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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Br J Haematol. Author manuscript; available in PMC Aug 1, 2010.
Published in final edited form as:
PMCID: PMC2747261
NIHMSID: NIHMS139957
Phase II study of a TLR-9 agonist (1018 ISS) with rituximab in patients with relapsed or refractory follicular lymphoma
Jonathan W Friedberg,1 Jennifer L Kelly,1 Donna Neuberg,2 Derick R. Peterson,1 Jeffery L Kutok,2 Rabih Salloum,1 Thomas Brenn,2 David C Fisher,2 Elizabeth Ronan,2 Virginia Dalton,2 Lynn Rich,1 Diana Marquis,1 Paul Sims,3 Paul G. Rothberg,1 Jane Liesveld,1 Richard I Fisher,1 Robert Coffman,3 Tim Mosmann,1 and Arnold S Freedman2
1 James P. Wilmot Cancer Center, University of Rochester Medical Center, Rochester, NY
2 Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA
3 Dynavax Technologies, Berkeley, CA
Corresponding Author: Jonathan W Friedberg, James P. Wilmot Cancer Center, University of Rochester, 601 Elmwood Ave., Box 704, Rochester, NY 14642; Jonathan_Friedberg/at/urmc.rochester.edu, phone: (585)275-5863, fax: (585)275-1051
Toll-Like Receptor-9 (TLR-9) agonists have pleotropic effects on both the innate and adaptive immune systems, including increased antigen expression, enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) and T helper cell type 1 shift in the immune response. We combined a TLR-9 agonist (1018 ISS, 0.2 mg/kg sc weekly × 4 beginning day 8) with standard rituximab (375mg/m2 weekly × 4) in patients (n=23) with relapsed/refractory, histologically confirmed follicular lymphoma, and evaluated immunological changes following the combination. Treatment was well-tolerated with no significant adverse events attributable to therapy. Clinical responses were observed in 48% of patients; the overall median progression-free survival was 9 months. Biologically relevant increases in ADCC and circulating CD-3 positive T cells were observed in 35% and 39% of patients, respectively. 45% of patients had increased T cells and dendritic cells in skin biopsies of 1018 ISS injection sites 24 h post-therapy. Pre-and post-biopsies of tumour tissue demonstrated an infiltration of CD8+ T cells and macrophages following treatment. This group of patients had favourable clinical outcome despite adverse prognostic factors. This study is the first to histologically confirm perturbation of the local immune microenvironment following systemic biological therapy of follicular lymphoma.
Keywords: lymphoma, CpG oligonucleotides, TLR-9, microenvironment, rituximab
Anti-CD20 monoclonal antibody therapy with rituximab has had a dramatic impact on the treatment of follicular lymphoma (Link & Friedberg, 2008). Improved overall survival has been observed in the past decade, due largely to the routine inclusion of rituximab in therapeutic regimens for follicular lymphoma (Fisher et al, 2005; Swenson et al, 2005). Despite this success, rituximab does not appear to be curative therapy for patients with follicular lymphoma. In a phase II trial (Hainsworth et al 2005), the majority of patients with follicular lymphoma became resistant to a single-agent rituximab regimen within three years of initiating this therapy. New approaches are therefore required to both enhance the cytotoxicity of rituximab, and overcome rituximab resistance, to further improve outcome for patients with follicular lymphoma(Friedberg, 2005).
The innate immune system represents the first line of defence for the human organism. Toll-like receptors (TLR) have evolved to recognize foreign material, such as lipopolysaccharide, RNA or DNA, and initiate an innate immune response (Akira et al, 2006). TLR-9 is stimulated by bacterial and viral DNA containing unmethylated CpG dinucleotides, and is expressed by a limited number of human immune cells, notably plasmacytoid dendritic cells, B-cells, and neutrophils (Krieg, 2008). Bacterial DNA containing these CpG sequences has multiple effects on the immune system. These direct and indirect effects include induction of B-cell proliferation and immunoglobulin production, secretion of interferon (IFN)-α, IFN-β, interleukin (IL)-12, and IL-18 by macrophages and dendritic cells, and IFN-γ secretion induced by IL-12 and IFN-α from natural killer (NK) cells. These cytokines can then induce the differentiation of naive CD4+ T cells into T-helper cell, type 1 (Th1) cells upon encountering specific antigens, resulting in an adaptive immune response (Mosmann & Coffman, 1989).
