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Biomedical subjects

M T Lotze

Publications and source records attributed to M T Lotze.

At least 127 records · Page 7Linked to original sources
PubMed1996

Interleukin-12 and B7.1 co-stimulation cooperate in the induction of effective antitumor immunity and therapy of established tumors.

Interleukin-12 (IL-12) promotes specific and long-lasting anti-tumor immunity mediated by T cells in a variety of murine tumor models. IL-12 also synergizes with B7.1 (CD80) co-stimulation to induce proliferation and cytokine production by both human and murine T cells in vitro. We evaluated the combined anti-tumor efficacy of IL-12 and B7.1 gene delivery in two apparently poorly immunogenic tumor models (TS/A and MCA207). In both of these models, expression of B7.1 and production of IL-12 in the inoculum led to improved anti-tumor immunity, with up to 80% long-term tumor-free animals (vs 0-20% of mice remaining tumor free when inoculated with either B7.1- or IL-12-transfected tumors alone). Tumor-free mice were capable of rejecting a subsequent rechallenge with the wild-type tumor in 66% of the cases. Cooperativity was dependent upon the level of IL-12 secreted by engineered cells. IL-12 delivery required B7 expression of therapeutic effects to be observed in these models. Vaccines provided at a site distal to a control, non-transfected tumor slowed (TS/A) or abrogated (MCA207) the progression of wild-type tumors. The synergistic anti-tumor effects associated with combined application of B7.1- and IL-12-transfected tumors were partially negated by systemic administration of the CD28-B7.1/B7.2 antagonist CTLA4-Ig or by inoculation with neutralizing antibodies directed against murine interferon-gamma or tumor necrosis factor-alpha, two cytokines elicited in response to IL-12 stimulation. These data support the potential clinical utility of combined gene therapy using IL-12- and B7.1-engineered autologous cells (tumor or fibroblasts) as a vaccine to elicit specific anti-tumor immunity.

Abatacept↗
PubMed1996

Amino acid substitutions at position 97 in HLA-A2 segregate cytolysis from cytokine release in MART-1/Melan-A peptide AAGIGILTV-specific cytotoxic T lymphocytes.

CD8+ T lymphocytes recognize antigenic peptides presented by major histocompatibility complex (MHC) class I molecules. Individual peptide termini appear to be fixed at the C- and N-terminal ends. In contrast, central peptide side chains residues may point in different directions and exhibit limited flexibility, dependent on the MHC class I structural variation. For instance, position 97 in HLA-A201 has been shown to shift individual peptide species into different coordinations, one oriented towards the peptide N terminus, or more towards the C-terminal end. The conformational shape of such non-anchor peptide residues may affect the affinity of MHC/peptide/TCR interaction, resulting in quantitative, or qualitative different T cell effector functions. To characterize the impact of different amino acid residues occupying position 97 in HLA-A2 on peptide binding and presentation to CTL, we generated a panel of mutated HLA-A2 molecules containing either M, K, T, V, G, Q, W, P or H at position 97. The HLA-A0201 presented melanoma-associated MART-1/Melan-A derived peptide AAGIGILTV was employed to assess the impact of such position-97 mutations on HLA-A2 in peptide binding measured in an HLA-A2 reconstitution assay and presentation to AAGIGILTV-specific polyclonal or clonal T lymphocytes as measured by cytotoxicity, or interferon (IFN)-gamma and granulocyte/ macrophage colony-stimulating factor (GM-CSF) secretion. The high-affinity AAGIGILTV peptide bound to all position-97 mutants, albeit with differential efficiencies, and elicited specific release of IFN-gamma and GM-CSF by CTL. CTL responses were triggered only by the HLA-A2 wild type, by HLA-A2-H97 (histidine position 97 mutant), and HLA-A2-W97. The HLA-A2-M97 presenting molecule elicited enhanced cytokine release and CTL effector functions by polyclonal and by clonal effector T cells. These results indicate that MHC class I-bound peptides can trigger specific cytokine release by effector T cells independently of their ability to induce cytolysis. We conclude that relatively minor changes in the MHC class I peptide binding groove, including substitutions at position 97, can affect recognition by antigen-specific T cells. Mutant MHC class I molecules, such as those described here, may act as partial peptide antagonists and could be useful for inducing T lymphocytes with qualitatively different effector functions.

Amino Acid Sequence↗
PubMed1996

A phase I trial of a synthetic mucin peptide vaccine. Induction of specific immune reactivity in patients with adenocarcinoma.

We tested a 105 amino acid synthetic mucin MUC-1 peptide that has 5 repeated immunodominant epitopes to evaluate toxicity and detect mucin-specific immune responses in patients with adenocarcinoma. We also studied the enhancement of these responses by vaccinating patients with the synthetic mucin peptide admixed with BCG. Mucins are glycoproteins present on the luminal surface of ductal epithelial cells and on tumors derived from them. The MUC-1 mucin is hypoglycosylated and nonpolarized on tumors and this exposes epitopes that can stimulate cytotoxic T-Cells (CTL). We vaccinated 63 patients with 100 micrograms of the 105aa mucin peptide mixed with BCG. Two additional vaccinations were given at 3-week intervals. All patients were able to tolerate vaccination, with most experiencing local ulceration at the vaccination site. All patients underwent hypersensitivity (DTH) testing with the 105aa and shorter mucin peptides, prior to vaccination. DTH responses were evaluated at 48 hr and the sites of highest peptide concentration were biopsied. Only 3 patients had a strong skin response to the long peptide. Examination of 55 biopsies showed intense T-Cell infiltration in 37 patients and lesser infiltration in 7. Seven of 22 patients tested had a 2- to 4-fold increase in mucin-specific CTLp. Serum levels of IL-6 were measured sequentially using the B9 hybridoma bioassay. Increasing serum levels of IL-6 correlated with constitutional symptoms (significance 0.001) and hypoalbuminemia (significance 0.007) but not with the extent of skin breakdown at vaccination sites. We conclude that mucin vaccination is safe and might serve to enhance specific responses to tumor antigens. IL-6 may be responsible for the constitution symptoms and hypoalbuminemia in these patients.

