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Biomedical subjects

B Gitlitz

Publications and source records attributed to B Gitlitz.

9 recordsLinked to original sources
PubMed2001

Induction of G250-targeted and T-cell-mediated antitumor activity against renal cell carcinoma using a chimeric fusion protein consisting of G250 and granulocyte/monocyte-colony stimulating factor.

Immunotherapy targeting for the induction of a T-cell-mediated antitumor response in patients with renal cell carcinoma (RCC) appears to hold significant promise. Here we describe a novel RCC vaccine strategy that allows for the concomitant delivery of dual immune activators: G250, a widely expressed RCC associated antigen; and granulocyte/macrophage-colony stimulating factor (GM-CSF), an immunomodulatory factor for antigen-presenting cells. The G250-GM-CSF fusion gene was constructed and expressed in Sf9 cells using a baculovirus expression vector system. The Mr 66,000 fusion protein (FP) was subsequently purified through a 6xHis-Ni2+-NTA affinity column and SP Sepharose/fast protein liquid chromatography. The purified FP retains GM-CSF bioactivity, which is comparable, on a molar basis, to that of recombinant GM-CSF when tested in a GM-CSF-dependent cell line. When combined with interleukin 4 (IL-4; 1000 units/ml), FP (0.34 microg/ml) induces differentiation of monocytes (CD14+) into dendritic cells (DCs) expressing surface markers characteristic for antigen-presenting cells. Up-regulation of mature DCs (CD83+CD19-; 17% versus 6%) with enhanced expression of HLA class I and class II antigens was detected in FP-cultured DCs as compared with DCs cultured with recombinant GM-CSF. Treatment of peripheral blood mononuclear cells (PBMCs) with FP alone (2.7 microg/10(7) cells) augments both T-cell helper 1 (Th1) and Th2 cytokine mRNA expression (IL-2, IL-4, GM-CSF, IFN-gamma, and tumor necrosis factor-alpha). Comparison of various immune manipulation strategies in parallel, bulk PBMCs treated with FP (0.34 microg/ml) plus IL-4 (1000 units/ml) for 1 week and restimulated weekly with FP plus IL-2 (20 IU/ml) induced maximal growth expansion of active T cells expressing the T-cell receptor and specific anti-RCC cytotoxicity, which could be blocked by the addition of anti-HLA class I, anti-CD3, or anti-CD8 antibodies. In one tested patient, an augmented cytotoxicity against lymph node-derived RCC target was determined as compared with that against primary tumor targets, which corresponded to an 8-fold higher G250 mRNA expression in lymph node tumor as compared with primary tumor. The replacement of FP with recombinant GM-CSF as an immunostimulant completely abrogated the selection of RCC-specific killer cells in peripheral blood mononuclear cell cultures. All FP-modulated peripheral blood mononuclear cell cultures with antitumor activity showed an up-regulated CD3+CD4+ cell population. These results suggest that GM-CSF-G250 FP is a potent immunostimulant with the capacity for activating immunomodulatory DCs and inducing a T-helper cell-supported, G250-targeted, and CD8+-mediated antitumor response. These findings may have important implications for the use of GM-CSF-G250 FP as a tumor vaccine for the treatment of patients with advanced kidney cancer.

Animals↗
PubMed1999

Presentation of renal tumor antigens by human dendritic cells activates tumor-infiltrating lymphocytes against autologous tumor: implications for live kidney cancer vaccines.

The clinical impact of dendritic cells (DCs) in the treatment of human cancer depends on their unique role as the most potent antigen-presenting cells that are capable of priming an antitumor T-cell response. Here, we demonstrate that functional DCs can be generated from peripheral blood of patients with metastatic renal cell carcinoma (RCC) by culture of monocytes/macrophages (CD14+) in autologous serum containing medium (RPMI) in the presence of granulocyte macrophage colony-stimulating factor and interleukin (IL) 4. For testing the capability of RCC-antigen uptake and processing, we loaded these DCs with autologous tumor lysate (TuLy) using liposomes, after which cytometric analysis of the DCs revealed a markedly increased expression of HLA class I antigen and a persistent high expression of class II. The immunogenicity of DC-TuLy was further tested in cultures of renal tumor infiltrating lymphocytes (TILs) cultured in low-dose IL-2 (20 Biologic Response Modifier Program units/ml). A synergistic effect of DC-TuLy and IL-2 in stimulating a T cell-dependent immune response was demonstrated by: (a) the increase of growth expansion of TILs (9.4-14.3-fold; day 21); (b) the up-regulation of the CD3+ CD56- TcR+ (both CD4+ and CD8+) cell population; (c) the augmentation of T cell-restricted autologous tumor lysis; and (d) the enhancement of IFN-gamma, tumor necrosis factor-alpha, granulocyte macrophage colony-stimulating factor, and IL-6 mRNA expression by TILs. Taken together, these data implicate that DC-TuLy can activate immunosuppressed TIL via an induction of enhanced antitumor CTL responses associated with production of Thl cells. This indicates a potential role of DC-TuLy vaccines for induction of active immunity in patients with advanced RCC.

