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M T Lotze

Publications and source records attributed to M T Lotze.

At least 145 records · Page 8Linked to original sources
PubMed1995

Host immune response in renal cell cancer: interleukin-4 (IL-4) and IL-10 mRNA are frequently detected in freshly collected tumor-infiltrating lymphocytes.

Human renal cell cancer (RCC) is clearly responsive to immunotherapy. Clinical responses may be mediated by "non-specific" (e.g. natural killer, NK, cells) or "specific" MHC-class-I-restricted tumor-specific CD8+ T lymphocytes. Typically RCC progresses, however, despite significant infiltration of various lymphoid cells. We examined freshly isolated RCC tumor-infiltrating lymphocytes (TIL) derived from seven RCC patients for cytokine expression by the polymerase chain reaction (PCR). Established RCC tumor cell lines derived from these RCC patients were negative for interleukin-2 (IL-2), IL-4, IL-10, and interferon gamma and found to be positive for tumor necrosis factor alpha (TNF alpha), IL-6, IL-1 beta, granulocyte/macrophage-colony-stimulating factor (GM-CSF), and transforming growth factor beta 1 (TGF beta 1) message as detected by PCR. An identical pattern of cytokine mRNA expression was identified in other long-term RCC lines and in normal human kidney cells upon culture, but not in two Wilms tumor cell lines tested. Short-term-, and long-term-established RCC lines, but not Wilms tumor lines, secreted substantial levels of GM-CSF, TNF alpha, IL-1 beta, and IL-6 as detected by enzyme-linked immunosorbent assay. Both RCC lines and Wilms tumor lines secreted TGF beta 1. In comparison, normal kidney cells secreted IL-6 and GM-CSF, but not IL-1 beta, or TFG beta 1 under identical in vitro cell culture conditions. We applied PCR-based methods to characterize the cytokine mRNA expression pattern in immune cells infiltrating into renal cell cancer without the need for expansion of such effector cells in vitro. Examining freshly collected RCC TIL by PCR from patients with primary cell cell cancer, we could demonstrate that such cells, but not lympho-mononuclear cells harvested from normal human kidney tissue, typically exhibit IL-4 and IL-10 mRNA expression.

Base Sequence↗
PubMed1995

Treatment of hepatic-metastatic colorectal cancer with a chemotherapeutic emulsion: interim results of a phase I trial.

BACKGROUND: Hepatic arterial infusion of 5-fluoro-2-deoxyuridine (FUdR) is associated with a 60% response rate among previously untreated patients who have hepatic-metastatic colorectal cancer. One obstacle to further dose escalation has been concomitant hepatic toxicity. We are evaluating a FUdR-containing chemotherapeutic emulsion to further dose intensify therapy without associated toxicity. METHODS: The in vitro pharmacokinetics of the emulsion were determined using high-pressure liquid chromatography (HPLC). The rate at which FUdR is released from emulsion into an overlying aqueous phase was determined in static and dynamic assays. Fifteen patients with hepatic-metastatic colorectal cancer were treated with intrahepatic arterial infusions of emulsion on a phase I dose-escalating clinical protocol. Serum collection determined systemic drug levels using HPLC. RESULTS: In vitro studies demonstrate that FUdR is slowly released from emulsion into overlying aqueous medium. The emulsion serves as a depot for FUdR. Therapy was well tolerated. Emulsion was sequestered in the liver after infusion in all treated patients. CONCLUSIONS: This Ethiodol-based, oil-in-water emulsion serves as a sustained-release preparation of FUdR. An Ethiodol-based oil-in-water emulsion is a clinically effective vehicle for delivering FUdR to hepatic-metastatic colorectal tumors.

Aged↗
PubMed1995

In situ interleukin-4 gene expression in cancer patients treated with genetically modified tumor vaccine.

