Dania Caron

Phase I Study of Recombinant Human CD40 Ligand in Cancer Patients

PURPOSE To determine the toxicity, maximum-tolerated dose (MTD), and pharmacokinetics of recombinant human CD40 ligand (rhuCD40L) (Avrend; Immunex Corp, Seattle, WA), suggested in preclinical studies to mediate cytotoxicity against CD40-expressing tumors and immune stimulation. PATIENTS AND METHODS Patients with advanced solid tumors or intermediate- or high-grade non-Hodgkins lymphoma (NHL) received rhuCD40L subcutaneously daily for 5 days in a phase I dose-escalation study. Subsequent courses were given until disease progression. RESULTS Thirty-two patients received rhuCD40L at three dose levels. A total of 65 courses were administered. The MTD was 0.1 mg/kg/d based on dose-related but transient elevations of serum liver transaminases. Grade 3 or 4 transaminase elevations occurred in 14%, 28%, and 57% of patients treated at 0.05, 0.10, and 0.15 mg/kg/d, respectively. Other toxicities were mild to moderate. At the MTD, the half-life of rhuCD40L was calculated at 24.8 +/- 22.8 hours. Two patients (6%) had a partial response on study (one patient with laryngeal carcinoma and one with NHL). For the patient with laryngeal cancer, a partial response was sustained for 12 months before the patient was taken off therapy and observed on no additional therapy. Three months later, the patient was found to have a complete response and remains biopsy-proven free of disease at 24 months. Twelve patients (38%) had stable disease after one course, which was sustained in four patients through four courses. CONCLUSION The MTD of rhuCD40L when administered subcutaneously daily for 5 days was defined by transient serum elevations in hepatic transaminases. Encouraging antitumor activity, including a long-term complete remission, was observed. Phase II studies are warranted.

Preoperative Mobilization of Circulating Dendritic Cells by Flt3 Ligand Administration to Patients With Metastatic Colon Cancer

PURPOSE To evaluate preoperative dendritic cell (DC) mobilization and tumor infiltration after administration of Flt3 ligand (Flt3L) to patients with metastatic colon cancer. PATIENTS AND METHODS Twelve patients with colon cancer metastatic to the liver or lung received Flt3L (20 microg/kg/d subcutaneously for 14 days for one to three cycles at monthly intervals) before attempted metastasectomy. The number and phenotype of DCs mobilized into peripheral-blood mononuclear cells (PBMCs) were evaluated by flow cytometry. After surgical resection, metastatic tumor tissue was evaluated for DC infiltration. In vivo immune responses to recall antigens were measured. RESULTS After Flt3L administration, on average, the total number of leukocytes in the peripheral blood increased from 5.9 +/- 1.0 x 10(3)/mm(3) to 11.2 +/- 3.8 x 10(3)/mm(3) (mean +/- SD, P: =. 0001). The percentage of CD11c(+)CD14(-) DCs in PBMCs increased from 2.4% +/- 1.8% to 8.8% +/- 4.7% (P: =.004). Delayed-type hypersensitivity (DTH) responses to recall antigens (CANDIDA:, mumps, and tetanus) showed marginally significant increases in reactivity after Flt3L administration (P: =.06, P: =.03, and P: =.08, respectively). An increase in the number of DCs was observed at the periphery of the tumors of patients who received Flt3L compared with those of patients who had not. CONCLUSION Flt3L is capable of mobilizing DCs into the peripheral blood of patients with metastatic colon cancer and may be associated with increases in DC infiltration in the peritumoral regions. Flt3L mobilization is associated with a trend toward increased DTH responses to recall antigens in vivo. The use of Flt3L to increase circulating DCs for cancer immunotherapy should be considered.

Blood dendritic cells generated with Flt3 ligand and CD40 ligand prime CD8+ T cells efficiently in cancer patients.

