B cell block: is rituximab a new possible treatment for systemic sclerosis?

There is no treatment for fibrotic diseases, including the autoimmune disease systemic sclerosis (sclerderma, SSc). Although broad spectrum immune suppressants have little to no effect on the fibrosis in SSc, agents targeting specific proinflammatory agents are currently being considered as possible therapeutic approaches to combating SSc. B cells are lymphocytes that proliferate and secrete antibody molecules which drive the autoimmune response. CD20 is a B cell marker; the agent rituximab is an antibody against CD20. In a recent report by Bosello and colleagues (Arthritis Res. Ther. 12(2): R54, 2010), rituximab was tolerated in SSc patients and appeared to result in an improvement of the skin score and of clinical symptoms of SSc. This report is one of a series of recent studies suggesting that rituximab may be a possible treatment for SSc. This commentary summarizes these observations.     

Expression and biological characterization of an anti-CD20 biosimilar candidate antibody: A case study

The CD20 molecule is a non-glycosylated protein expressed mainly on the surface of B lymphocytes. In some pathogenic B cells, it shows an increased expression, thus becoming an attractive target for diagnosis and therapy. Rituximab is a chimeric antibody that specifically recognizes the human CD20 molecule. This antibody is indicated for the treatment of non-Hodgkin lymphomas and autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus. In this work, we describe the stable expression and biological evaluation of an anti-CD20 biosimilar antibody. While rituximab is produced in fed-batch culture of recombinant Chinese hamster ovary (CHO) cells, our biosimilar antibody expression process consists of continuous culture of recombinant murine NS0 myeloma cells. The ability of the purified biosimilar antibody to recognize the CD20 molecule on human tumor cell lines, as well as on peripheral blood mononuclear cells from humans and primates, was demonstrated by flow cytometry. The biosimilar antibody induced complement-dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity and apoptosis on human cell lines with high expression of CD20. In addition, this antibody depleted CD20-positive B lymphocytes from peripheral blood in monkeys. These results indicate that the biological properties of the biosimilar antibody compare favorably with those of the innovator product, and that it should be evaluated in future clinical trials.     

Expression and biological characterization of an anti-CD20 biosimilar candidate antibody

The CD20 molecule is a non-glycosylated protein expressed mainly on the surface of B lymphocytes. In some pathogenic B cells, it shows an increased expression, thus becoming an attractive target for diagnosis and therapy. Rituximab is a chimeric antibody that specifically recognizes the human CD20 molecule. This antibody is indicated for the treatment of non-Hodgkin lymphomas and autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus. In this work, we describe the stable expression and biological evaluation of an anti-CD20 biosimilar antibody. While rituximab is produced in fed-batch culture of recombinant Chinese hamster ovary (CHO) cells, our biosimilar antibody expression process consists of continuous culture of recombinant murine NS0 myeloma cells. The ability of the purified biosimilar antibody to recognize the CD20 molecule on human tumor cell lines, as well as on peripheral blood mononuclear cells from humans and primates, was demonstrated by flow cytometry. The biosimilar antibody induced complement-dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity and apoptosis on human cell lines with high expression of CD20. In addition, this antibody depleted CD20-positive B lymphocytes from peripheral blood in monkeys. These results indicate that the biological properties of the biosimilar antibody compare favorably with those of the innovator product, and that it should be evaluated in future clinical trials.     

Rituximab inhibits Kv1.3 channels in human B lymphoma cells via activation of FcγRIIB receptors

Kv1.3 channels play an important role in modulating lymphocyte proliferation and apoptosis. We hypothesized that Kv1.3 channels in B lymphocytes might be regulated by rituximab, an antibody to CD20, a drug for treatments of B-cell lymphomas and autoimmune diseases. Using both whole-cell and cell-attached patch-clamp techniques, we found that rituximab inhibited Kv1.3 channels in Daudi human B lymphoma cells by promoting the channel inactivation at a concentration which was much greater than that required for activation of CD20. The effect of rituximab on Kv1.3 channels was abolished after selective blockade of FcγRIIB receptors with anti-FcγRIIB antibody. Western blot experiments showed that Daudi B cells expressed both Kv1.3 channel and the low affinity Fc receptor, FcγRIIB, which could be activated by the Fc region of rituximab. In contrast, normal lymphocytes expressed less Kv1.3 channels with faster inactivation. Confocal microscopy and flow cytometry data showed that rituximab induced apoptosis of Daudi B cells and that the effect was attenuated by blockade of FcγRIIB receptors and partially mimicked by inhibition of Kv1.3 channels. These results suggest that in addition to previously described complement-dependent cytotoxicity, rituximab also induces apoptosis of malignant B lymphocyte by stimulating FcγRIIB receptors and inhibiting Kv1.3 channels.     