Patients with advanced malignancies, participating in our ongoing phase I interleukin-4 (IL-4) gene therapy protocol at the Pittsburgh Cancer Institute, were vaccinated with irradiated autologous tumor cells together with IL-4 gene-transduced irradiated autologous fibroblasts. The level of expression of the IL-4 gene in cultured transduced and selected fibroblasts and in biopsies obtained from vaccination sites was evaluated using quantitative reverse transcription-polymerase chain reaction (RT-PCR). The number of copies of IL-4 mRNA/ng of total cellular RNA was determined in the transduced fibroblasts. Good agreement was observed between IL-4 message expression, as determined by RT-PCR, and IL-4 production, as determined by enzyme-linked immunosorbent assay (ELISA) in the fibroblast supernatants. Tissue biopsies of multiple vaccination sites were obtained from the patients to determine the level of gene expression in situ for IL-4 and Neo-r. The Neo-r gene was used as a marker for transduced fibroblasts. Two weeks after the first vaccination, mRNA for the IL-4 gene was still detectable in all tissue biopsies. The Neo-r gene was also detectable, indicating the presence of transduced fibroblasts in the biopsy. After the second vaccination, expression of the IL-4 and Neo-r genes was generally the highest on day 1 after vaccine administration and was considerably lower but still detectable on day 14 in all biopsies tested. These data indicate that autologous dermal fibroblasts transduced with the IL-4 and Neo-r genes and used as a source of IL-4 in tumor vaccine are able to express the IL-4 gene in vivo.

Base Sequence↗
PubMed1995

HLA-DQ1 is associated with clinical response and survival of patients with melanoma who are treated with interleukin-2.

AIM: The risk of developing melanoma, the natural history of this disease, and the response to therapy with biological reagents may be determined, in part, by a patient's human leukocyte antigen (HLA) phenotype. In order to study this, the relationship between HLA type and clinical response to therapy with interleukin-2 (IL-2) was evaluated. METHODS: We retrospectively determined the HLA phenotype of 82 patients with metastatic melanoma who were treated with IL-2-based therapy. Fresh or frozen lymphocytes were serologically typed by standard lymphocytotoxicity techniques (NIH or Amos modified). The treatment regimens included IL-2 alone or with tumour infiltrating lymphocytes, lymphokine activated killer cells, cyclophosphamide, interferon-alpha, and tumour necrosis factor-alpha. Initially, the relationship between clinical response and each HLA antigen was evaluated by performing a two-tailed Fisher exact test. Associations with a P-value less than or equal to 0.10 without adjustment for multiple comparisons were considered worthy of further study. Independent confirmation of these apparent associations was obtained by studying the relationship between HLA phenotype and patient survival using Cox proportional hazards models. RESULTS: In the initial screening, a statistically significant association between clinical response and the expression of HLA-DQ1 was observed (unadjusted P2 = 0.0017). HLA-DQ1 was also independently associated with prolonged survival (P2 = 0.026). This positive association with survival was evident both for patients who responded to therapy and those who did not respond, as defined by > 50% tumour regression. CONCLUSIONS: Among patients with metastatic melanoma, HLA-DQ1 appears to be associated with clinical response to therapy using IL-2. This apparent association is confirmed by the observation that HLA-DQ1 is independently associated with prolonged survival in this group of patients.

Alleles↗
PubMed1995

Increased in vitro and in vivo tumoricidal activity of a macrophage cell line genetically engineered to express IFN-gamma, IL-4, IL-6, or TNF-alpha.

Genetically engineered monocytes and macrophages may have potential as effector cells for the adoptive immunotherapy of cancer. As a first step, we have transfected the genes encoding either mouse interferon (IFN)-gamma, human interleukin (IL)-6, mouse IL-4, or mouse tumor necrosis factor (TNF)-alpha into the mouse macrophage cell line, J774A.1 cells using retroviral vectors. In vitro activation of J774A.1 cells by gene modification was assessed by morphological changes, proliferative activity was determined by [3H]-TdR uptake, and cytolytic activity was assessed using an 18-hour chromium-51 (51Cr) release assay. In vivo tumoricidal activity was studied by means of local adoptive immunotherapy using intratumoral injection of transfected effector cells. IFN-gamma gene-transfected J774A.1 [J7(IFN-gamma)] cells developed filamentous processes, increased doubling times, and enhanced tumoricidal activity against three tumor cell lines: the TNF-sensitive fibrosarcoma line WEHI 164 and the TNF-alpha-resistant cell lines B16 melanoma and C1300 neuroblastoma. IL-6-, TNF-alpha-, and IL-4-gene-transfected J774A.1 cells also had augmented tumoricidal activity but did not display any changes in morphology or growth. Cytolytic activity was markedly reduced after the addition of anti-TNF-alpha antibodies. Cytolytic J7(IFN-gamma) cells showed upregulated expression of TNF-alpha messenger RNA. After intratumoral injection of J7(IL-4) and J7(IFN-gamma) cell mixtures, 50% of established B16 melanomas were rejected by C57BL/6 mice, thereby demonstrating synergistic killing. Further studies on gene-transfected macrophages should better define their potential usefulness in tumor immunotherapy.