The effects of bacterial DNA on TLR-9 can be mimicked using synthetic oligodeoxyribonucleotides containing CpG motifs and a stabilizing phosphorothioate backbone. One such immunostimulatory oligodeoxyribonucleotide, 1018 ISS, is under clinical development as a hepatitis vaccine adjuvant, and for allergy indications. We previously conducted a phase I trial combining 1018 ISS with rituximab in patients with relapsed or refractory non-Hodgkin lymphoma (NHL), demonstrating safety of the combination and significant immunological activity through a dose-related increase in the expression of several IFN-inducible genes following 1018 ISS (Friedberg et al, 2005).
Rituximab kills both lymphoma cells and normal B cells by several distinct mechanisms, including binding of antibody to CD20, resulting in cell death by antibody-dependent cell-mediated cytotoxicity (ADCC), complement-mediated cytotoxicity, direct apoptosis, or through a “vaccinal effect” leading to T-cell mediated death (Friedberg, 2005). TLR-9 agonists have pleotropic effects on both the innate and adaptive immune systems, including increased antigen expression, enhanced ADCC and shifting the immune response in a Th1 direction. These agents therefore have significant potential to manipulate the immune response to FL, and alter the malignant microenvironment of follicular lymphoma in a favourable way. We therefore hypothesized that 1018 ISS would have additive or synergistic effects with rituximab on follicular lymphoma through enhancement of cytotoxic effector mechanisms, as well as through stimulation of anti-tumour immunity. To study this, we conducted a phase II trial in patients with relapsed or refractory follicular lymphoma that combined rituximab with 1018 ISS with the schedule previously evaluated in our phase I trial, to demonstrate the effect of the combination on progression-free survival (Friedberg et al, 2005). In the context of this current trial, we performed numerous immunological studies on blood and tumour tissue before and after combination therapy, and investigated the association between these results and clinical outcome.
This trial was registered in the clinicaltrials.gov database; identifier: NCT00251394. This study was conducted jointly at the University of Rochester Cancer Center and the Dana Farber Cancer Institute, approved by the Institutional Review Board at both Dana Farber Cancer Institute and James P. Wilmot Cancer Center at the University of Rochester, and conducted in accordance with the Declaration of Helsinki. All patients provided written informed consent.
Eligible patients included adults (≥18), with pathological evidence of CD20+, B-cell follicular lymphoma (Grades 1 – 3), treated with at least one prior chemotherapy regimen. Patients had to meet the following additional criteria for inclusion: Eastern Cooperative Oncology Group performance status ≤2, WBC > 2 × 109/l, absolute neutrophil count > 1 × 109/l, platelet count > 100 × 109/l, and an expected survival >4 months. Patients receiving chemotherapy or radiation therapy within 30 days prior to screening visit, patients receiving radioimmunotherapy, autologous stem cell transplantation, or fludarabine within 6 months prior to screening visit, as well as patients with documented clinical heart failure, severe pulmonary disease or clinically significant pulmonary symptoms, uncontrolled infection, known Hepatitis B infections, pregnant or lactating women, and patients with clinically apparent central nervous system lymphoma were excluded.
Rituximab was given on days 1, 8, 15 and 22 (weekly × 4 doses) at a dose of 375 mg/m2. Patients received a subcutaneous injection of 1018 ISS 0.2 mg/kg given 30–60 min after completion of each of the second, third, and fourth Rituximab infusions (days 8, 15, and 22) as well as a fourth injection of 1018 ISS 0.2 mg/kg 1 week after the fourth rituximab infusion, as previously described (Friedberg et al, 2005). There were no dose modifications of rituximab or 1018 ISS for toxicities. Patients were monitored for active Hepatitis B virus infection or hepatitis while under treatment with rituximab.