Antigen Presentation↗
PubMed1998

Adenovirus-mediated interleukin-2 production by tumors induces growth of cytotoxic tumor-infiltrating lymphocytes against human renal cell carcinoma.

Combination therapy with interleukin-2 (IL-2) and tumor-infiltrating lymphocytes (TILs) demonstrates significant clinical activity in patients with metastatic renal cell carcinoma (RCC). To investigate whether local delivery of IL-2 via gene transfer is capable of improving the potency and efficacy of in vitro propagated TILs as compared with standard growth conditions [400 BRMP U (BU)/ml], a replication-deficient adenovirus expressing the human IL-2 gene under control of the cytomegalovirus (CMV) promoter (Ad-IL-2) has been constructed in our laboratory. RCC-TIL cultures were initiated by directly infecting RCC tumor suspension with Ad-IL-2 at a multiplicity of infection of 10:1. Subsequently the TIL cultures were restimulated with nonirradiated autologous RCC infected with Ad-IL-2 (RCC-Ad-IL-2) every 10 days (TIL/tumor = 50:1). Cell growth, phenotype, cytotoxicity, and cytokine messenger RNA (mRNA) expression were analyzed and compared with TIL growth stimulated with exogenous IL-2 (400 BU/ml). All five TILs tested responded to RCC-Ad-IL-2 activation, and a completed clearance of tumor cells was observed in cultures within 7-10 days. Lysis of nonirradiated RCC-Ad-IL-2 cells by TILs also was observed in cultures 3-5 days after restimulation. The IL-2 concentration in cell culture supernatants was maintained between 10 BU and 35 BU/ml (2 and 7 ng/ml), respectively. When compared with exogenous IL-2, RCC-Ad-IL-2 induced less growth expansion of TILs whereas a reduced CD56+ (23 +/- 14% vs. 44 +/- 13%; p < 0.05) but increased CD3+CD4+ cell population (32 +/- 11% vs. 15 +/- 6%; p < 0.05) with enhanced T cell-receptor use (59 +/- 10% vs. 42 +/- 7%; p < 0.005) was determined. An augmented human leukocyte antigen (HLA)-restricted and tumor-specific cytotoxicity was detected in RCC-Ad-IL-2-expanded TILs (day 35, 15.3 +/- 4.2 LU vs. 4.6 +/- 1.8 LU; p < 0.005). These properties were mediated by the CD8+ and CD4+ T-cell populations, as demonstrated by antibody-blocking assays. A unique cytokine profile also was detected in RCC-Ad-IL-2-induced TILs, which demonstrated an upregulation of both GM-CSF and IL-6 mRNA as compared with TILs expanded in the presence of exogenous IL-2. These data suggest that RCC-Ad-IL-2 is a potent immune stimulant that can be used in vitro as an immunogen to propagate cytotoxic RCC-TILs for adoptive immunotherapy or potentially in vivo by direct injection as a live tumor vaccine.

Adenoviridae↗
PubMed1997

Treatment of metastatic renal cell carcinoma with nephrectomy, interleukin-2 and cytokine-primed or CD8(+) selected tumor infiltrating lymphocytes from primary tumor.

