The present trial was designed to assess the feasibility of subcutaneous low-dose interleukin-2 (IL-2) given for 3 months in an outpatient setting. Twenty patients with advanced cancers (16 metastatic renal cell carcinoma) were included in this phase I study at the following three dose levels: 1, 3, and 6 x 10(6) IU/day (groups of 6, 6, and 8 patients, respectively). IL-2 was administered once daily 6 days a week for 12 weeks. Complete therapy was achieved in 13 of 20 patients, whereas 5 of 20 received at least 5 weeks of IL-2. Minor dose-dependent toxicities were observed including fatigue, transient grade 2-3 fever (11 of 18), and grade 1-2 digestive disorders (6 of 18) without significant biologic modifications but two cases of hypothyroidism. Doses were decreased from 6 to 3 x 10(6) IU/day in one patient (fever and allergic edema). All patients developed transient subcutaneous nodules at the injection sites. These side effects never required hospitalization nor discontinuation of therapy. A dose-dependent and sustained increase in peripheral blood eosinophils and lymphocytes was observed, demonstrating that subcutaneous injections in this low-dose range could have similar biologic effects to those achieved with more intensive schedules. Because it is safe, practicable, and low in cost, we conclude that s.c. low-dose IL-2 could be useful for the design of immunomodulation trials with potential new application fields.
The recent cloning of tumor-associated antigens (TAAs) recognized by CD8+ T lymphocytes (TCD8+) has made it possible to use recombinant and synthetic forms of TAAs to generate TCD8+ with anti-tumor activity. To explore new therapeutic strategies in a mouse model, we retrovirally transduced the experimental murine tumor CT26(H-2d), with the lacZ gene encoding our model TAA, beta-galactosidase (beta-gal). The transduced cell line, CT26.CL25, grew as rapidly and as lethally as the parental cell line in normal, immunocompetent animals. In an attempt to elicit TCD8+ directed against our model TAA by using purely recombinant and synthetic forms of our model TAA, we synthesized a nine-amino-acid long immunodominant peptide of beta-gal (TPH-PARIGL), corresponding to amino acid residues 876-884, which was known to be presented by the Ld major histocompatibility complex (MHC) class I molecule, and a recombinant vaccinia virus encoding the full-length beta-gal protein (VJS6). Splenocytes obtained from naïve mice and co-cultured with beta-gal peptide could not be expanded in primary ex vivo cultures. However, mice immunized with VJS6, but not with a control recombinant vaccinia virus, yielded splenocytes that were capable of specifically lysing CT26.CL25 in vitro after co-culture with beta-gal peptide. Most significantly, adoptive transfer of these cells could effectively treat mice bearing 3-day-old established pulmonary metastases. These observations show that therapeutic TCD8+ directed against a model TAA could be generated by using purely recombinant and synthetic forms of this antigen. These findings point the way to a potentially useful immunotherapeutic strategy, which has been made possible by the recent cloning of immunogenic TAAs that are expressed by human malignancies.
Interferons (IFNs) are known to have antiviral effects and have been shown to enhance the expression of tumor-associated antigens (TAA) on different target cells. In our current study, we investigated the potential of IFN-alpha or IFN-gamma to enhance the expression of the TAAs recognized by monoclonal antibodies (MAbs) 19-9, B72.3, 17-1A, and BR55-2 on pancreatic cancer cell lines and the potential of IFN-gamma to modulate the expression of a single TAA, BR55-2, on nonpancreatic cancer cell lines. Expression of these TAAs, percentage of positive cells and mean fluorescence intensity, was measured by flow cytometry. In these studies, we provide evidence that one prostate (DU 145) and two pancreatic (HPAF and BxPC-3) cancer cell lines that moderately express BR55-2 can be upregulated by IFN-gamma treatment, with optimal enhancement occurring between 48 and 72 h with 1,000 IU/ml. Cell lines that highly expressed BR55-2 could not be further upregulated by the doses of IFNs tested during the various periods used. IFN-alpha or IFN-gamma treatments did not significantly change the levels of TAA expression on pancreatic cancer cell lines that bound MAbs 17-1A or 19-9. Cell lines that did not bind MAbs 17-1A, 19-9, B72.3, or BR55-2 before IFN treatments could not be induced to express these antigens after treatment. Although antigen expression does not ensure detectable therapeutic benefit, increased antigen expression on tumor tissues may augment the efficacy of MAbs bearing radionuclides, toxins, or effector cells to the tumor site. In each of these situations, the use of IFNs to enhance TAA expression, particularly IFN-gamma, may merit consideration.