Flt3 ligand mobilizes dendritic cells (DCs) into blood, allowing generation in vivo of large numbers of DCs for immunotherapy. These immature DCs can be rapidly activated by soluble CD40 ligand (CD40L). We developed a novel overnight method using these cytokines to produce DCs for cancer immunotherapy. Flt3 ligand-mobilized DCs (FLDCs) were isolated, activated with CD40L, loaded with antigenic peptides from influenza matrix protein, hepatitis B core antigen, NY-ESO-1, MAGE-A4, and MAGE-A10, and injected into patients with resected melanoma. Three injections were given at 4-week intervals. Study end points included antigen-specific immune responses (skin reactions to peptides alone or peptide-pulsed FLDCs; circulating T-cell responses), safety, and toxicity. No patient had a measurable tumor. Six patients were entered. FLDCs were obtained, enriched, and cultured under Good Manufacturing Practice grade conditions. Overnight culture with soluble CD40L caused marked up-regulation of activation markers (CD83 and HLA-DR). These FLDCs were functional and able to stimulate antigen-specific T cells in vitro. No significant adverse events were attributable to FLDCs. Peptide-pulsed FLDCs caused strong local skin reactions up to 60 mm diameter with intense perivascular infiltration of T cells, exceeding those seen in our previous peptide-based protocols. Antigen-specific blood T-cell responses were induced, including responses to an antigen for which the patients were naive (hepatitis B core antigen) and MAGE-A10. MAGE-A10–specific T cells with a skewed T-cell receptor repertoire were detected in 1 patient in blood ex vivo and from tumor biopsies. Vaccination with FLDCs pulsed with peptides is safe and primes immune responses to cancer antigens.

Multiple signals are required for maturation of human dendritic cells mobilized in vivo with Flt3 ligand

The ligand for the receptor tyrosine kinase fms‐like tyrosine kinase 3(Flt3L) is a growth factor for hematopoietic progenitors and inducesexpansion of the two distinct lineages of dendritic cells (DC) thathave been described in humans. These two lineages, DC1 and DC2, havebeen described according to their ability to induce naive T celldifferentiation to T helper cell type 1 (Th1) and Th2 effector cells, respectively. The immunoregulatory potential of DC1 and DC2 depends ontheir state of maturation and activation, which can be mediated byseveral molecules. Because monocyte‐derived DC1 produce interleukin‐12(IL‐12) when stimulated with CD40 ligand (CD40L), we hypothesized thatsimilar results would be obtained with DC1 mobilized by Flt3L. Unexpectedly, we found that immature DC expanded in vivo by Flt3Ltreatment could not be stimulated to produce IL‐12 in vitro using CD40Land/or interferon‐γ (IFN‐γ) alone. Instead, we found thatFlt3L‐mobilized DC from cancer patients require a sequence of specificsignals for maturation, which included initial treatment withgranulocyte macrophage‐colony stimulating factor followed by acombination of maturation signals such as CD40L and IFN‐γ. Flt3L‐mobilized DC matured in this manner possessed greater Tcell‐stimulatory function than nonmatured DC. The ability to generatephenotypically mature, IL‐12‐producing DC1 from peripheral bloodmononuclear cells mobilized by Flt3L will have important implicationsfor the development of effective cancer immunotherapystrategies.

Safety and Biological Activity of Repeated Doses of Recombinant Human Flt3 Ligand in Patients with Bone Scan-Negative Hormone-Refractory Prostate Cancer

