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New-Generation Anti-CD20 Monoclonal Antibody (GA101) Evokes Homotypic Adhesion and Actin-Dependent, Lysosome-Mediated Cell Death in B-Cell Lymphoma.
Abstract
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The addition of the anti-CD20 monoclonal antibody (mAb) rituximab to chemotherapy has substantially improved the clinical outcome of patients with a wide range of B-cell malignancies. Despite this success, many patients are not cured by standard approaches and there is intense investigation into the development of new-generation anti-CD20 mAbs with further improved therapeutic efficacy. Although Fc-FcgR interactions appear to underlie much of the therapeutic success with Rituximab, certain Type II anti-CD20 mAbs, can directly induce programmed cell death (PCD), whereas rituximab-like Type I anti-CD20 mAbs do not (Chan et al. Cancer Res 63: 5480-5489, 2003). We have demonstrated that Type II mAbs are more effective at B-cell depletion in syngeneic human CD20 transgenic mice (Beers et al. Blood 112: 4170-4177, 2008). Recently, we elucidated the mechanism underlying PCD induced by the Type II anti-CD20 mAb Tositumomab, demonstrating a novel non-apoptotic mode of cell death, defined by homotypic adhesion, peripheral relocalization of actin and lysosomal activity (Ivanov et al. J Clin Invest, doi: 10.1172/JCI37884, 2009). Here we confirm that the humanized anti-CD20 mAb GA101 and derivatives harboring non-glycomodified human IgG1 or mouse IgG2a Fc regions are bone fide Type II reagents, lacking the ability to translocate CD20 into lipid rafts or initiate calcium flux. Furthermore, GA101 initiates extensive non-apoptotic cell death in a range of B-lymphoma cell lines in contrast to rituximab (e.g. in Raji cells 48 ± 1.8% versus 13 ± 0.2%, p<0.001 by Student's t-test) quantified using the Annexin V/propidium iodide cell death assay. Inhibitors of actin polymerization (latrunculin B and cytochalasin D) inhibited cell death elicited by GA101 from 45 ± 1.5% to 15 ± 3.1% (p<0.01). The importance of cell to cell contact in this form of antibody induced cell death was confirmed by the addition of low-melting point agarose which physically blocked cell to cell contact and markedly attenuated cell death induced by GA101. The role of lysosomal activity in GA101-induced PCD was assessed using an inhibitor of the lysosomal cysteine protease cathepsin B, which significantly inhibited cell death induced by GA101 from 53 ± 4.3% to 18 ± 1.9%, (p<0.001). To confirm that this mode of death is non-apoptotic, we demonstrated that GA101-induced PCD occurred independently of BCL-2 over-expression and caspase activation. Complement-dependent cytotoxicity (CDC) assays using human serum as a source of complement reveal that GA101 has significantly weaker CDC activity than rituximab, consistent with our previous work on Type II anti-CD20 mAbs (Cragg et al Blood 101: 2738-2743, 2003). Taken together, these findings demonstrate that GA101 is the first humanized anti-CD20 mAb with Type II properties, potently eliciting a novel mode of cell death in B-cell malignancies, which potentially can lead to improved B-cell depletion over rituximab. Furthermore, we are currently investigating the relative ability of GA101 and rituximab to delete B cells in vivo using directly comparable versions of these mAb with human or mouse Fc regions in human CD20 transgenic mice and will present these data.
Disclosures
No relevant conflicts of interest to declare.
... 110 GA101 demonstrates properties consistent with a bone fide type II anti-CD20 mAb, potently evoking the previously described nonapoptotic PCD and lacking the ability to translocate CD20 into membrane lipid rafts or activate complement. 100,101 GA101 has demonstrated superior tumor growth inhibition compared with rituximab in subcutaneous lymphoma xenograft models as monotherapy, 101 or in combination with cyclophosphamide in an FL xenograft model, 102 and greater B-cell depletion than rituximab in nonhuman primates 101 and hCD20 transgenic mice. 100 The first phase 1 dose-escalation trials of GA101 were performed in patients with relapsed/refractory B-cell malignancies and B-NHL who were previously exposed to rituximab. ...
... 100,101 GA101 has demonstrated superior tumor growth inhibition compared with rituximab in subcutaneous lymphoma xenograft models as monotherapy, 101 or in combination with cyclophosphamide in an FL xenograft model, 102 and greater B-cell depletion than rituximab in nonhuman primates 101 and hCD20 transgenic mice. 100 The first phase 1 dose-escalation trials of GA101 were performed in patients with relapsed/refractory B-cell malignancies and B-NHL who were previously exposed to rituximab. ORR values of 58% (7 of 12 patients) and 43% (9 of 21 patients) were seen, respectively, and responses occurred across all Fc␥RIIIa polymorphisms. ...