Patient Evaluation
All patients had pre-treatment histories, physical examinations, baseline tumour assessment (chest x-ray or computed tomography (CT) scans and abdominal/pelvic CT or magnetic resonance imaging (MRI) scan), bone marrow biopsy, lymph node biopsy, and routine blood counts and chemistries. In addition, each patient had a baseline extremity skin punch biopsy and additional blood drawn (approximately 40 ml) for correlative laboratory evaluation of biologic response. Patients were seen during the study on days 1, 8, 15, 22, and 29 for physical examination, laboratory evaluation, and toxicity assessment. The National Cancer Institute Common Toxicity Criteria (NCI CTC Version 3, http://ctep.cancer.gov/protocolDevelopment/electronic_applications/docs/ctcaev3.pdf) were used for all symptoms. Additional blood samples (approximately 40 ml at each visit) were taken on days 8, 9, 29, 30, and 90, and repeat lymph node (when possible), skin (at injection site and a distant site), and bone marrow biopsies were repeated on Day 30 (for those patients with bone marrow involvement with lymphoma at baseline), for correlative evaluation of biological response.
Response evaluation
Physical examination, tumour assessment (chest x-ray or CT scan and abdominal/pelvic CT or MRI), and bone marrow biopsy (if baseline bone marrow biopsy was positive) were obtained at Day 90 for response evaluation using the 1999 Cheson International workshop response criteria (Cheson et al, 1999). Long-term follow-up evaluation and tumour assessment continued every 3 months for the first 2 years and then every 6 months until disease progression. Serum human anti-murine antibody (HAMA) and anti-DNA antibody levels were measured at baseline and on Day 90, and repeated in follow-up visits only if the previous assessments were positive.
FCGR3A genotyping
The genotype at codon 158 of the FCGR3A (FcγRIIIa)_gene was determined by polymerase chain reaction (PCR) and nucleotide sequencing (Wu et al, 1997). DNA was prepared using the QIAamp Blood DNA extraction kit (Qiagen Inc., Valencia, CA). The PCR was done in a volume of 25 μl with upstream primer (5′-ATTTTCATCATAATTCTGACCACT-3′) and downstream primer (5′-CCTTGAGTGATGGTGATGTTCA-3′) each at a final concentration of 1 μmol/l, 50μmol/l of each dNTP, 2 units of HotStar Taq DNA polymerase, and 2.5 μl of the 10X buffer provided by the enzyme manufacturer (Qiagen). The sequencing reactions were purified using the CleanSeq system (Agencourt Bioscience, Beverly, MA) and then resolved by capillary electrophoresis on the ABI 3100 Prism Genetic Analyzer.
Correlative Laboratory Studies
Immunophenotyping
Flow cytometry was performed using fresh peripheral blood mononuclear cells (PBMC) isolated using CPT tubes. Normal control PBMC (freshly isolated) was run concurrently with every patient sample. The following antibodies were used for this analysis: CD3, CD4, CD8, CD56, CD19, CD20, CD16, HLA-DR, CD25, CD45RA, CD45RO, CD45, CD80, and CD14. Data analysis was performed by FloJo software (Copyright, Tree Star, Inc.). Pre and post-treatment values for patient were evaluated for an increase in peripheral blood cell populations in response to 1018 ISS. A difference ≥10 in each cell marker, as a percentage of PBMC (Day 29 – baseline), was considered an increase.