3T3 Cells↗
PubMed1995

Genetically marking human cells--results of the first clinical gene transfer studies.

The rapid development of both knowledge and techniques in molecular biology have made it possible to engineer genetic constructs and transfer them into cells of individuals with various diseases. Such gene therapies may alleviate or perhaps even cure diseases for which no adequate treatment now exists. One potential application is to treat genetic disease by inserting a normal gene into cells in individuals with a "malfunctioning" gene. The added genetic information could allow these cells to function properly and might reduce or eliminate the sequelae of the disease. Such genetic manipulation could also be used to combat other diseases using the same general technique. For example in cancer patients, various cytokine genes inserted into tumor cells may serve as components of a tumor vaccine because such cytokine-secreting tumor cells can induce a significant T-cell response in experimental animal models when compared with non-gene-modified tumors, ultimately leading to a systemic immune response. In addition to treating patients, transferred genes also can serve as markers to obtain important information about the fate of otherwise indistinguishable cells. For example, we used a genetic marker to label tumor-infiltrating lymphocytes (TILs) to monitor their in vivo survival and ability to "home" to tumor sites. Gene markers also were transferred into autologous bone marrow cells to study the mechanism of tumor relapse. This review will focus primarily on studies using gene markers to track TILs after transfer. We will focus on the following issues: (a) that TILs are potent antitumor cells, mediating partial and complete responses in patients with melanoma; (b) the importance of the initial gene marked TIL study; (c) safety considerations in the use of gene marking/gene therapy; (d) results of gene-marked TIL studies; and (e) other gene-marked cells.

Animals↗
PubMed1995

Antitumor effects of interleukin-12 (IL-12): applications for the immunotherapy and gene therapy of cancer.

Interleukin-12 (IL-12) is a pleiotropic cytokine, formerly termed cytotoxic lymphocyte maturation factor (CLMF) or natural killer cell stimulatory factor (NKSF), which is produced primarily by stimulated macrophages. IL-12 is a disulfide-linked heterodimeric cytokine composed of a 35-kDa light chain (p35) and a 40-kDa heavy chain (p40). Unlike most other cytokines, simultaneous transfection of mammalian cells with two different genes is necessary for the production of biologically active IL-12. IL-12 exerts a variety of biological effects on human T and natural killer (NK) cells in vitro, in addition to its ability to promote cytolytic activity, including direct stimulation of the production of IFN-gamma and other cytokines from peripheral blood T and NK cells. The recent finding that IL-12 directs the development of a TH1 type immune response from naive T cells demonstrates the critical role of IL-12 in regulating the immune response. The characteristics of IL-12 function described above strongly suggest its potential usefulness in cancer therapy. Indeed, our studies demonstrate that IL-12 exerts potent antitumor effects following systemic or local administration. We have shown that IL-12 delivered by retroviral vectors allows high-level expression and effective eradication of established tumor in multiple murine tumor models including MCA207 sarcoma. Successful therapy is associated with acquisition of a state of long-term, specific and protective immunity to subsequent challenge with tumor. We have recently received approval from the Recombinant DNA Advisory Committee to proceed with IL-12 gene therapy in humans.

3T3 Cells↗
PubMed1995

Addition of interleukin 12 to low dose interleukin 2 treatment improves antitumor efficacy in vivo.