Interleukin-21 Enhances Rituximab Activity in a Cynomolgus Monkey Model of B Cell Depletion and in Mouse B Cell Lymphoma Models

Rituximab, a monoclonal antibody targeting CD20 on B cells, is currently used to treat many subtypes of B cell lymphomas. However, treatment is not curative and response rates are variable. Recombinant interleukin-21 (rIL-21) is a cytokine that enhances immune effector function and affects both primary and transformed B cell differentiation. We hypothesized that the combination of rIL-21 plus rituximab would be a more efficacious treatment for B cell malignancies than rituximab alone. We cultured human and cynomolgus monkey NK cells with rIL-21 and found that their activity was increased and proteins associated with antibody dependent cytotoxicity were up-regulated. Studies in cynomolgus monkeys modeled the effects of rIL-21 on rituximab activity against CD20 B cells. In these studies, rIL-21 activated innate immune effectors, increased ADCC and mobilized B cells into peripheral blood. When rIL-21 was combined with rituximab, deeper and more durable B cell depletion was observed. In another series of experiments, IL-21 was shown to have direct antiproliferative activity against a subset of human lymphoma cell lines, and combination of murine IL-21 with rituximab yielded significant survival benefits over either agent alone in xenogeneic mouse tumor models of disseminated lymphoma. Therefore, our results do suggest that the therapeutic efficacy of rituximab may be improved when used in combination with rIL-21.     

SLE - Rituximab in lupus

B cells are essential to the development of systemic lupus erythematosus (SLE). The chimeric monoclonal antibody rituximab depletes B cells by targeting the pan-B-cell surface marker CD20. Preliminary experience with this agent in SLE and other autoimmune diseases has been encouraging. Controlled trials in SLE will be necessary to determine whether rituximab is useful therapy in this disease, and will teach us more about the roles of B cells in its pathogenesis.     

Depletion of B cells in murine lupus: efficacy and resistance

In mice, genetic deletion of B cells strongly suppresses systemic autoimmunity, providing a rationale for depleting B cells to treat autoimmunity. In fact, B cell depletion with rituximab is approved for rheumatoid arthritis patients, and clinical trials are underway for systemic lupus erythematosus. Yet, basic questions concerning mechanism, pathologic effect, and extent of B cell depletion cannot be easily studied in humans. To better understand how B cell depletion affects autoimmunity, we have generated a transgenic mouse expressing human CD20 on B cells in an autoimmune-prone MRL/MpJ-Fas(lpr) (MRL/lpr) background. Using high doses of a murine anti-human CD20 mAb, we were able to achieve significant depletion of B cells, which in turn markedly ameliorated clinical and histologic disease as well as antinuclear Ab and serum autoantibody levels. However, we also found that B cells were quite refractory to depletion in autoimmune-prone strains compared with non-autoimmune-prone strains. This was true with multiple anti-CD20 Abs, including a new anti-mouse CD20 Ab, and in several different autoimmune-prone strains. Thus, whereas successful B cell depletion is a promising therapy for lupus, at least some patients might be resistant to the therapy as a byproduct of the autoimmune condition itself.     

Exhaustion of cytotoxic effector systems may limit monoclonal antibody-based immunotherapy in cancer patients

The CD20 mAb ofatumumab (OFA) induces complement-mediated lysis of B cells. In an investigator-initiated phase II trial of OFA plus chemotherapy for chronic lymphocytic leukemia (CLL), OFA treatment promoted partial CLL B cell depletion that coincided with reduced complement titers. Remaining CLL B cells circulated with bound OFA and covalently bound complement breakdown product C3d, indicative of ongoing complement activation. Presumably, neither complement- nor effector cell-based mechanisms were sufficiently robust to clear these remaining B cells. Instead, almost all of the bound OFA and CD20 was removed from the cells, in accordance with previous clinical studies that demonstrated comparable loss of CD20 from B cells after treatment of CLL patients with rituximab. In vitro experiments with OFA and rituximab addressing these observations suggest that host effector mechanisms that support mAb-mediated lysis and tumor cell clearance are finite, and they can be saturated or exhausted at high B cell burdens, particularly at high mAb concentrations. Interestingly, only a fraction of available complement was required to kill cells with CD20 mAbs, and killing could be tuned by titrating the mAb concentration. Consequently, maximal B cell killing of an initial and secondary B cell challenge was achieved with intermediate mAb concentrations, whereas high concentrations promoted lower overall killing. Therefore, mAb therapies that rely substantially on effector mechanisms subject to exhaustion, including complement, may benefit from lower, more frequent dosing schemes optimized to sustain and maximize killing by cytotoxic immune effector systems.     