The current study was designed to characterize nitric oxide (NO.) synthesis during interleukin-2 (IL-2) treatment of mice, and to determine whether NO. mediated IL-2-induced "vascular leak." We developed a technique for chronic subcutaneous infusion of the NO. synthase inhibitor N omega monomethyl-L-arginine (MLA) via osmotic minipump to aid in further study of these processes. After IL-2 administration to C3H/HeN mice (180,000 IU i.p. b.i.d. for 5 days), NO. synthesis increased two-to-three fold, peaking on days 5-8. Administration of MLA reduced NO. synthesis in both IL-2-treated mice (from 2.7 to 1 microM/mouse/day), and normal mice (from 1 to 0.5 microM/mouse/day). This agent decreased IL-2-induced radiolabeled albumin accumulation in the liver after i.p. IL-2 administration (p < 0.02). MLA infusions resulted in minimal systemic toxicity in mice, as reflected by complete blood counts or serum chemistries. MLA also did not impair lymphokine-activated killer cell induction in vitro or in vivo, or alter IL-2-induced tumor responses in a 3-day pulmonary metastasis model. These experiments demonstrated that NO. is a mediator involved in the genesis of vascular permeability induced by IL-2 treatment. Studies designed to further evaluate the toxicity and usefulness of MLA infusions to modify this IL-2 induced toxicity appear to be warranted.
The thymus is the site of T-cell maturation and contains T-cell precursors that differentiate into cytolytic T lymphocytes (CTLs) in vitro in the presence of interleukin-2 (IL-2). Malignant thymoma is often associated with a lymphocytic infiltration of these precursors. The antitumor effects of IL-2 are mediated in part by activated CTLs. Based on these considerations and anecdotal reports of its anti-tumor activity in thymoma, we conducted a Phase II trial of IL-2 in 14 patients with thymoma. IL-2 was administered s.c. at a dose of 12 x 10(6) IU/m2/day for 5 days for 4 weeks followed by a 2-week rest period. Patients were evaluated for response after each 6-week cycle, and those tolerating therapy with no disease progression were eligible for a maximum of 4 cycles. All patients had failed prior standard chemotherapy and 12 had received prior radiotherapy. All 14 patients were evaluable for toxicity and response. The median number of cycles received was two. One patient was removed from study during cycle 1 because of severe bronchospasm. Five patients required dose reductions for grade 3 toxicity (anorexia, nausea, hyperbilirubinemia, elevated SGPT, and skin desquamation, one patient each). Two patients developed new symptoms of myasthenia gravis while in the study and were removed (one for progressive disease, one for steroid requirement). There were no objective responses. The one patient who required steroids for newly diagnosed myasthenia gravis had a minor response. We conclude that subcutaneously administered IL-2, although it has acceptable toxicity, has no significant clinical activity in previously treated patients with advanced thymoma.
Because the requirement for long-term cell culture can make adoptive cellular immunotherapy cumbersome, experiments were designed to determine whether smaller numbers of tumor-sensitized T cells activated briefly with bryostatin 1 and ionomycin (B/I) could be returned immediately to recipient mice without in vitro expansion and still have an anti-tumor effect in vivo. Popliteal tumor-draining lymph nodes (DLNs) from mice bearing progressive MCA-105 and MCA-203 footpad sarcomas were harvested and treated for 18 h with B/I. These cells were then washed and transferred immediately to naive C57B1/6 mice. In some experiments, these mice were irradiated (500 rads) before adoptive transfer and were given interleukin-2 (IL-2, 7,500 IU i.p., b.i.d. for 3 days) after receiving the activated lymphocytes. Recipient mice were challenged with sarcoma cells (4 x 10(5) i.v.) 6 to 32 days after receiving the activated lymphocytes. Mice receiving 10(6) B/I-activated lymphocytes before tumor challenge had significantly fewer metastases than did controls. This protective effect did not require exogenous IL-2 or host irradiation. Using Thy-1 congenic donors, it was shown that B/I-activated T cells expanded in recipients when IL-2 was also given, and these cells were a prominent component (15% of total cells) in the infiltrates found in the lungs of mice 7 days after i.v. tumor challenge. Combining these B/I-"pulsed" cells with cyclophosphamide (CYP) and IL-2 to treat mice with established (3-day) metastases resulted in significant reduction in pulmonary nodules, with complete regression in many of the treated mice, which was rarely seen with CYP alone or with CYP + IL-2. Thus, adoptive transfer of tumor-sensitized, B/I-activated DLN cells confers protection against i.v. tumor challenge, without prior in vitro expansion of the effector cells. Phenotyping studies demonstrate that donor cells activated with B/I do expand in recipient mice after adoptive transfer and can move to sites of tumor. Moreover, these cells can mediate a therapeutic effect on established tumor metastases, when combined with chemotherapy.