Interleukin 12 (IL-12) enhances lysis mediated by NK- and lymphokine activated killer (LAK) cells. It also causes proliferation of IL-2 stimulated T and NK cells in vitro. For these IL-2 complementing properties murine pulmonary metastases of a coloncarcinoma line were treated with IL-12 and IL-2 or with the individual agents. Results were compared to sham treated controls. IL-2 alone mediated significant tumor reduction but provoked pulmonary edema and concomittand toxicity, graded in three steps. IL-12 combined with an IL-2 dose reduced by 81% still resulted in significant antitumoral activity. Toxicity, however, was not discernable from sham treated controls. IL-12 thus appears as an attractive cytokine for combination with IL-2 in antitumor therapy. Particularly treatment of tumors, like gastrointestinal tract cancers, so far mainly resistant to cell mediated antitumor therapy, might profit from this approach.

Animals↗
PubMed1995

Human intestinal V delta 1+ T cells obtained from patients with colon cancer respond exclusively to SEB but not to SEA.

The function and activation requirements for gamma delta T cells residing in the human intestine are still poorly defined. We have established two gamma delta + T cell tumor-infiltrating lymphocyte (TIL) lines derived from a primary colorectal cancer (gamma delta TIL No. 3481), and from a colorectal cancer lesion metastatic to the liver (gamma delta TIL No. 7279). Both gamma delta TIL lines used exclusively the V delta 1 segment and predominantly the V gamma 2 segments of the T cell receptor (TCR) variable regions and lysed allogeneic colorectal cancer cell lines, e.g. HCT 116, but not natural killer/lymphokine-activated-killer-sensitive target cell lines, e.g. K562 or Daudi. gamma delta T cell effector functions were evaluated on the basis of their recognition and cytolysis of colorectal cancer cell lines, T cell proliferation, and interferon (IFN)-gamma release. Both gamma delta T cell lines exhibited similar responses to the staphylococcal superantigens (SE) A and B. SEA and SEB did not influence target cell cytolysis of colon cancer targets. Neither gamma delta + T cell line responded to SEA as measured by IFN-gamma release of T cell proliferation. In marked contrast, SEB induced T cell proliferation and IFN-gamma release in the absence of stimulator cells. SEB induced secretion of IFN-gamma by gamma delta T cells which could be augmented if stimulator cells (HCT116) were also added to gamma delta T cells. On the basis of these data, we suggest that intestine-derived V delta 1/V gamma 2+ T cells respond preferentially to SEB and not to SEA. This disparity may reflect the inherently higher affinity of individual gamma delta TCR subsets for SEB but not to SEA and/or indicate that a subset of gamma delta + TILs in patients with colon cancer may be preferentially expanded with a TCR rearrangement favoring the interaction with SEB. The induction of IFN-gamma release and proliferative gamma delta + T cell responses by SEB suggests a pivotal role for intestinal gamma delta T cells in mediating immune responses against bacteria and bacterial products, or potentially in anti-tumor-directed immunity. Such immune responses mediated by gamma delta + T cells may take place prior to the maturation of antigen-specific MHC-restricted alpha beta + T cell responses.

Antigens, Bacterial↗
PubMed1994

Partial purification of murine tumor-associated peptide epitopes common to histologically distinct tumors, melanoma and sarcoma, that are presented by H-2Kb molecules and recognized by CD8+ tumor-infiltrating lymphocytes.

The B16/BL6 melanoma is a relatively nonimmunogenic tumor expressing low levels of MHC class I molecules. BL6 clones expressing transfected H-2Kb class I molecules were, by contrast, highly immunogenic in immunocompetent mice. Tumor-infiltrating lymphocytes (TILs) generated from the H-2Kb+ BL6 lesions (Thy 1.2+, CD8+, CD4-) efficiently lysed H-2Kb+ melanoma (CL8-1 and 2Kb38) and the H-2Kb+ nonrelated 3-methylcholanthrene (MCA)-105 sarcoma, but not the H-2Kb- parental melanoma BL6-8. This strongly suggests that CL8-1, 2Kb38, and MCA-105 express identical or cross-reactive T cell epitopes recognized by CL8-1 TILs in the context of the H-2Kb class I allele. To identify the T cell epitopes, peptides were acid-eluted from various cells, and fractionated by HPLC. Five HPLC fractions (F1mel-F5mel) of 70 tested contained peptides derived from H-2Kb+ CL8-1 melanoma (but not H-2Kb- melanomas) that were capable of conferring susceptibility to CL8-1 TIL lysis on H-2b-expressing target cells (EL4, C1R.Kb), but not on H-2d-expressing P815 target cells. CL8-1 TILs failed to recognize peptides derived from H-2Kb+ nonmelanoma targets such as EL4 or normal B6 splenocytes. Interestingly, CL8-1 TILs appeared to recognize peptide species contained in two HPLC fractions derived from the MCA-105 sarcoma (F1sar and F5sar). Conversely, TILs derived from MCA-105 lesions recognized MCA-105 and CL8-1 tumor cells, as well as F5mel and F5sar peptides presented by EL4 targets. These data support common murine tumor-associated peptide epitopes presented by H-2Kb and recognized by CD8+ CTLs derived from histologically distinct tumors, melanoma and sarcoma.

