1 Department of Medicine, Section of Hematology/Oncology; 2 Department of Surgery, Section of Urology; 3 University of Chicago Cancer Research Center; 4 Department of Pathology, University of Chicago, Chicago, IL, USA
*Correspondence to: Dr T. F. Gajewski, University of Chicago, 5841 South Maryland Ave, MC 2115, Chicago, IL 60637, USA. Tel: +1-773-702-4601; Fax: +1-773-702-3701; Email: tgajewsk{at}medicine.bsd.uchicago.edu
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Abstract |
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Patients and methods: Rituximab (375 mg/m2) weekly for 4 weeks. IL-2 [11 (million units) daily] s.c., 4 days a week for weeks 58, followed by a 2-week rest (weeks 9 and 10). Patients without disease progression continued on IL-2. Disease re-evaluation was performed after rituximab and after every course of IL-2.
Results: Fifteen patients with RCC and six with melanoma were enrolled. One patient had a partial response and seven patients had stable disease. Toxicities were similar to those expected with IL-2 alone, and there were no grade 4 events. Circulating B cells were depleted in all patients. The subsequent low-dose IL-2 increased absolute numbers of natural killer cells, activated CD4+ and activated CD8+ T cells. Expanded T cells produced interferon-, but not IL-4. Proliferation of peripheral blood lymphocytes to phytohemagglutinin was diminished following rituximab treatment, suggesting that B cells participate in this response in vitro.
Conclusions: Our results suggest that depletion of circulating B cells with rituximab does not increase the response rate, alter the toxicity profile or change the biological activity in response to low-dose IL-2 in patients with RCC or melanoma.
Key words: clinical trial, cytokines, immunoregulation, immunotherapy
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Introduction |
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There is accumulating preclinical evidence that normal host B lymphocytes may interfere with T-cell-mediated tumor rejection. Brodt and colleagues reported that mice depleted of immunoglobulin (Ig)-bearing lymphocytes had increased antitumor immunity in vivo [8]. Schreiber and colleagues found that mice reconstituted to lack B cells showed slower tumor growth and higher rejection rates [9
]. Qin et al. observed superior tumor rejection in B-cell-deficient mice compared with control mice [10
]. Our laboratory has confirmed this notion using TCR transgenic T cells. 2C/RAG2/ T cells that recognize the alloantigen Ld [11
] were adoptively transferred into either RAG2-deficient mice (which lack T cells and B cells) or TCR
-knockout mice (which lack T cells but contain B cells). Upon subcutaneous challenge with Ld-expressing P1.HTR tumor cells [12
], RAG2/ but not TCR
/ mice successfully rejected the tumor (Markiewicz and Gajewski, unpublished data). These observations support a negative regulatory role for B cells in countering T-cell-mediated tumor elimination.
Previous studies in patients with lymphoma have shown that treatment with rituximab, in addition to causing regression of malignant CD20+ lymphomas, also results in a rapid and sustained depletion of circulating B cells, without significant toxicity [1315
]. These observations enabled the design of a phase II trial of B-cell depletion with rituximab in patients with RCC and melanoma before treatment with low-dose IL-2, in an attempt to eliminate the putative negative regulatory effect of B cells on T-cell-mediated tumor rejection.
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Patients and methods |
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Exclusion criteria included: previous IL-2 therapy; use of chemotherapy, radiotherapy or biological therapy within 4 weeks before enrolment; concurrent systemic corticosteroids (except physiologic replacement doses) or other immunosuppressive medications; and clinically significant autoimmune disease. Patients with active infections including HIV, chronic active hepatitis, or known hepatitis B or C were excluded. Lactating or pregnant women were excluded and patients of reproductive age must have agreed to use an acceptable method of birth control.
Treatment
This was an open-label, non-randomized, single-institution study. Patients were treated on an outpatient basis. Treatment consisted of four, weekly doses of rituximab (375 mg/m2 per dose, weeks 14), followed by IL-2 administration. IL-2 was given subcutaneously (11 x 106 IU daily, 4 days a week) for 4 weeks (weeks 58), followed by a 2-week rest (weeks 9 and 10). If the total B-cell number recovered to 50% of pre-treatment values during weeks 9 or 10 of any treatment cycle, rituximab was to be readministered in four, weekly doses, as above, before further administration of IL-2.
If at the time of scheduled treatment a patient experienced grade 3 toxicity, treatment with rituximab or IL-2 therapy was withheld for up to 2 weeks until the toxicity decreased to grade
1 or less. A single 50% reduction in the dose of IL-2 was allowed at the discretion of the treating physician. If any toxicity did not return to grade
1 within 3 weeks, the patient was removed from the study.
Response and toxicity
Evaluation of clinical response was the primary end point. Response evaluation was carried out after rituximab alone and after every two cycles of IL-2 therapy. Tumor measurements were analyzed according to RECIST (response evaluation criteria in solid tumors) criteria. Toxicity was graded according to standard National Cancer Institute common toxicity criteria (NCI-CTC version 2.0).