T cell interferon gamma (IFN-γ) secretion
The immune status of T cells in PBMC was evaluated using enzyme-linked immunosorbent spot (ELISPOT) at Day 0, 7, and 28 for all patients. T cell secretion of IFN-γ was assessed in triplicate, at 3x105 cells per ELISPOT well, using thawed PBMC stimulated for 20 h with or without an allogeneic Epstein-Barr virus (EBV)-transformed B cell line (EBV Mann cells). An ELISPOT ratio of ≥2, when comparing day 28 to baseline secretion, was the threshold for determining a positive increase in proinflammatory cytokines secretion by T cells in peripheral blood.
ADCC
PBMC were isolated from heparinized blood by Ficoll-Paque (GE Healthcare, Piscataway, NJ) separation and frozen in media containing 90% Fetal Bovine Serum (Hyclone, Logan, UT) and 10% Dimethyl Sulfoxide (DMSO, Baker Inc. Phillipsburg, NJ). The lytic activity of effector cells was determined by 51Cr-release of target cells using well-established methods, as we previously described (Liesveld et al, 1991). The source of effector cells was PBMC from the treated patients. The target cells were a CD20+ cell line DB (ATCC). The target cells were treated with and without rituximab for 1 h. Target cells were then incubated with effector cells at different effector:target ratios (50:1,25:1,12.5:1,6:1). Supernatants were assessed using a gamma counter to determine percent lysis. A difference ≥10 in % specific cell lysis (Day 29 – baseline) was considered enhancement of cytotoxic effector mechanisms in peripheral blood.
Interferon-inducible Gene Expression
Quantitative PCR analysis of PBMC was used to evaluate changes in mRNA expression in a panel of interferon-inducible genes (IFIT2, CCL2 and CXCL10) between PBMC isolated before and 24 h after the first dose of 1018 ISS, using methods previously described (Friedberg et al, 2005). Changes in response to 1018 ISS therapy were measured as fold change, comparing day 8 to day 7. A greater than 2-fold increase in gene expression was considered the threshold for a biologically relevant change in gene expression.
Immunohistochemistry
We evaluated the tumor and injection site microenvironment using immunohistochemistry against the following antigens, in lymph node and skin biopsy samples: CD3, CD4, CD8, CD1a, CD68, S100, CD123, CD20, and FOXP3. Immunohistochemistry was performed using 4μm thick formalin-fixed, paraffin-embedded tissue sections. Two independent haematopathologists reviewed these specimens blinded to clinical outcome data, and scored degree of infiltration using a numeric scale. Tissue samples were scored on a relative qualitative scale comparing sites across all specimens for the range of scores, with the following designations: 0+ (rare to absent positive cells), 1+ (sparse infiltrate, scattered cells), 2+ (moderate infiltrate with more clustered perivascular cells), 3+ (dense perivascular infiltrate that cuffed vessels). Increased cells in the injection sites (change in ≥ 1 category) were determined relative to both the pre-injection and distant site. Only those with more frequent cells at injection sites relative to both pre-injection and distant sites were considered to have significant infiltrates.
Statistical Analysis
The primary endpoint of this study was the proportion of patients alive without progressive disease one year after study entry. This was estimated to be 50% in patients treated with rituximab alone, based on published reports at the time of study planning and initiation. Our study sought to enrol 30 patients, and was planned to have power of 0.89 to identify an increase from 50% to 75%, testing at the one-sided 0.05 level of significance, with the addition of 1018 ISS. The criterion for declaring efficacy required that at least 20 of the 30 enrolled patients be alive without progression at one year. Because of one case of early censoring, this proportion was ultimately reported using a Kaplan-Meier estimator, with standard deviation calculated by Greenwood’s formula. Changes from baseline in laboratory parameters were assessed as specified in the protocol; mean, standard deviation, median, and range of changes were provided as descriptive statistics. The proportion of patients experiencing significant changes was estimated, with 90% exact binomial confidence intervals (CIs). Statistical analysis was performed using SAS statistical software (Cary, NC), and the progression-free survival (PFS) curve was generated in R.