Animals↗
PubMed1994

Fibroblasts genetically engineered to secrete interleukin 12 can suppress tumor growth and induce antitumor immunity to a murine melanoma in vivo.

Interleukin 12 (IL-12), a disulfide-linked heterodimeric cytokine produced primarily by macrophages, is composed of light (p35) and heavy (p40) chains. It binds to a receptor on T-cells and natural killer cells, promoting the induction of primarily a TH1 response in vitro and in vivo. To determine whether paracrine IL-12 secretion can alter tumor cell growth or promote antitumor immunity, we have developed a delivery system using genetically engineered fibroblasts in murine tumor models. NIH3T3 cells were stably transfected to express 100-240 units/10(6) cells/48 h of IL-12 using expression plasmids carrying both the murine p35 and p40 genes of murine IL-12. The effects of paracrine secretion of IL-12 on tumor establishment and vaccination models were examined using the poorly immunogenic murine melanoma cell line (BL-6) in C57BL/6 mice. To determine the effects of IL-12 on tumor formation, nonirradiated BL-6 cells were inoculated s.c. into C57BL/6 mice admixed with NIH3T3 cells transfected with both subunits of mIL-12 (3T3-IL-12) or with cells transfected with only the neomycin phosphotransferase gene (3T3-Neo). Compared to mice given injections of BL-6 alone, the day of emergence of detectable tumors was significantly delayed in mice given injections of BL-6 admixed with 3T3-IL-12, but not in mice with BL-6 admixed with 3T3-Neo. Effectiveness in this system was related to the amount of IL-12 expressed by the 3T3-IL-12. To determine the ability of locally secreted IL-12 at the tumor site to induce antitumor immunity, 10(6) irradiated tumor cells mixed with 3T3-IL-12 or 3T3-Neo were injected as a vaccine, and the response to a tumor challenge was subsequently examined. With a tumor challenge of less than 1 x 10(5) nonirradiated BL-6 cells, significant delay of establishment of tumor was noted with a relatively small amount of IL-12 secretion (1.2 units/5 x 10(5) cells/48 h). Larger amounts of secreted IL-12 provided no additional therapeutic benefit. Histological examination of tumor inoculum with 3T3-IL-12 secreting a high level of IL-12 showed peritumoral accumulation of macrophages, a characteristic capsule around the tumor composed of palisades of fibroblasts, and decreased numbers of CD4+ cells in the tumor. These results suggest that local delivery of IL-12 inhibits tumor growth in a dose dependent manner but leads to the development of an antitumor immune response when IL-12 is expressed at the tumor site at the relatively small amount indicated above.(ABSTRACT TRUNCATED AT 400 WORDS)

3T3 Cells↗
PubMed1994

Identification of wild-type and mutant p53 peptides binding to HLA-A2 assessed by a peptide loading-deficient cell line assay and a novel major histocompatibility complex class I peptide binding assay.