Isolation and characterization of the B-cell marker CD20

The integral membrane protein CD20 has been identified as an important therapeutic target in the treatment of non-Hodgkin's lymphoma (NHL). CD20 binding of many antibodies including the therapeutic antibody, rituximab, has been shown to be critically dependent upon the conformation of a loop structure between the third and fourth helical transmembrane regions. In this work, human and murine CD20 proteins expressed in Escherichia coli are shown to be localized with the cell membrane and are purified in nondenaturing detergent solutions. The purified human and murine CD20 proteins have a substantial helical structure as measured by circular dichroism spectroscopy. Only small changes in the secondary structure are observed following the reduction of CD20, with the addition of SDS, or after heating. The rituximab antibody is shown to bind to purified human CD20 with nanomolar affinity. Rituximab binding is abolished by reduction and alkylation of CD20, with data consistent with the proposed antibody epitope being within the disulfide-bonded loop formed between cysteine residues 167 and 183. Disulfide-bond-dependent antibody binding is partially recovered following reoxidation of reduced CD20. Antibody binding is unaffected by mutations of cysteines proposed to be in the intracellular domain of CD20. The affinities of intact rituximab and its Fab fragment to the isolated and purified CD20 are similar to the observed affinity of rituximab Fab for CD20 on the surface of B cells. However, the intact rituximab antibody shows much higher affinity for CD20 on B cells. This suggests that B cells display CD20 in such a way that allows for marked avidity effects to be observed, perhaps through cross-linking of CD20 monomers into lipid rafts, which limits receptor diffusion in the membrane. Such cross-linking may play a role in partitioning CD20 into lipid rafts and in enhancing antibody-dependent B-cell depletion activities of rituximab and other therapeutic anti-CD20 antibodies.     

Poster Sessions

Approval of an anti-CD20 chimeric monoclonal antibody, rituximab, has revolutionized cancer treatment and also validated CD20 targeting for providing benefit and improvement of overall response rate in B cell malignancies. Although many patients have benefited from the treatment of rituximab, there are still significant numbers of patients who are refractory or develop resistance to the treatment. Here we discuss pre-clinically well-defined potential mechanisms of action for rituximab and review the ways next generation anti-CD20 monoclonal antibodies can potentially exploit them to further enhance the treatment of B cell malignancies. Although the relative importance of each of these mechanism remains to be established in the clinic, well-designed clinical trials will help to define the efficacy and understanding of which effector activity of modified next generation anti-CD20 mAb will be important in the treatment of B-cell malignancies.     

Evolution of anti-CD20 monoclonal antibody therapeutics in oncology

Approval of an anti-CD20 chimeric monoclonal antibody, rituximab, has revolutionized cancer treatment and also validated CD20 targeting for providing benefit and improvement of overall response rate in B cell malignancies. Although many patients have benefited from the treatment of rituximab, there are still significant numbers of patients who are refractory or develop resistance to the treatment. Here we discuss pre-clinically well-defined potential mechanisms of action for rituximab and review the ways next generation anti-CD20 monoclonal antibodies can potentially exploit them to further enhance the treatment of B cell malignancies. Although the relative importance of each of these mechanism remains to be established in the clinic, well-designed clinical trials will help to define the efficacy and understanding of which effector activity of modified next generation anti-CD20 mAb will be important in the treatment of B-cell malignancies.Key words: CD20, NHL, CLL, monoclonal antibody, next generation anti-CD20 antibodies, ADCC, CDC, ADCP, PCD, rituximab     

Anti-CD22/CD20 Bispecific Antibody with Enhanced Trogocytosis for Treatment of Lupus