Purpose: The purpose of this study was to evaluate the safety, biological activity, and feasibility of repeated doses of the dendritic cell (DC)-stimulating agent Flt3 ligand (FL) in patients with bone scan-negative hormone-refractory prostate cancer. Experimental Design: Thirty-one patients with hormone-refractory prostate cancer who had elevated prostate-specific antigen (PSA) levels and negative bone scans were enrolled. Six cycles (28 days each) were planned. In the first cycle, patients were randomized to FL or placebo. All patients received open-label FL during the next five courses. DC, anti-FL antibody, and PSA levels were measured every 15 days to assess biological activity. Results: DCs increased markedly in FL-treated patients from precycle to day 15, and the increase was consistent in each cycle. Mean percentages of DCs in peripheral blood ranged from 1.4% to 1.9% precycle and from 10.1% to 13.9% on day 15, and after the first cycle, absolute counts on day 15 were approximately 29-fold higher than precycle levels. Natural killer cell counts (CD56+) were found to be elevated after cycle 1 (154% increase versus 2.8% decrease in placebo group at day 22). Twenty-two of 27 patients tested developed nonneutralizing anti-FL antibody. The most frequently experienced toxicity was injection site reaction, followed by asthenia, rash, and diarrhea. Although median PSA levels did not vary during any cycle, a significant slowing in velocity of PSA was observed while patients were on-study (relative velocity = 0.002) compared with prestudy PSA velocity (relative velocity = 0.007). Conclusions: FL was well tolerated. FL consistently produced an increase in DC count without any evidence of decreasing response with continued exposure. The expansion of DCs and the slowing of PSA velocity after administration of FL suggest potential clinical applications in the immunotherapy of prostate cancer.

The use of Flt3 ligand as an adjuvant for hepatitis B vaccination of healthy adults

A phase I/II clinical trial was carried out to determine the safety of Flt3 ligand used as a vaccine adjuvant when administered to healthy human volunteers on two different schedules. In the first phase of this study, Flt3 ligand was administered SQ at a dose of 20 microg/kg (to a maximum of 1500 microg) every day (N=10) or every other day (N=10) for 1 week. The Flt3 ligand injection series was followed 1 day later by the first of three vaccinations with the licensed hepatitis B vaccine. In the second phase of the trial, 30 volunteers received either Flt3 ligand or placebo on the alternate day schedule in a randomized, double-blind design. The Flt3 ligand injections were safe and very well-tolerated. The number of lineage negative, HLA-DR(hi), CD11c(+), CD123(-) dendritic cells (DCs) increased 23-fold, and the lineage negative, HLA-DR(hi), CD11c(-), CD(123 bright) pre-DCs increased 6-fold. There was an associated increase in monocytes and WBCs in the Flt3 ligand recipients. Despite the marked increase in peripheral circulating dendritic cells, no increase was observed in the hepatitis B antibody titers induced after vaccination.

Biologic activity of interleukin 1 (IL-1) alpha in patients with refractory malignancies.

Interleukin Iα (IL-Iα) is a cytokine with pleiotropic effects, including cytotoxic–cytostatic activity against some tumor cell lines. We have conducted a phase I study of recombinant human IL-Iα (rhIL-Iα) in 17 patients with refractory malignancies to examine its toxicity and biologic activity. rhIL-Iα was given as a 2-h IV infusion daily for 5 days at five dose levels (0.08, 0.2, 0.8, 2.0, and 5.0 μg/m2). Seventeen patients with malignancies were treated, with no objective tumor responses noted. Common toxicities included: fever (100%), rigors and/or chills (96%), myalgia (54%), and headache (48%). Three patients developed grade III hypotension. The maximum tolerated dose was 2.0 μg/m2. rhIL-Iα induced a significant increase in absolute neutrophil count over baseline (p < 0.05), and delayed but significant increase in platelet count over baseline (p < 0.05), and there was a marked increase in the number of progenitors [colony-forming units (CFU)-G, CFU-M, CFU-GM, CFU-GEMM and burst-forming units (BFU-E)] observed in the peripheral blood. Nine of 12 evaluable patients showed an increase in bone marrow cellularity or myeloid:erthyroid ratio. Immunophenotyping did not demonstrate an increase in peripheral blood or bone marrow CD34+ cells. Interferon-γ-mediated monocyte cytotoxicity (MCCTX) was significantly enhanced from baseline (p < 0.001), although an increase in direct MCCTX did not reach statistical significance. In summary. rhIL-Iα administration is well tolerated at a close of 2.0 μg/m2 with fever, rigors, myalgia, and headache being the most frequent toxicities. Although there were no objective tumor responses, we have demonstrated significant biologic activity with increased neutrophil and platelet counts, increased peripheral blood progenitor cells, and enhanced interferon-γ-mediated MCCTX.