A multicenter, phase II trial of continuous-infusion interleukin 2 (IL-2) was done in the Southwest Oncology Group to evaluate the efficacy and safety of this treatment in a broad-based population of patients with metastatic renal-cell carcinoma. Forty-seven patients from 11 different institutions were entered in this study, with 43 eligible. Two technically ineligible patients who received treatment and for whom records are available are included in the data analysis. Thus, there are 45 analyzable patients. Of these patients, performance status was 0 in 58% and 1 in 42%. Thirty-one patients had a prior nephrectomy, and 12 patients had received prior therapy. IL-2 was initially given at a dose of 4.5 x 10(6) Roche U/m2/day, 4 days a week, for 4 weeks in a row, followed by a 3-week rest period. Because of the difficulty in obtaining reimbursement for the hospitalization required on the days of IL-2 administration, after 10 patients had been entered, the treatment regimen was changed to 6 x 10(6) Roche U/m2/day for 4 days as an inpatient, followed by 2 weeks of potential outpatient treatment at a dose of 3 x 10(6) Roche U/m2/day for 4 days each week. This was followed by a 2-week rest period. Within the 45 analyzable patients, there were 0 complete responses and 6 partial responses, for a response rate of 13% (95% confidence interval 5.1-27%). Responses occurred in lung metastases, nodal disease, and in one patient with bone metastases and the primary kidney tumor. Response durations were 1 month, 1 month, 14+ months, 19 months, 26+ months, and 27 months. Of 12 patients with a nephrectomy and only lung metastases, 4 showed partial responses. Medial survival for all analyzable patients is 15 months (95% confidence interval 8-20 months). Toxicity was significant, with nausea and vomiting, diarrhea, fever and chills, dermatologic changes, and fatigue the most frequent. There were 18 instances of grade 4 toxicity, with the most common grade 4 toxicity, respiratory, found in 8 patients. There were two early deaths of probable heart-related causes while receiving treatment. A continuous-infusion IL-2 regimen that allows some potential outpatient treatment shows effectiveness and toxicity similar to that in other multicenter IL-2 infusion trials and high-dose intravenous bolus regimens.
Previous reports of autologous bone marrow transplant (auto-BMT) have demonstrated that myeloablative therapy followed by cyclosporin A (CsA), with and without interferon (IFN), can generate autoreactive cytotoxic T lymphocytes (auto-CTL) with potential therapeutic benefit. This is the first report of an attempt to generate auto-CTL using CsA and IFN after a non-myeloablative regimen. Cyclophosphamide (CTX) 1,200 mg/m2 i.v. day 1 was followed by CsA and IFN-alpha days 2-28, administered in a sequential three-step Phase I dose-escalation scheme. Patients were evaluated twice weekly for clinical evidence of graft-versus-host (GVH) reaction. Peripheral blood mononuclear cells (PBMCs) were obtained before treatment, at time of clinical GVH reaction, and days 21 and 28, and analyzed for auto-CTL, natural killer (NK) cell, and lymphokine-activated killer (LAK) cell activity. Patients also underwent punch skin biopsy at the time of clinical GVH reaction or day 21 to identify histologic evidence of GVH. Fourteen patients completed therapy and were evaluable for immunologic studies and anti-tumor response. No increase in auto-CTL, NK cell, or LAK cell activity was seen. Clinical or histologic evidence of GVH reaction did not occur. We conclude that this myelosuppressive dose of CTX combined with CsA and IFN is unable to generate clinical or immunologic evidence of an auto-GVH reaction. Further efforts are warranted to evaluate other therapeutic attempts to generate auto-CTL with anti-tumor activity based on preliminary results of clinical benefit in auto-BMT.
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