... It is however interesting to note that a nonglycoengineered version of GA101 showed superior therapeutic efficacy compared with rituximab in preclinical mouse models. 100,108 In addition to the glycoengineered GA101, several novel type I anti-CD20 mAbs have been developed with Fc arms engineered to enhance Fc␥RIIIa binding and ADCC ability, including AME-133v and PRO131921 (a modified version of ocrelizumab), and are now undergoing early phase 1/2 trials in relapsed B-NHL. 98,99 Currently, it appears that the Fc optimization in the next-generation anti-CD20 mAbs is entirely focused on enhancing Fc␥RIIIa binding. ...
The anti-CD20 monoclonal antibody (mAb) rituximab has revolutionized the treatment of B-cell malignancies. This unprecedented success has not only substantially changed the mindset of the clinical community about the ability of mAb to improve outcomes but has catalyzed the interest in the pharmaceutical industry to develop the next generation of anti-CD20 mAbs. Since the introduction of rituximab 15 years ago, we have learned much about the potential mechanisms underlying the therapeutic efficacy of anti-CD20 mAbs. In parallel, many novel anti-CD20 mAbs have entered the clinic, each designed with modifications to structure aimed at further improving efficacy. On review of the newer generation of anti-CD20 mAbs entering clinical trials, it appears that the link between the novel mechanistic insights and the development of these next-generation anti-CD20 mAbs is unclear. As we move into an era of personalized medicine, it will become increasingly important for us to develop closer links between the emerging mechanistic insights and the clinical development, to further enhance the potency of anti-CD20 mAbs beyond that achieved with rituximab.
... A recent study demonstrated that obinutuzumab, a novel type II anti-CD20 mAb, induces higher levels of direct, non-complement dependent cell death in various B-lymphoma cell lines and primary B-cell malignancies compared to rituximab [29,30]. Obinutuzumab-induced cell death is mediated by lysosomes and critically depends on its ability to promote homotypic adhesion in an actin-dependent manner [31]. As PCLP1 is linked to actin cytoskeleton [32], we investigated whether this protein could modulate obinutuzumab-induced cell death. ...
Podocalyxin (PCLP1) is a CD34-related sialomucin expressed by some normal cells and a variety of malignant tumors, including leukemia, and associated with the most aggressive cancers and poor clinical outcome. PCLP1 increases breast tumor growth, migration and invasion; however, its role in hematologic malignancies still remains undetermined. The purpose of this study was to investigate the expression and function of PCLP1 in mature B-cell lymphoma cells. We found that overexpression of PCLP1 significantly increases proliferation, cell-to-cell interaction, clonogenicity, and migration of B-cell lymphoma cells. Furthermore, PCLP1 overexpression results in higher resistance to death induced by dexamethasone, reactive oxygen species and type II anti-CD20 monoclonal antibody obinutuzumab. Strikingly, enforced expression of PCLP1 enhances lipid droplet formation as well as pentose phosphate pathway and glutamine dependence, indicative of metabolic reprogramming necessary to support the abnormal proliferation rate of tumor cells. Flow cytometry analysis revealed augmented levels of PCLP1 in malignant cells from some patients with mature B-cell lymphoma compared to their normal B-cell counterparts. In summary, our results demonstrate that PCLP1 contributes to proliferation and survival of mature B-cell lymphoma cells, suggesting that PCLP1 may promote lymphomagenesis and represents a therapeutic target for the treatment of B-cell lymphomas.
... In contrast to rituximab and ofatumumab, obinutuzumab (GA101) is a glycoengineered type 2 antibody targeting CD20 with increased antibody-dependent cellular cytotoxicity and direct, non-apoptotic cell death induction mediated through lysosomes [22]. Phase I trials in patients with refractory disease showed promising results, with 62% of patients responding overall (overall response rate, or ORR) [23]. ...
Chronic lymphocytic leukemia (CLL) is the most prevalent type of leukemia, affects mostly elderly CLL patients, and is incurable without allogeneic transplantation. Although classic chemo(immuno)therapy is still the standard of care for patients in need of treatment, this paradigm might change in the near future with the advent of new therapeutic agents targeting major pathogenic pathways in CLL.
Purpose of review:
Passive immunotherapy with therapeutic monoclonal antibodies (mAbs) has revolutionized the treatment of cancer, especially hematological malignancies over the last 20 years. While use of mAbs has improved outcomes, development of resistance is inevitable in most cases, hindering the long-term survival of cancer patients. This review focuses on the available data on mechanisms of resistance to rituximab and includes some additional information for other mAbs currently in use in hematological malignancies.
Recent findings:
Mechanisms of resistance have been identified that target all described mechanisms of mAb activity including altered antigen expression or binding, impaired complement-mediated cytotoxicity (CMC) or antibody-dependent cellular cytotoxicity (ADCC), altered intracellular signaling effects, and inhibition of direct induction of cell death. Numerous approaches to circumvent identified mechanisms of resistance continue to be investigated, but a thorough understanding of which resistance mechanisms are most clinically relevant is still elusive. In recent years, a deeper understanding of the tumor microenvironment and targeting the apoptotic pathway has led to promising breakthroughs. Resistance may be driven by unique patient-, disease-, and antibody-related factors. Understanding the mechanisms of resistance to mAbs will guide the development of strategies to overcome resistance and re-sensitize cancer cells to these biological agents.