Patients
Between July 2004 and November 2006, 25 patients underwent baseline evaluation, and a total of 23 patients were enrolled and treated. We halted enrolment at this point (short of the planned 30 patients), as it was clear that it was not possible to achieve the minimum number of responses required to conclude that the efficacy of rituximab was enhanced by 1018 ISS. All patients enrolled completed the full treatment regimen, and were followed until evidence of disease progression through to study closure in May 2008. At study closure, the median follow-up time among the 4 patients that had not progressed was 31 months. Baseline clinical characteristics and prior therapies are detailed in Table 1. Of the 23 patients, 13 (57%) were male; all were white and non-Hispanic. The median age was 57 years, range 28 to 77 years. The majority of patients were diagnosed with grade 1 (52%) or 2 (38%) follicular lymphoma, presenting with advanced stage disease (96% stage III/IV) at screening. Most patients were asymptomatic at study entry (2 patients with documented B symptoms). Seven patients enrolled (30%) were rituximab naïve. Ten (43%) patients were FF genotype for the FCGR3A gene.
Table 1
Table 1
Baseline demographic and clinical patient characteristics
Adverse Events
Adverse events were minimal and limited to grade 1 and 2 toxicities with the exception of a single episode of grade III menorrhagia and a single episode of grade III elevated alanine transaminase, both thought to be unrelated to 1018 ISS. Commonly observed mild toxicities included fatigue, rituximab infusion reactions, and erythema at the 1018 ISS injection sites.
Clinical Response and Patient Outcome
Of the 23 patients, 11 (48%, 90% CI 30%, 66%) achieved a partial response (PR), complete response (CR) or unconfirmed CR (CRu) to therapy. Six additional patients (26%) maintained stable disease at day 90. Thus, the rate of being alive without progressive disease at day 90, was 74% (90% CI 55%, 88%). Thirteen of the patients who were without progressive disease at day 90 have subsequently progressed, including the 1 CR patient; 6 of the 8 patients who had a PR to this regimen; and all of the 6 patients who had stable disease at day 90. One patient who achieved a CRu was lost to follow-up (and thus censored) shortly after day 90, and one-year assessment of the patient could not be made.
The percentage of patients alive without progression at one year from study entry was estimated to be 41% (90% CI 23.7%, 58.3%). Median PFS is 8.8 months (Figure 1), and the 95% CI for the median was 5 months to 19.4 months. The 4 cases that remain alive without progressive disease have been followed for 2.7, 23.4, 38.0, and 44.2 months. Using a criterion that required statistical significance at the 0.05 level, none of the demographic or clinical variables investigated were associated with differential PFS. Additionally, there was also no evidence of an association between PFS and FCGR3A genotype.
Figure 1
Figure 1
Progression-free survival (N=23).
Correlative Studies
The results of these studies are summarized in Tables 2 and and3.3. There was no evidence of an association between PFS and biological response measures in any of these correlative studies.
Table 2
Table 2
Summary of Systemic Immunologic Response to 1018 ISS
Table 3
Table 3
Summary Tumour Microenvironment Response to 1018 ISS
Immunophenotyping
As expected, CD19 and CD20 cells were depleted in peripheral blood following rituximab therapy (Anolik et al, 2007). CD3 positive cells (as a percentage of PBMC) increased in 39% of patients following 1018 ISS compared to baseline. Evaluation of CD4, CD8, CD56, CD19, CD16, HLA-DR, CD25, CD45RA, CD45RO, CD45, CD80, and CD14 failed to show a signal in response to 1018 ISS.
T cell interferon gamma (IFN-γ) secretion
When compared to baseline, 25% of patients had a > 2-fold increase in IFN-γ secretion after stimulation as measured by ELISPOT following 1018 ISS therapy.
ADCC
The results of the chromium release assay showed that 35% of patients had more than a 10% increase in specific cell lysis on day 29 compared to baseline.