PURPOSE: Metastatic renal cell carcinoma is a disease with a mean survival of 6 to 10 months. Interleukin-2 (IL-2), the only approved therapy for metastatic renal cell carcinoma, is associated with a 14% response rate and durable remissions in some patients with high performance status. We performed a series of trials of IL-2 plus tumor infiltrating lymphocyte cell therapy and report the clinical results from 62 patients enrolled in these trials. MATERIALS AND METHODS: Patients were eligible if they had metastatic renal cell carcinoma with the primary tumor in place and an Eastern Cooperative Oncology Group performance status of 0 or 1. Patients were treated with cytokines before nephrectomy and preparation of cytokine primed tumor infiltrating lymphocytes or CD8(+) tumor infiltrating lymphocytes were isolated for infusion into patients. Of 62 patients enrolled 55 were treated with tumor infiltrating lymphocytes and IL-2, and were evaluable for toxicity, response and survival. RESULTS: There were no postoperative mortalities. Of the patients 7 (11%) could not undergo systemic therapy. No unexpected IL-2 related toxicities or significant toxicities related to cell infusion were noted. Overall 5 patients (9.1%) achieved a complete response and 14 (25.5%) achieved a partial response. The responses were durable with a median duration of 14 months (range 0.8+ to 64+). The actuarial survival was 65% at 1 year and 43% at 2 years from the time of nephrectomy, with an overall median survival for all patients of 22 months (range 2 to 70+). The median survival for the responding patients has not yet been reached (range 2 to 63+). CONCLUSIONS: These results demonstrate that immunotherapy with radical nephrectomy, tumor infiltrating lymphocytes, and IL-2 provides substantial clinical benefit in the majority of patients. Component cellular therapy with enriched cell fractions allows the administration of a more standardized cell product. The present results with nephrectomy, tumor infiltrating lymphocytes and IL-2 are encouraging, and a randomized clinical trial of nephrectomy, CD8(+) tumor infiltrating lymphocytes, plus IL-2 versus nephrectomy and IL-2 alone is currently in progress.

Adult↗
PubMed1997

Interleukin-2-based immunotherapy for the treatment of metastatic renal cell carcinoma: an analysis of 203 consecutively treated patients.

PURPOSE: This article analyzes the long-term results of 203 consecutive patients with metastatic renal cell carcinoma who were treated with various recombinant interleukin-2 (rIL-2) -based immunotherapy regimens, and describes factors that may influence response to therapy and long-term survival. PATIENTS AND METHODS: The response and survival of 203 patients with metastatic renal cell carcinoma treated consecutively between July 1987 and October 1995 at the UCLA Medical Center Kidney Cancer Program with rIL-2-based immunotherapy were analyzed. Patients were divided into four groups: (1) no prior nephrectomy (n = 24), (2) nephrectomy > 6 months prior to rIL-2 therapy (n = 76), (3) nephrectomy < or = 6 months prior to rIL-2 therapy (n = 47), and (4) nephrectomy followed by treatment with rIL-2 and tumor-infiltrating lymphocytes +/- interferon-alpha (n = 56). Response and survival for each of these patient groups and survival per response to therapy were compared. RESULTS: The overall median survival for all patients was 18 months, and survival at 1, 2, and 3 years after therapy was 61%, 40%, and 31% percent, respectively. A total of 12 patients (6%) achieved a complete response, and all were alive at 3 years. Of 36 patients (18%) who achieved a partial response and 41 patients (20%) with stable disease, 3-year survival was 37% and 50%, respectively. The survival of patients with a partial response or stable disease was significantly better than that of patients who exhibited progressive disease. Patients with nephrectomy > 6 months prior to rIL-2 therapy had a 46% 3-year survival rate, compared with a 9% 3-year survival rate for patients with nephrectomy < or = 6 months prior to rIL-2 therapy and a 4% 3-year survival rate for patients with no nephrectomy. Patients treated with tumor-infiltrating lymphocytes had a 38% 3-year survival rate, which was also significantly better than patients treated with nephrectomy < or = 6 months prior to rIL-2 therapy or with no nephrectomy. CONCLUSION: This analysis demonstrated that rIL-2-based therapy offers a significant survival benefit to patients with advanced metastatic renal cell carcinoma, compared with historical controls. Furthermore, we have shown that nephrectomy > 6 months prior to rIL-2 therapy and nephrectomy followed by treatment with tumor-infiltrating lymphocytes/rIL-2 +/- interferon-alpha was associated with the greatest survival benefit. Tumor response to rIL-2-based therapy and time from nephrectomy to treatment were the most important predictors of survival. Randomized studies in a large group of patients are needed to confirm these observations.

Adult↗
PubMed1996

Cytoreductive surgery in the management of metastatic renal cell carcinoma: the UCLA experience.

