Modifying the immune system to treat chronic lymphocytic leukemia

Despite recent advances, chronic lymphocytic leukemia (CLL) remains incurable with conventional chemotherapy [1]. Conversely, a clear graft-versus-leukemia effect exists in CLL, and some patients are cured following allogeneic stem cell transplant [2]. However, allogeneic stem cell transplant is associated with significant morbidity and mortality, and is applicable to only a minority of patients with CLL. Therefore, manipulation of the patient's own immune system in order to exert control over CLL is an attractive treatment strategy.

Attempts at stimulating an autologous immunological response to CLL have included dendritic cell-based vaccines [3], vaccination with autologous CLL cells manipulated to express immunogenic molecules [4,5] and the use of immunomodulatory drugs [6,7]. None of these strategies have replicated the potent graft-versus-leukemia effect of allogeneic stem cell transplant, but they have all shown promising immunological changes that add to our fledging understanding of the CLL/immune system interaction.

A novel mechanism of immune manipulation in patients with CLL is through stimulation of the toll-like receptor (TLR) pathway. The human TLR family includes 10 members homologous to the toll gene in Drosophila [8]. TLRs directly recognize microbial antigens, and are an integral part of the innate immune response against infectious agents. The signaling through these receptors in immune and inflammatory responses is diverse [9]. The extra- or intracellular localization of these receptors is also varied, but they are expressed widely on cells of the innate immune system (myeloid cells, natural killer [NK] cells and dendritic cells), and are expressed in a restricted manner on T-cells and B-cells [10]. Of relevance to CLL, TLR9 (which recognizes unmethylated cytosine-phosphate-guanosine [CpG] motifs) is expressed on CLL cells and plasmacytoid dendritic cells [10], and ligation of this receptor causes immune activation of CLL cells, resulting in increased expression of co-stimulatory and antigen-presenting molecules (CD40, CD54, CD80, CD86 and major histocompatibility complex [MHC] I) [11]. What this means is that the CLL cell itself becomes a better antigen-presenting cell, hopefully resulting in stimulation of CLL-directed immunity. Second, ligation of TLR9 increases CD20 expression in the CLL cell, potentially increasing its susceptibility to treatment with rituximab (anti-CD20) [12]. Third, although TLR9 ligation results in proliferation of CLL cells, it can also induce apoptosis [11]. Lastly, stimulation of plasmacytoid dendritic cells by TLR9 ligation may augment the generalized immune response against CLL.

There is therefore considerable rationale for the use of drugs stimulating TLR9 in CLL. In general for TLRs, several agonists and antagonists have been proposed or used [13]. The most widely evaluated agent in the context of TLR9 is CpG oligonucleotide 7909 (PF-03512676), a synthetic 24-mer oligonucleotide with four unmethylated CpG motifs. This class B oligonucleotide showed promising effects in CLL cells in culture and in animal CLL xenograft models [14]. Moreover, it has been tested in a number of human cancers including B-cell non-Hodgkin lymphoma (NHL) [12], where it was combined with rituximab in order to take advantage of CD20 up-regulation. Doses up to 0.24 mg/kg (subcutaneous (sc) route) or 0.48 mg/kg (intravenous (iv) route) were given, with many patients experiencing flu-like side effects or sc injection site reactions [12].

In this issue, Zent and colleagues report the results of a phase I study of CpG 7909 in patients with CLL [15]. Forty-one patients with predominantly early relapse of CLL received treatment. Both the iv route and sc route were evaluated. The maximal tolerated dose in the sc group was 0.45 mg/kg, with flu-like symptoms being the dose-limiting toxicity. In the iv group, no grade 3–4 toxicity was observed, at doses up to 0.75 mg/kg. Based on these safety data, six patients went on to receive 1.05 mg/kg intravenously, followed by eight weekly sc doses of 0.45 mg/kg.

Although no objective clinical responses were observed with CpG 7909 treatment, the correlative science studies identified interesting treatment-related immunological effects. For example, in the iv group, treatment with CpG 7909 induced changes in the CLL cell, including increased expression of CD20, CD86 and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL). In addition, circulating T-cells and NK cells were increased. Circulating T-cells showed evidence of activation, with expression of CD38. On the other hand, patients in the sc group had a decrease in circulating lymphocyte count and increased CD38 expression in T-cells, but their CLL cells did not change phenotype. There was evidence of a potent immune response in the sc group, with local inflammation and cytokine side-effects following drug injection. Unfortunately, the authors did not evaluate the effect of CpG 7909 and TLR9 signaling such as nuclear factor κB (NFκB) activation, NFκB-dependent production of interleukins and activation of the JAK–STAT (Janus kinase–signal transducer and activator of transcription) pathway, as demonstrated previously in CLL lymphocytes [14].

What does this mean for future studies using CpG 7909? First, we believe that the current study showed clearly that i.v. administration and s.c. administration of this drug are not the same, giving impetus to rational sequencing of doses. Clearly, if one wants an alteration in the CLL cell in order to stimulate an autologous immune response, CpG 7909 should be used intravenously (where the CLL cells are). If one wants to use CpG 7909 as an immunoadjuvant targeting the host immune system, then the sc route is more logical. We agree with the authors that an iv dose followed by ongoing sc doses may be the most efficient way of stimulating CLL-specific immunity. Second, we feel that additional studies should evaluate chemotherapeutic partners to combine with this oligonucleotide. The current standard of care for CLL includes alkylating agent and purine nucleoside analogs. These agents affect the immune system, further complicating the use and timing of CpG 7909. Finally, based on an increase in CD20 in malignant B-cells, investigations are needed to determine the synergistic combination of rituximab with CpG 7909.

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