Mutations of the p53 gene are the most frequently observed genetic changes in human cancers; often leading to an overexpression of the wild-type (wt) p53 protein. Demonstrable T cell reactivity against tumor cells overexpressing wt or mutant p53-derived peptides could support the application of such epitopes in cancer immunotherapies. As the binding of peptide to MHC class I molecules is a prerequisite for antigen-specific T cell recognition, we evaluated the ability of wt and mutant p53 peptides to bind to HLA-A2.1 using two independent flow cytometry-based assay systems, the T2 major histocompatibility complex (MHC) class I peptide stabilization assay (stabilization assay) and the peptide-induced MHC class I reconstitution assay (reconstitution assay). The twenty selected wt sequences each conformed to the previously reported HLA-A2.1 peptide binding motif. Seven of the wt p53 and 2/13 mutant p53 peptides derived from the previously chosen wt peptides bound to HLA-A2.1 in both the stabilization and the reconstitution assays. An additional six wt and six mutant p53 peptides, presumably exhibiting lower affinity for HLA-A2.1, were identified only in the reconstitution assay. Those p53 peptides binding HLA-A2.1 may provide useful immunogens for the generation of HLA-A2.1-restricted cytolytic T lymphocytes in vitro and in vivo.

Amino Acid Sequence↗
PubMed1994

Flow-cytometric determination of peptide-class I complex formation. Identification of p53 peptides that bind to HLA-A2.

A novel class I-peptide-binding assay was developed and used to identify a series of peptides derived from the human p53 tumor-suppressor gene product capable of binding the HLA-A2 class I allele. Brief pH 3.3 acid treatment of human cell lines rapidly denatures pre-existing class I complexes, as detected by loss of binding of conformation-dependent mAbs, leaving only free class I heavy chains associated with the viable cell surface. These heavy chains may be induced to refold and be recognized by antibodies (in 2-4 hours) when acid-treated cells are coincubated with exogenous beta 2-microglobulin and peptides capable of binding the relevant class I allele examined. This assay, with a detection limit of 1-10 nM peptide, was used to screen the capacity of a panel of nine peptides bearing HLA-A2-binding motifs and derived from the human p53 tumor-suppressor protein sequence. Eight of the nine peptides bound to, and reconstituted, HLA-A2 on acid-treated cells. This assay system will enable the rapid identification of peptides binding to any class I allele, which is the initial prerequisite for elucidating potential CD8+ T-cell epitopes.

Amino Acid Sequence↗
PubMed1994

Construction and characterization of retroviral vectors expressing biologically active human interleukin-12.

Interleukin-12 (IL-12) is a heterodimeric cytokine originally defined by its ability to induce the maturation of cytolytic lymphocytes and by its capacity to effectively synergize with IL-2 in the induction of cytolytic activity. Recent studies in mice have demonstrated the ability of IL-12 to cause tumor regression and stimulate long-term antitumor immunity in treated animals. To examine the antitumor effect of direct gene transfer of IL-12 into tumors, we have developed retroviral vectors that coordinately express both subunits of IL-12. An MFG-based retroviral vector was used to generate a recombinant retrovirus in which a long terminal repeat (LTR)-driven polycistronic transcript encodes both subunits of human IL-12: hp35 and hp40 cDNAs are linked and coexpressed using the internal ribosome entry site (IRES) from the encephalomyocarditis virus (DFG-hIL-12). In addition, two IRES sequences were used to express both subunits of IL-12 and a neomycin resistance (neoR) selectable marker gene from the same polycistronic message (TFG-hIL-12). The amphotropic DFG-hIL-12 and TFG-hIL-12 viruses were used to infect both human and murine cell lines as well as primary tumor cultures. The production of human IL-12 by the nonselected, infected cells was measured in both a PHA blast proliferation bioassay and an ELISA and ranged from 15 to 40 ng/10(6) cells per 24 hr. Following G418 selection of TFG-hIL-12-infected cells, the level of expression of IL-12 was significantly higher (up to 120 ng/10(6) cells per 24 hr). The IL-12 protein secreted by the infected cells exhibited all of the biologic activities of recombinant hIL-12: proliferation of activated natural killer (NK) and T cells, stimulation of interferon-gamma (IFN-gamma) induction by NK and T cells, and enhancement of lymphokine-activated killer (LAK) activity. These retroviral vectors expressing human IL-12 should be useful in evaluating the biological properties of IL-12 as well as for use in clinical trials for gene therapy of patients with cancer.

Animals↗