The humanized anti-CD22 antibody, epratuzumab, has demonstrated therapeutic activity in clinical trials of lymphoma, leukemia and autoimmune diseases, treating currently over 1500 cases of non-Hodgkin lymphoma, acute lymphoblastic leukemias, Waldenström’s macroglobulinemia, Sjögren’s syndrome, and systemic lupus erythematosus. Because epratuzumab reduces on average only 35% of circulating B cells in patients, and has minimal antibody-dependent cellular cytotoxicity and negligible complement-dependent cytotoxicity when evaluated in vitro, its therapeutic activity may not result completely from B-cell depletion. We reported recently that epratuzumab mediates Fc/FcR-dependent membrane transfer from B cells to effector cells via trogocytosis, resulting in a substantial reduction of multiple BCR modulators, including CD22, CD19, CD21, and CD79b, as well as key cell adhesion molecules, including CD44, CD62L, and β7 integrin, on the surface of B cells in peripheral blood mononuclear cells obtained from normal donors or SLE patients. Rituximab has clinical activity in lupus, but failed to achieve primary endpoints in a Phase III trial. This is the first study of trogocytosis mediated by bispecific antibodies targeting neighboring cell-surface proteins, CD22, CD20, and CD19, as demonstrated by flow cytometry and immunofluorescence microscopy. We show that, compared to epratuzumab, a bispecific hexavalent antibody comprising epratuzumab and veltuzumab (humanized anti-CD20 mAb) exhibits enhanced trogocytosis resulting in major reductions in B-cell surface levels of CD19, CD20, CD21, CD22, CD79b, CD44, CD62L and β7-integrin, and with considerably less immunocompromising B-cell depletion that would result with anti-CD20 mAbs such as veltuzumab or rituximab, given either alone or in combination with epratuzumab. A CD22/CD19 bispecific hexavalent antibody, which exhibited enhanced trogocytosis of some antigens and minimal B-cell depletion, may also be therapeutically useful. The bispecific antibody is a candidate for improved treatment of lupus and other autoimmune diseases, offering advantages over administration of the two parental antibodies in combination.     

Rituximab treatment in rheumatoid arthritis: how does it work?

Treatment with the chimerical monoclonal antibody rituximab results in CD20-directed B cell depletion. Although this depletion is almost complete in the peripheral blood of nearly all patients with rheumatoid arthritis, a proportion of patients does not exhibit a clinical response. The paper by Nakou and colleagues suggests that a decrease in CD19+CD27+ memory B cells in both peripheral blood and bone marrow precedes the clinical response to rituximab. This finding adds to the emerging evidence that lack of response to rituximab is associated with persistence of B lineage cells in specific body compartments.     

Loss of CD20 and bound CD20 antibody from opsonized B cells occurs more rapidly because of trogocytosis mediated by Fc receptor-expressing effector cells than direct internalization by the B cells

We previously reported that 1 h after infusion of CD20 mAb rituximab in patients with chronic lymphocytic leukemia (CLL), >80% of CD20 was removed from circulating B cells, and we replicated this finding, based on in vitro models. This reaction occurs via an endocytic process called shaving/trogocytosis, mediated by FcγR on acceptor cells including monocytes/macrophages, which remove and internalize rituximab-CD20 immune complexes from B cells. Beers et al. reported that CD20 mAb-induced antigenic modulation occurs as a result of internalization of B cell-bound mAb-CD20 complexes by the B cells themselves, with internalization of ～40% observed after 2 h at 37°C. These findings raise fundamental questions regarding the relative importance of shaving versus internalization in promoting CD20 loss and have substantial implications for the design of mAb-based cancer therapies. Therefore, we performed direct comparisons, based on flow cytometry, to determine the relative rates and extent of shaving versus internalization. B cells, from cell lines, from patients with CLL, and from normal donors, were opsonized with CD20 mAbs rituximab or ofatumumab and incubated for varying times and then reacted with acceptor THP-1 monocytes to promote shaving. We find that shaving induces considerably greater loss of CD20 and bound mAb from opsonized B cells in much shorter time periods (75-90% in <45 min) than is observed for internalization. Both shaving/trogocytosis and internalization could contribute to CD20 loss when CLL patients receive rituximab therapy, but shaving should occur more rapidly and is most likely to be the key mechanism of CD20 loss.     

B cell targeted therapies

Although the precise pathogenesis of rheumatoid arthritis (RA) remains unclear, many cell populations, including monocytes, macrophages, endothelial cells, fibroblasts and B cells, participate in the inflammatory process. Ongoing research continues to evaluate the critical roles played by B cells in sustaining the chronic inflammatory process of RA. These findings have contributed to the development of targeted therapies that deplete B cells, such as rituximab, as well as inhibitors of B lymphocyte stimulation, such as belimumab. In a phase I trial, belimumab treatment significantly reduced CD20+ levels in patients with systemic lupus erythematosus. Phase I and phase II trials of rituximab found that rituximab plus methotrexate achieved significantly better American College of Rheumatology 50% responses for patients with RA than those patients receiving monotherapy with methotrexate. These clinical trial data present promising evidence for B cell targeted therapies as future therapeutic options for RA.     

