ABSTRACT: Despite many therapeutic options for chronic lymphocytic leukemia (CLL), the disease remains incurable. Since monoclonal antibodies and recombinant toxins that bind surface antigens expressed on the malignant lymphocytes have been developed, targeted therapy has become a vital option in treating CLL. Rituximab (Rituxan), a chimeric human-mouse anti-CD20 antibody, and alemtuzumab (Campath), a humanized anti-CD52 monoclonal antibody, have both shown activity in CLL—as single agents and in combination with conventional chemotherapy. The possibility of combining antibodies has been explored as well, with some efficacy. In this review, we discuss the clinical data on the activity of commercially available antibodies in CLL, both as monotherapy and in combination with other agents.
Chronic lymphocytic leukemia (CLL) is the most common leukemia in the western hemisphere.[ 1] Early-stage CLL (Rai stage I and II) usually requires no therapy and has a median survival of almost 10 years. However, advanced-stage disease (Rai stage III and IV) has a worse prognosis with a median survival of 18 months. Traditionally, CLL has been treated with alkylating agents, steroids, or fludarabine (Fludara), although there is recent evidence demonstrating that initial fludarabine is associated with im proved response rates and progression- free survival. Once patients fail purine analog-based therapy, other therapeutic options are usually limited as they produce modest efficacy and significant toxicity. This mandates the search for innovative approaches that have activity in this disease and can be administered safely in a heavily pretreated older patient population.
Targeted therapy has become an important paradigm for cancer treatment. The selective cytotoxicity of monoclonal antibodies against malignant cells allows for enhanced efficacy with fewer adverse events. Chronic lymphocytic leukemia is an ideal therapeutic model with the identification of a spectrum of well-char- acterized cell surface antigens. It is anticipated that the introduction of monoclonal antibodies in the treatment of CLL will allow for improved outcome, and would permit combinations of biochemotherapy in an effort to cure this devastating leukemia.
Antigens and Antibodies
Ideally, targeted therapy should be directed against tumor-specific antigens that are expressed on malignant cells and not on normal tissue. However, antigens with a restricted range of expression on normal cells that are not critical for survival are acceptable. It is clear that the nature of the target antigen plays an important role in determining the likelihood of therapeutic success. For instance, having a target antigen present in sufficient concentration is essential.
Monoclonal antibodies that are generated from rodents can be immunogenic, with patients generating a human antimouse antibody response. In CLL, the patient's perturbed immune system often precludes this response. This dilemma, however, has been virtually eliminated by humanizing parts of the monoclonal antibody through genetic engineering. In addition, the efficacy of monoclonal antibodies, when used alone, might be limited by their large size, which could prevent adequate penetration in the case of large bulky adenopathy.
The theoretical basis for combining monoclonal antibodies with conventional chemotherapeutic agents is based on evidence of in vitro synergy. Animal model data support this observation. Also, the timing of anti- antibody administration can be crucial. When monoclonal antibodies are combined with conventional approaches, maximal activity may be witnessed with pretreatment, simultaneous therapy, or after the reduction of bulky disease. Table 1 summarizes the critical issues contributing to therapeutic efficacy with monoclonal antibodies.
Mechanisms of Action
The mechanisms of action of monoclonal antibodies are not fully understood, but antibody-dependent cellular cytotoxicity (ADCC) and complement-mediated cytotoxicity (CMC) are attractive theories. In fact, the immunoglobulin heavychain isotype may be critical, as it interacts with both human complement and Fc receptors to permit cell lysis. Recently, Cartron and colleagues showed that the FcgR3a receptor polymorphism (158V/F) plays an important role in the therapeutic response to rituximab (Rituxan). The FCGR3A-158V/F genotype was determined in 49 patients who had received rituximab for a previously untreated follicular non-Hodgkin's lymphoma. The clinical response and the disappearance of the BCL2-JH gene rearrangement in both peripheral blood and bone marrow were evaluated at 2 months and at 1 year.
The study population consisted of 20% FCGR3A-158V homozygous patients, 35% FCGR3A-158F homozygous patients, and 45% heterozygous patients (FCGR3A-158F carriers). The objective response rates at 2 months and 1 year were 100% and 90%, respectively, in FCGR3A- 158V homozygous patients, compared with 67% (P = .03) and 51% (P = .03), respectively, in FCGR3A- 158F carriers. A disappearance of the BCL2-JH gene rearrangement in both peripheral blood and marrow was observed at 1 year in 5 of 6 homozygous FCGR3A-158V patients compared with 5 of 17 FCGR3A-158F carriers (P = .03)
The homozygous FCGR3A-158V genotype was confirmed to be the single parameter associated with clinical and molecular responses by multivariate analysis. This study- showed an association between the FCGR3A genotype and clinical and molecular responses to rituximab. In addition, although in vitro complement activation clearly enhances the cytotoxicity of rituximab and alemtuzumab (Campath-1H) against hematologic malignant cell lines, the evidence for complement activation in vivo has been less definitive. This issue was addressed in one investigation where there was no clinical correlation between rituximab activity in lymphoma and the expression of inhibitors of complement activators (CD46, CD55, and CD59).