Immunologic monitoring
Immunologic assessments were performed before therapy, after the fourth dose of rituximab, and every 2 weeks while on IL-2. The clinical chemistry laboratory measured quantitative immunoglobulin levels. The numbers of T, B and NK cells, as well as of HLA-DR+ CD4+ and CD8+ T cells were analyzed by flow cytometry as described previously [16]. Numbers of CD4+ and CD8+ T cells capable of producing IL-2, IFN-
and IL-4 were analyzed following stimulation with phorbol myristate acetate (PMA) + ionomycin and intracellular cytokine staining. Briefly, heparinized blood was centrifuged and plasma was removed. Cells were resuspended in RPMI-1640 medium with L-glutamine and monensin
Half the sample was activated by the addition of PMA
and ionomycin
the other half was set aside as a control. Cells were incubated for 4 h at 37°C, stained with fluorescein isothiocyanate-conjugated anti-CD8 and PerCP-conjugated anti-CD3, and the red blood cells were lysed with fluorescence-activated cell sorter (FACS) Lysing Solution (BD Biosciences, San Jose, CA, USA). The remaining cells were washed, permeabilized and stained for intracellular cytokines using phycoerythrin-conjugated anti-IL-2, anti-IL-4 or anti-IFN-
(BD Biosciences). Data were acquired using a FACScanTM flow cytometer (Becton Dickenson Immunocytometry Systems, San Jose, CA, USA) and analyzed with FlowJo software© (Stanford University, 19951996; Tree Star, Inc., 19972002). The number of CD8+ T cells producing cytokine was determined after subtracting background staining.
Ex vivo proliferation in response to phytohemagglutinin (PHA) was measured in batch fashion on cryopreserved peripheral blood mononuclear cells (PBMCs). PBMCs were isolated using LymphoprepTM (Axis-Shield, Oslo, Norway) and cryopreserved. For each patient, cryopreserved PBMCs from all time points were thawed and resuspended in RPMI-1640 medium supplemented with penicillin/streptomycin, L-glutamine and 10% fetal bovine serum. PBMCs were plated in triplicate on 96-well plates with PHA (Sigma, St Louis, MO, USA) at 5 µg/ml. After 72 h incubation at 37°C, the cells were pulsed with [3H]thymidine (1 µCi/well). After another 18 h, the cells were harvested using a FiltermateTM harvester and incorporated [3H]thymidine was detected using a TopCount® NXTTM microplate scintillation and luminescence counter (Packard BioScience, Meriden, CT, USA).
Statistical considerations
Previous studies had documented response rates of 15% with IL-2 in melanoma and RCC. A total of 15 melanoma patients and 15 renal cell carcinoma patients were intended to be treated. If two or more of the 15 patients in either group responded, the regimen would be considered active, warranting further study in that disease. However, when only a single clinical response was seen in the first total of 21 patients, the study was closed to accrual. Estimates of time to progression and survival were determined using the KaplanMeier method. Immunologic parameters were characterized with descriptive statistics.
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Results |
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No objective responses were observed after treatment with rituximab alone. Among the 21 evaluable patients, there was one partial response following IL-2 administration in a patient with RCC. That response remained durable for at least 28 months with no further therapy. Seven patients with RCC and two patients with melanoma had stable disease.
The median time to progression was 15.4 weeks (range 3.3115.7 weeks) and the median overall survival was 77 weeks (range 14.9135 weeks). The median duration of stable disease was 26.7 weeks (range 8.971 weeks) (data not shown).
Adverse events
Table 2 lists the observed adverse events. The majority of adverse events were grade 1 or 2, and there were no grade 4 toxicities. Anemia was the most common hematologic toxicity and grade 2 neutropenia was observed in two patients (10%). The most common non-hematological toxicity was grade 12 fever and/or chills. Other common toxicities were fatigue, anorexia and injection-site reactions. None of the 21 patients developed opportunistic infections. The observed toxicities were similar to those seen in other clinical trials using similar doses of IL-2. No significant toxicities were observed after treatment with rituximab.
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Discussion |
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Although multiple experimental preclinical models were encouraging, our clinical trial did not show any benefit of B-cell depletion with rituximab as an adjunct to IL-2 therapy. Rituximab is directed against the CD20 antigen expressed by normal pre-B- and mature B-lymphocytes. CD20 is not found on the surface of plasma cells, and whether rituximab effectively depletes tissue-based B cells is unclear. The lack of diminution in circulating Ig levels suggests that incomplete depletion of B-lineage cells is achieved with rituximab. The potentiation of antitumor immunity noted in murine models by the absence of B cells may be mediated by a feature of B-lineage cells not negated by rituximab.
Several models have been proposed to explain the inhibitory effect of B cells on antitumor immunity in murine studies. These include regulation by antigenantibody complexes [18], linkage of specific IgG to latent transforming growth factor-ß (TGF-ß) [19
], secretion of IL-10 and consequent inhibition of Th1 responses [20
], and Th2 promotion through antigen presentation by B cells [17
]. Of note, B-cell depletion with rituximab did not alter the predominant Th1 cytokine profile seen, arguing against the latter mechanism.
Although the use of rituximab to deplete B cells did not augment clinical activity in response to IL-2, it could theoretically potentiate responses to other immunotherapeutic interventions, such as vaccines or adoptive T-cell therapy. B-cell depletion might favor antigen presentation by dendritic cells, and also could facilitate homeostatic proliferation of transferred T cells. Future studies will be required to explore these possibilities.
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Acknowledgements |
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Received for publication March 9, 2004. Revision received March 17, 2004. Accepted for publication March 23, 2004.
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References |
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