IFN-inducible gene expression
Based upon our experience in the phase I trial, three interferon alpha/beta-inducible genes, IFIT2, CCL2 and CXCL10, were evaluated as pharmacodynamic markers. As indicated in Table 2, over half of patients achieved a “doubling” of gene expression as measured by qPCR following 1018 ISS therapy, as compared to baseline. Patients whose CXCL10 expression at least doubled after 1018 ISS were more likely to have a response to therapy at Day 90, compared to those who did not demonstrate a doubling of CXCL10expression (OR=14, p=0.03).
Immunohistochemistry
Immunohistochemistry was performed on skin biopsy at baseline, and following therapy in both injection sites and in a remote skin site. Details of skin infiltration are summarized in Table 3. Almost half of the patients had an infiltrate consisting of cells positive for CD4, CD8, or CD68. Additionally, CD3 staining in the skin biopsy samples was evaluated in 11 patients. Seven samples (64%) demonstrated an increase in CD3 infiltration following 1018 ISS and rituximab therapy at the injection site while only 3 (27%) samples demonstrated an increase in CD3 infiltration at the distant skin biopsy site following treatment with 1018 ISS and rituximab.
Three patients had paired (pre-post) lymph node biopsies. While none of these pairs demonstrated a change in CD4 or S100 infiltration, they all demonstrated an increase in CD3 and CD8 infiltration following 1018 ISS as compared to before treatment. FOXP3 appeared to be decreased in one of the 3 samples, as shown in the tissue stain images in Figure 2. Three patients had paired pre-post bone marrow for immunohistochemistry analysis (all of which had bone marrow involvement with lymphoma at baseline screening). One showed CD4 increase; one showed CD8 increase; there was no change in either S100 or CD3; full CD20 depletion was observed in all three samples; and a FOXP3 increase in one patient and decrease in one patient was observed.
Figure 2
Figure 2
Immunohistochemical evaluation of tumour microenvironment response
Our study confirms the safety observed in our phase I trial of the combination of 1018 ISS and rituximab in patients with relapsed follicular lymphoma. Although our observed response rate of 48% was similar to published reports of single-agent rituximab for indolent lymphoma (Davis et al, 2000a), many of our patients had received prior rituximab in the context of chemotherapy combinations, rendering a more heavily pretreated group of patients than in the initial trials of single-agent rituximab. Randomized studies with contemporary controls are required to definitively determine the contribution of the 1018 ISS to outcome in combination with rituximab, particularly in an era where extended schedules of rituximab can provide PFS of significant duration (Friedberg, 2004; Ghielmini et al, 2004). However, in our study, polymorphisms for the FCGR3A receptor, which generally predict for poor short PFS following rituximab in patients with follicular lymphoma (Ghielmini et al, 2005; Weng & Levy, 2003), were not associated with PFS. The FCGR3A receptor mediates ADCC, and the poor prognosis variant (FF polymorphism) results in decreased effector cell binding to antibody. As patients in our trial with this variant were able to respond to the rituximab/TLR-9 agonist combination, it is possible that enhanced ADCC from 1018 ISS resulted in improved outcome for this subgroup of patients. We prospectively assessed ADCC in vitro using a standard chromium release assay, and demonstrated that the majority of patients had increases in ADCC of PBMC following 1018 ISS compared to baseline; including 5 (50%) patients with FF genotype.
Laboratory studies support this potential clinical observation, that ADCC, in the setting of monoclonal antibody therapy, may be enhanced by TLR-9 agonists. A recently published in vitro study activated peripheral blood mononuclear cells with CpG oligonucleotides, and co-cultured them with B cell lymphoma cells in the presence of rituximab (Moga et al, 2008). Rituximab-mediated ADCC was enhanced when PBMC were activated with CpG compared with control. NK cells appeared to be the main effector cells in this experiment, which supported the evaluation of the rituximab and CpG combination immunotherapeutic approach. In another experiment of a non-obese diabetic severe combined immunodeficient mouse model of acute lymphoblastic leukemia, a TLR-9 agonist induced a significant reduction in leukaemia burden through indirect effects, including the production of IL-12, IFN-α and IFN-γ, with NK cells appearing to be the main effector cell in this model (Fujii et al, 2007).