In addition, emerging evidence suggests a role for antibody-induced apoptosis. Byrd and colleagues studied the mechanisms of action of rituximab in patients with B-cell chronic lymphocytic leukemia (B-CLL) and demonstrated activation of caspase 3 and caspase 9, as well as poly(ADPribose) polymerase cleavage in blood leukemia cells immediately following rituximab infusion. These investigators have also shown that significant downmodulation of antiapoptotic proteins occurs. Other groups have provided complementary evidence that apoptosis is one of the mechanistic pathways for monoclonal antibody cytotoxicity.[4,9] In addition, the mechanism of action may be dictated by the targeted tissue compartment (for example, blood vessels, spleen, lymph nodes).
Rituximab in CLL
Rituximab is a chimeric monoclonal anti-CD20 antibody that consists of the murine variable regions of the parent 2B8 murine ant-CD20 grafted onto a human immunoglobulin G1-constant region. The CD20 antigen is an excellent immunotherapy target as it is expressed only on malignant and mature B cells and not on precursor B cells, and the antigen is not shed, internalized, or modulated to a significant degree once antigen-antibody binding has occurred. It is a 33-kD protein, the gene of which is located on chromosome 11q12-q13 and encodes a 297-amino acid, nonglycosylated, tetraspan molecule with intracellular N and C termini and short loops protruding from the cell membrane.[ 12] The precise function of CD20 is still undetermined, although some have suggested that this protein is involved in regulation of calcium flux and cell signaling that affects B-cell growth and differentiation.[ 14] CD20 is expressed at high density (100,000 molecules/cell) on most malignant mature B cells, although this density is lower in B-CLL (8,000 molecules/cell).
The initial pivotal trial that led to rituximab approval showed 6% complete response and 42% partial response, for an overall response of 48% in patients with relapsed indolent non-Hodgkin's lymphoma (NHL). The median duration of response in this trial was 11.6 months. Of the patients treated on that study, 33 had a diagnosis of small lymphocytic lymphoma (SLL). These patients had remarkably lower responses than those with other histologic diagnoses (13% compared with 58%). It is believed that the lower CD20 antigen density and altered pharmacokinetics cause rapid clearance of the drug that may have accounted for the lower response rate. Since then, rituximab has shown efficacy in bulky indolent NHL, previously untreated patients, and in combination with chemotherapy, as discussed later in this article.[16,17]
Refractory or Relapsed Patients
As the CD20 antigen is also expressed on circulating tumor cells in CLL, several investigators have explored the efficacy of this agent in relapsed or refractory patients. Winkler and colleagues reported on 12 patients with fludarabine-resistant CLL or leukemic variants of other low-grade NHLs that were treated with rituximab at 375 mg/m2 once weekly for 4 weeks. The response rates in the CLL patients were modest. There was one complete response (duration of 9 weeks), one partial response (duration of greater than 22 weeks), seven cases of stable disease, and one case of progressive disease. The authors concluded that there is some efficacy for rituximab in CLL, and that further studies were warranted. Since then, Huhn and colleagues reported minimal activity of rituximab in CLL, showing an overall response rate of 25% in 28 previously treated patients, with all responses being partial.
Some investigators explored in- creasing the frequency or the dose of rituximab in order to overcome the poor pharmacokinetic profile in CLL. To this regard, O'Brien et al recently reported the results of a phase I escalation study of rituximab in CLL patients who failed prior traditional therapies. In this study, eligible patients were treated with rituximab in four weekly infusions with escalated doses ranging from 375 mg/m2 to 2,250 mg/m2. The response rate was 36% among 39 evaluable patients, with a suggestion of better response at higher dose levels. All responses were partial. Adverse events were fever, chills, and hypotension, with more side effects at higher dose levels. The median time to disease progression was 8 months. Although there was a suggestion of good activity of rituximab in CLL at these high dose levels, the cost was substantial, and the authors concluded that a better approach would be combining rituximab with other effective agents.
Byrd et al reported another important study in CLL patients who were treated with thrice-weekly rituximab for a total of 12 doses. To assess toxicity, the first cohort of patients received rituximab at 250 mg/m2 three times weekly for 4 weeks. All other treated patients received a dose of 375 mg/m2. Among 29 patients who were assessable for efficacy, one patient achieved a complete response (3%); the overall response rate was 45%. Of the patients who did not respond to therapy, 11 had stable disease, and 3 had evidence of disease progression. Previously untreated patients had a better response rate (83%) compared to patients who had previously received alkylator therapy (30%) or who were refractory to fludarabine (41%), yet this was not statistically significant. The median duration of response in this study was 10 months.
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