We assessed the role of cytoreductive surgery in patients with metastatic renal cell carcinoma (RCC) selected for interleukin-2 (IL-2)-based immunotherapy. Sixty-three consecutive newly diagnosed patients with metastatic RCC were treated at our institution between April, 1990 and October, 1994. The patients were selected based on their ability to undergo a radical nephrectomy and to receive a combination of interleukin-2 and interferon alfa (IFN-alpha). The mean age was 58.7 years (range, 34-74 years). All but one patient had an Eastern Cooperative Oncology Group performance status of 0 or 1, and presented with metastatic disease and locally advanced primary tumors. All patients successfully underwent cytoreductive nephrectomy, but 6 patients (10%) required concomitant resection of caval thrombus, 3 (5%) required partial hepatectomy, 2 (3%) needed duodenal repairs, and 1 (2%) required a splenectomy. Postoperative complications were observed in 8 patients (12.7%). There were no postoperative mortalities. Seven patients (11%) could not undergo immunotherapy because of myocardial infarctions (n = 2), no growth of tumor infiltrating lymphocytes (TILs) (n = 1), deterioration of performance status (n = 1), transient ischemic attack (n = 1), chronic renal failure (n = 1), and a diagnosis other than RCC (n = 1). Overall, 56 of 63 (88%) patients selected underwent immunotherapy. Among these 56 patients, a response rate of 33.9% [7 (12.5%) complete, and 12 (21.4%) partial] was observed. Moreover, the 2- and 3-year survival rates were 43% and 38%, respectively. Our results support the argument for an aggressive approach (surgery combined with IL-2-based immunotherapy including TILs) in the management of metastatic RCC. Further studies are needed to elucidate the individual contributions of these therapeutic processes.

Adult↗
PubMedDate not supplied

Androgen deprivation induces selective outgrowth of aggressive hormone-refractory prostate cancer clones expressing distinct cellular and molecular properties not present in parental androgen-dependent cancer cells.

PURPOSE: The mechanism of progression of human prostate cancer (CaP) cells under androgen ablation therapy remains unclear. To study the alternative pathways of CaP cell growth under conditions of androgen deprivation, androgen-independent CaP variants were selected and expanded from an androgen-dependent CaP line via an in vitro androgen deprivation treatment. Cellular and molecular properties of these androgen-independent variants were characterized both in vitro and in vivo and compared with those of their parental androgen-dependent cells. METHODS: Androgen deprivation treatment of an androgen-dependent CaP cell line, LNCaP, was carried out by replacing culture medium with RPMI 1640 medium plus 10% charcoal-stripped serum. Cells that survived through the androgen deprivation treatment were harvested and expanded in the androgen-deficient culture medium and were designated CL-1. The CL-1 cells were also recultured in androgen-containing medium and designated CL-2. The growth (cell cycle analysis, 3H-thymidine incorporation assay, growth expansion, and colonization efficiency), expression of CaP-associated markers (semiquantitative reverse transcriptase polymerase chain reaction), interaction with endothelial and bone marrow stromal cells, sensitivity to anticancer agents and radiation (growth inhibition), and tumorigenicity of CL-1 and CL-2 cells were determined and compared with these characteristics in parental LNCaP cells. RESULTS: CL-1 and CL-2 cells are fast-growing cells when compared with parental LNCaP cells. They were capable of potentiating the growth of endothelial and bone marrow stromal cells in co-culture experiments and acquired significant resistance to radiation and to anticancer cytotoxic agents (Taxol paclitaxel, vinblastine, and etoposide). In contrast to the poorly tumorigenic parental LNCaP cells, CL-1 and CL-2 lines proved highly tumorigenic, exhibiting invasive and metastatic characteristics in intact and castrated mice or in female mice within a short period of 3 to 4 weeks. No growth supplements (e.g., Matrigel) were needed. When transfected with the green fluorescence protein (GFP) gene and transplanted orthotopically in the accessory sex gland, extensive metastatic disease from the primary CL tumor could be identified in bone, lymph nodes, lung, liver, spleen, kidney, and brain. Semiquantitative reverse transcriptase polymerase chain reaction analysis revealed a markedly distinct molecular expression profile in the CL lines: overexpression of basic fibroblast growth factor, interleukin-6, interleukin-8, vascular endothelial growth factor, transforming growth factor-beta, epidermal growth factor receptor, caveolin, and bcl-2 messenger RNAs and marked down-regulation of E-cadherin, p-53, and pentaerythritol tetranitrate. CONCLUSIONS: Early administration of hormonal therapy after failure of first-line treatment is associated with a profound clonal selection of aggressive AI variants, such as CL-1 and CL-2 lines. These tumor lines, with their parental counterparts, can serve as valuable tools for studying the cellular and molecular mechanisms of CaP progression and metastasis under hormonal therapy. CL-1 and CL-2 offer a unique and reproducible model for the evaluation of drug sensitivity and for other therapeutic modalities for advanced prostate cancer.

Androgens↗