Inhibition of Increased Circulating Tfh Cell by Anti-CD20 Monoclonal Antibody in Patients with Type 1 Diabetes

Objectives

Follicular helper T (Tfh) cells exert an important role in autoimmune diseases. Whether it might be involved in type 1 diabetes (T1D) is unknown. Our aim was to investigate the role of Tfh cells in patients with T1D and the effect of anti-CD20 monoclonal antibody (rituximab) on Tfh cells from T1D patients.

Patients and Methods

Fifty-four patients with T1D and 37 healthy controls were enrolled in the current study. 20 of those patients were treated with rituximab. The frequencies of circulating CD4⁺CXCR5⁺ICOS⁺T cells were analyzed by flow cytometry. The serum autoantibodies were detected by radioligand assay. The levels of IL-21, IL-6 and BCL-6 were assessed using ELISA and/or real-time PCR.

Results

Increased frequencies of circulating Tfh cells together with enhanced expression of IL-21 were detected in patients. The correlation between the frequencies of circulating Tfh cells and the serum autoantibodies or C-peptide level was comfirmed. After rituximab therapy, follow-up analysis demonstrated that the frequencies of circulating Tfh cell and serum IA2A were decreased. The levels of IL-21, IL-6 and Bcl-6 mRNA were decreased after treatment. Furthermore, beta cell function in 10 of 20 patients was improved.

Conclusions

These data indicate Tfh cells may participate in the T1D-relatede immune responses and B cells might play a role in the development of Tfh responses in the disease progression.     

The dual effects of B cell depletion on antigen-specific T cells in BDC2.5NOD mice

B cells play a critical role in the pathogenesis of autoimmune diabetes. To investigate the mechanisms by which B cell depletion therapy attenuates islet β cell loss and particularly to examine the effect of B cells on both diabetogenic and regulatory Ag-specific T cells, we generated a transgenic BDC2.5NOD mouse expressing human CD20 on B cells. This allowed us to deplete B cells for defined time periods and investigate the effect of B cell depletion on Ag-specific BDC2.5 T cells. We depleted B cells with anti-human CD20 Ab using a multiple injection protocol. We studied two time points, before and after B cell regeneration, to examine the effect on BDC2.5 T cell phenotype and functions that included antigenic response, cytokine profile, diabetogenicity, and suppressive function of regulatory T (T(reg)) cells. We found unexpectedly that B cell depletion induced transient aggressive behavior in BDC2.5 diabetogenic T cells and reduction in T(reg) cell number and function during the depletion period. However, after B cell reconstitution, we found that more regenerated B cells, particularly in the CD1d(-) fraction, expressed immune regulatory function. Our results suggest that the regenerated B cells are likely to be responsible for the therapeutic effect after B cell depletion. Our preclinical study also provides direct evidence that B cells regulate both pathogenic and T(reg) cell function, and this knowledge could explain the increased T cell responses to islet Ag after rituximab therapy in diabetic patients in a recent report and will be useful in design of future clinical protocols.     

Rituximab infusion promotes rapid complement depletion and acute CD20 loss in chronic lymphocytic leukemia

Complement plays an important role in the immunotherapeutic action of the anti-CD20 mAb rituximab, and therefore we investigated whether complement might be the limiting factor in rituximab therapy. Our in vitro studies indicate that at high cell densities, binding of rituximab to human CD20(+) cells leads to loss of complement activity and consumption of component C2. Infusion of rituximab in chronic lymphocytic leukemia patients also depletes complement; sera of treated patients have reduced capacity to C3b opsonize and kill CD20(+) cells unless supplemented with normal serum or component C2. Initiation of rituximab infusion in chronic lymphocytic leukemia patients leads to rapid clearance of CD20(+) cells. However, substantial numbers of B cells, with significantly reduced levels of CD20, return to the bloodstream immediately after rituximab infusion. In addition, a mAb specific for the Fc region of rituximab does not bind to these recirculating cells, suggesting that the rituximab-opsonized cells were temporarily sequestered by the mononuclear phagocytic system, and then released back into the circulation after the rituximab-CD20 complexes were removed by phagocytic cells. Western blots provide additional evidence for this escape mechanism that appears to occur as a consequence of CD20 loss. Treatment paradigms to prevent this escape, such as use of engineered or alternative anti-CD20 mAbs, may allow for more effective immunotherapy of chronic lymphocytic leukemia.