Single-agent 1018 ISS has been evaluated in a murine model of B-cell lymphoma (Ponzio et al, 2006). Growth inhibition of B lymphoma cells (RCS line) was observed, as well as the demonstration of tumour-specific cytotoxic T lymphocyte (CTL) responses, suggesting significant immunostimulatory activity. In another murine model, direct injection of TLR-9 agonist into B cell tumours resulted in eradication of lymphoma through a CD8 T-cell dependent mechanism (Li et al, 2007). TLR-9 expression was required either by the tumour or by the host, suggesting that antigen presentation by cells within the tumour through TLR-9 stimulation is an effective form of immunotherapy. Further work on this same model system has demonstrated that the addition of specific antibodies against different functional T-cell targets greatly enhances the anti-tumour activity of TLR-9 stimulation, and can “cure” murine lymphoma without chemotherapy (Houot et al, 2008).
Taken together, these studies suggest that the microenvironment of lymphoma may be critical in the response to TLR-9 agonists. In studies of follicular lymphoma treated with standard approaches, gene expression profiling analysis has revealed two major clusters of expressed genes, IR-1 and IR-2. The genes in IR-1 have a signature of infiltrating T cells, while IR-2 has genes expressed in macrophages and dendritic cells. Patients with an IR-1 signature had a more favourable prognosis than IR-2, suggesting that cells in the neoplastic microenvironment may predict clinical outcome (Dave et al, 2004). A follow up study reported that the presence of significant numbers of CD68+ macrophages within the tumour was associated with a poor outcome and, in a multivariate analysis, added to the International Prognostic Index (IPI)( The International Non-Hodgkin’s Lymphoma Prognostic Factors Project 1993) as a predictor of overall survival in patients with follicular lymphoma (Farinha et al, 2005). Other studies have also related prognosis to presence or absence of normal infiltrating immune cells (Alvaro et al, 2006), specifically high levels of CD8+ T cells (Wahlin et al, 2007), and high levels of FOXP3+ (Treg) T cells (Carreras et al, 2006).
In our study, we evaluated the microenvironmental effects of TLR-9 agonist in combination with rituximab. Skin biopsies were obtained at baseline, and following 1018 ISS therapy at both the injection site and a distant site. Following 1018 ISS, 9 patients (42%) had significant infiltration with CD8+ lymphocytes; and 9 patients had increased numbers of CD68+ cells at injection sites. Pre- and post-biopsies of tumour tissue (including lymph nodes and bone marrow) were performed in 6 patients, and in all cases a significant infiltration of T cells (CD8 > CD4) and macrophages were observed following treatment with 1018 ISS and rituximab, recapitulating the importance of CD8 T cells observed in the aforementioned mouse model. However, in the absence of data for rituximab alone, it remains unclear whether the local immune response changes are related to rituximab or the combination of rituximab plus 1018 ISS therapy. Our findings are in keeping with the recent publication by Haining et al (2008), who utilized 1018 ISS as a cancer vaccine adjuvant, and demonstrated a plasmacytoid dendritic cell-mediated chemokine response which resulted in T-cell migration to peripheral tissues. To our knowledge, our study is the first clinical trial to histologically confirm perturbation of the local immune malignant microenvironment following systemic biological therapy of follicular lymphoma. Additional trials are ongoing to further explore this observation in patients with lymphoma. Brody et al (2008) reported preliminary results of a clinical trial treating patients with indolent lymphoma with direct, intratumoural injection of another TLR-9 agonist (PF-3512676) following very low dose local radiation therapy. Systemic responses have been observed, suggesting the TLR-9 agonist-induced microenvironmental immunological effect enhanced an anti-tumour systemic immune response (Brody et al, 2008).
