mechanisms, but the exact mechanism of rituximab-mediated cell kill is still unknown. Importantly, no data have been reported on the combined contribution of CDC and ADCC. We have developed a system of clonally related CEM-CD20 cells by retroviral transfer of the human CD20 cDNA (n = 90). This set of cells, with the CD20 molecule as the only variable, was used to study the importance of CD20 expression level on rituximab-mediated CDC, ADCC, and the combination. We show a sigmoidal correlation of CD20 expression level and rituximab-mediated killing via CDC but not ADCC. On both high and low CD20-expressing cells, all CD20 molecules were translocated into lipid rafts after rituximab binding. Furthermore, CDC and ADCC act simultaneously and CDC-resistant cells are sensitive to ADCC and vice versa. These findings suggest that CDC depends on CD20 expression level and that both CDC and ADCC act complementary. These data give new insights into novel strategies to improve the efficacy of CD20-specific antibodies for the treatment of CD20+ tumors.

B cells as therapeutic targets for rheumatic diseases.

Trials treating human rheumatic diseases with biologic agents and drugs that selectively affect B cells will be reviewed. Rituximab, an anti-CD20 monoclonal mouse/human antibody, will be the primary focus of the review because it has been widely used in several autoimmune and rheumatic conditions, but the limited studies on other reagents such as anti-BlyS, anti-CD154, and B cell tolerogens will also be covered. The single most important recent development was the completion of a randomized, double-blind, placebo-controlled trial of rituximab in methotrexate-resistant rheumatoid arthritis. In this trial, B cell depletion with rituximab led to a sustained clinical response with an impressive improvement in America College of Rheumatology 50% response (ACR 50) at both 24 and 48 weeks. Additional open studies of rituximab showing clinical benefit in systemic lupus erythematosus, cryoglobulinemia, antineutrophil cytoplasmic antibodies+ vasculitis, and dermatomyositis are noteworthy but must be interpreted with caution until randomized control trials are available. Two well-designed studies of anti-CD154 antibodies in systemic lupus erythematosus were reported. Unfortunately, one was halted because of unexpected vascular complications, and the other failed to show any beneficial clinical effect. A phase I study using anti-BlyS in SLE demonstrated a selective effect on B cells and no overt toxicity, but in this very short-term study no effect on serology or clinical activity was seen. Two B cell tolerogens have been used in human trials. The first tolerogen, directed at anti-dsDNA responses in SLE, did significantly decrease titers of high-affinity anti-dsDNA antibodies but had no clinically beneficial effect overall. A phase I trial of a tolerogen directed at anti-beta2-glycoprotein I antibodies demonstrated a decrease in antibody titers after a single injection. Several therapeutic agents targeting B cells have now been tested or are being tested in human trials. The success of rituximab in a well-controlled trial confirms previous preliminary reports indicating that B cell depletion can treat established autoimmune disease.