Purpose of Review
Since its initial approval in 1997, rituximab has revolutionized the treatment of CD20-positive lymphoproliferative disorders. Now, over two decades later, second-generation molecules are emerging that may have key biological advantages compared to rituximab, as well as biosimilars that may be more cost-effective. Clinicians, health policy makers, and payers will now need to critically appraise the available evidence for these competitors and decide which anti-CD20 to use.
Recent Findings
Evidence has emerged directly comparing rituximab IV to a subcutaneous preparation, and head-to-head comparisons of rituximab versus next-generation anti-CD20 monoclonal antibodies have also been published. Trials comparing rituximab with newly developed biosimilars have also allowed for registration of these agents.
Summary
In this review, we will present an overview of anti-CD20 monoclonal antibody development, discuss the mechanistic and clinical evidence for rituximab, as well as the novel compounds, and provide commentary on the possible advantages and limitations of these agents.
The anti-CD20 mAb rituximab has substantially improved the clinical outcome of patients with a wide range of B-cell malignancies. However, many patients relapse or fail to respond to rituximab, and thus there is intense investigation into the development of novel anti-CD20 mAbs with improved therapeutic efficacy. Although Fc-FcγR interactions appear to underlie much of the therapeutic success with rituximab, certain type II anti-CD20 mAbs efficiently induce programmed cell death (PCD), whereas rituximab-like type I anti-CD20 mAbs do not. Here, we show that the humanized, glycoengineered anti-CD20 mAb GA101 and derivatives harboring non-glycoengineered Fc regions are type II mAb that trigger nonapoptotic PCD in a range of B-lymphoma cell lines and primary B-cell malignancies. We demonstrate that GA101-induced cell death is dependent on actin reorganization, can be abrogated by inhibitors of actin polymerization, and is independent of BCL-2 overexpression and caspase activation. GA101-induced PCD is executed by lysosomes which disperse their contents into the cytoplasm and surrounding environment. Taken together, these findings reveal that GA101 is able to potently elicit actin-dependent, lysosomal cell death, which may potentially lead to improved clearance of B-cell malignancies in vivo.
To the editor:
Two groups have recently proposed that type II anti-CD20 monoclonal antibodies (mAbs) B1 (tositumomab) and GA101, as well as anti–HLA-DR antibody (L243), induce strong homotypic adhesion and direct cell death. They claim, on the basis of annexin V, propidium iodide (PI), and flow
Chronic lymphocytic leukemia (CLL) represents the most prevalent adult leukemia. Treatment with chemotherapy over the past 3 decades has been palliative. The introduction of therapeutic antibodies has increased the number of treatment options for this disease. Despite this increase, our true understanding of the mechanism of action of antibody therapy in CLL remains limited. Rituximab, a CD20 antibody, is currently widely used in combination-based strategies for both previously untreated symptomatic CLL and as salvage therapy. Recent data suggest that the addition of rituximab to fludarabine with or without cyclophosphamide prolongs survival in younger patients with CLL. Other improved CD20 antibodies with promising clinical activity, including ofatumumab and GA-101, are coming forward. Alemtuzumab, a CD52 antibody, likewise has demonstrated benefit in both symptomatic, previously untreated CLL and in patients with relapsed disease but has less selectivity. Development of other therapeutic antibodies targeting alternative B-cell-specific antigens in CLL has been less successful, although many promising candidate antibodies and/or small modular immune pharmaceuticals (SMIPs) are coming forward. In addition, recent efforts to combine currently applied therapeutic antibodies with other biologic and targeted therapies with efficacy in CLL offers the potential to move toward alternative non-chemotherapy-based treatment approaches.
Limitations of therapeutic options for chronic lymphocytic leukemia (CLL) and non-Hodgkin lymphoma (NHL) have necessitated the development of novel treatments/strategies. Rituximab (chimeric anti-CD20 monoclonal antibody [mAb]) considerably improved therapeutic outcomes for patients with B-cell malignancies, particularly when combined with chemotherapy; outcomes, however, are limited by rituximab resistance or reduced response upon re-treatment. Novel anti-CD20 mAbs are in development that may enhance mAb therapy. Ofatumumab (human anti-CD20 mAb) induces highly potent cell lysis, including in cells with low CD20 expression, and is the most clinically advanced new anti-CD20 mAb. Positive phase III interim data for ofatumumab in fludarabine-refractory CLL that is also refractory to alemtuzumab or less suitable for alemtuzumab due to bulky (>5 cm) lymphadenopathy has led to FDA approval of this agent in this population. Preclinical and early clinical assessment of other novel anti-CD20 mAbs include: ocrelizumab, veltuzumab, GA101, AME-133v, and PRO131921; data suggest potential for improved efficacy over rituximab that will require substantiation in large-scale clinical trials. New treatment strategies and novel anti-CD20 mAbs have the potential to enhance long-term outcomes for CLL and NHL.
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.
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