A concern over the use of TLR-9 agonists in the context of lymphoma therapy has been the potential for malignant B-cell proliferation. Similar to our phase I experience, we observed no evidence of normal or malignant B cell proliferation. A series of in vitro studies also confirmed no statistically significant increase in proliferation of follicular lymphoma cells in response to CpG oligonucleotides (Jahrsdorfer et al, 2005). The TLR-9 agonist PF-3512676 has been evaluated in vivo as a single agent administered intravenously to patients with relapsed lymphoma (Link et al, 2006); in a phase I trial for safety that enrolled 23 patients with previously treated NHL, enhanced ADCC was observed, and no evidence of disease progression was detected in this trial. Leonard et al (2007) published results of a phase I trial of this same TLR-9 agonist in combination with rituximab for patients with both indolent and aggressive NHL. In this study, 50 patients were enrolled, and the TLR-9 agonist was given either intravenously or subcutaneously for 4 or 8 weeks. Two patients with rituximab-refractory disease responded to the combination therapy, suggesting a synergistic effect. At doses of 0.24 mg/kg given in an extended schedule, increased adverse events, including neutropenia were noted, which was different from our experience. PF-3512676 has also demonstrated antineoplastic activity in combination with standard chemotherapy for advanced-stage non-small-cell lung cancer, suggesting an improved response rate and possible survival enhancement, with late responses suggesting an antitumor immune response (Manegold et al, 2008).
In our study, there was no single immunological response profile that predicted for favourable outcome. The interferon-inducible genes indentified in our phase I trial again appeared to be surrogates of CpG activity; the group of patients that exhibited the greatest fold induction of these genes had a higher response rate compared with other patients. ELISPOTS evaluating T-cell activity, and peripheral blood flow cytometry failed to detect significantly enhanced T-cell responses in the time period evaluated. Given the provocative findings in the few tumours we successfully obtained for study after exposure to 1018 ISS, we suggest that, in future trials, focused evaluation on local immune responses to TLR-9 agonists and other agents designed to manipulate the microenvironment (Friedberg et al, 2008) will be more informative than evaluating PBMC. Other biological approaches with cytokines to improving rituximab therapy in follicular lymphoma have been studied in humans, including IL-2 (Friedberg et al, 2002; Gluck et al, 2004), IL-12 (Ansell et al, 2002), granulocyte-macrophage colony-stimulating factor (Cartron et al, 2008) and interferon-alpha (Davis et al, 2000b). All of these studies have been limited by lack of randomization, and heterogeneous patient groups. Despite these limitations, most of these trials suggest improved PFS compared to historical controls of rituximab alone (Friedberg & Freedman, 2006). However, these agents all have substantially more toxicity than what we observed with the 1018 ISS/rituximab combination (LaCasce & Freedman, 2008). Moreover, the pleotropic effects of TLR-9 agonists have significant advantages over these cytokines, affecting many arms of both the innate and adaptive immune systems, and the ability to directly target the malignant microenvironment in a favourable way. In this light, further development of TLR-9 agonists with rituximab is warranted, which should include focused translational investigation on optimal dose, schedule, and route to fully realize the immunological potential of this rational combination.
Acknowledgments
Supported by CA-102216 (to JWF); CA-103244 (to ASF); HL-007152 (to JK), A1054953 (to TM) and RR 024160. JWF is a Clinical Scholar of the Leukemia and Lymphoma Society.
We thank the patients who participated in this study, and all of the nurses and physicians involved in their care. We thank Sally Quataert for assistance in sample organization and preparation, Matt Cochran for performing flow cytometric analysis, and Shelley Secor-Socha for ELISPOT analysis, and Karen Rosell for ADCC studies. Supported by CA-102216 (to JWF); CA-103244 (to ASF); HL-007152 (to JK), A1054953 (to TM) and RR 024160. JWF is a Clinical Scholar of the Leukemia and Lymphoma Society.
Footnotes
Conflict of Interest Statements
Conflict-of-interest disclosure: R.C and P.S. are employed by Dynavax Technologies; J.W.F. is on the Genentech Advisory Board.
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