Adenocarcinoma↗
PubMed1996

Postoperative abdominal wall defects with enterocutaneous fistulae.

BACKGROUND: Abdominal wall dehiscence with an associated enterocutaneous fistula is a surgical complication with high morbidity and mortality. Management of the abdominal wall defect is complicated by the continued emergence of liquid bowel contents. PATIENTS AND METHODS: Large abdominal wall wounds of 10 patients with postoperative abdominal wall dehiscence and active enterocutaneous fistulae were managed with early skin grafting directly onto the granulated abdominal viscera. RESULTS: Skin graft take averaged 93 +/- 12%, and there were no perioperative complications related to the skin grafting procedure. Overall mortality was 1 out of 10 patients. Enterocutaneous fistula output did not prove overly injurious to the skin grafts. Wound care was simplified in all but 1 patient with fitting of an ostomy appliance. CONCLUSION: Temporary abdominal wall wound closure with skin grafts improved patient comfort and simplified wound care in a staged reconstructive approach to this surgical complication.

Abdominal Injuries↗
PubMed1996

Gene therapy.

Explore the source record for details and available documents.

Arterial Occlusive Diseases↗
PubMed1996

Recognition of human renal cell carcinoma and melanoma by HLA-A2-restricted cytotoxic T lymphocytes is mediated by shared peptide epitopes and up-regulated by interferon-gamma.

Cytotoxic T lymphocytes (CTL) have previously been isolated from peripheral blood of patients with renal cell carcinoma (RCC). The CD8-positive CTL line MZ1257-CTL-5 (CTL-5) has been shown to lyse autologous cultured RCC cells in an HLA-A2 restricted fashion. Allogeneic, HLA-A2-matched RCC and melanoma cell lines were also lysed by CTL-5, suggesting that melanoma and renal cancer share antigenic determinants. The aim of the study was to determine whether RCC and melanoma share peptide epitopes that are recognized by CTL-5 in the context of HLA-A2 molecules. Peptides were acideulated from various cell lines, separated by reversed phase high performance liquid chromatography (RP-HPLC), and assessed for their ability to reconstitute the CTL-5-defined epitope by pulsing the peptides on HLA-A2 positive antigen-processing mutant cell line CEM x 721.174.T2 (T2). Peptides eluted from allogeneic HLA-A2-matched RCC and melanoma cell lines exhibited the CTL-5-defined epitope in the same HPLC fractions as peptides derived from the autologous RCC line. Renal cancer and melanoma cells preincubated with interferon-gamma (IFN-gamma) resulted in an additional peak of reconstitution activity in both cell types. This second lytic peak was also observed when high amounts of autologous RCC cells were used for peptide preparation without IFN-gamma pretreatment, indicating that IFN-gamma increases the amount of MHC class I/peptide complexes per cell, rather than inducing a neo-epitope.

B-Lymphocytes↗
PubMed1996

New treatment options for patients with melanoma: review of melanoma-derived T-cell epitope-based peptide vaccines.

Human melanoma represents the principal cause of death in patients with skin cancer in the United States and Europe. Tumour infiltrating lymphocytes recognizing melanoma have been used to identify the tumour antigens recognized by T-cells in the context of MHC class I or class II molecules. Such antigens include MAGE-1, MAGE-3, MART-1/Melan-A, gp100, tyrosinase, the tyrosinase-related antigen gp75, the antigen gp15 and the mutated CDK4 and beta-catenin gene-products. The identification of these T-cell epitopes provides us with novel reagents for the development of state-of-the-art treatments and for the (immuno-)monitoring of patients with melanoma. In order for treatments, including peptide-based vaccines, to be successful, several conceptual criteria must be met: (1) The patient's tumour must present the relevant epitope(s) integrated into the vaccine, (2) the tumour should express the appropriate restricting major histocompatibility complex (MHC) molecule(s) required for patient cytotoxic T lymphocyte (CTL) reactivity, and (3) the patient's T-cell repertoire should be able to react productively against the melanoma antigens present in the vaccine. Clinical trials implementing peptide-based vaccines or whole protein therapies have been initiated in the United States and Europe. We suggest that such treatments should include the careful monitoring of anti-tumour T-cell responses. This should include examination of melanoma antigen and MHC class I allele expression in the individual patient's tumour, assessment of the status of the peptide transporter molecules TAP1/TAP2 and evaluation of T-cell mediated immune responses reactive against peptides and autologous melanoma. Evaluation of clinical parameters (such as disease-free survival) in conjunction with an examination of immunological parameters may facilitate our understanding of the immune responses against T-cell antigens that are shared among melanoma and normal melanocytes, and may ultimately help to identify the most effective immunotherapy for patients with melanoma.

Amino Acid Sequence↗
PubMed1996

Inhibition of nitric oxide synthesis does not improve interleukin-2-mediated antitumor effects in vivo.

Nitric oxide (NO) decreases cytotoxicity and proliferation of cytotoxic lymphocytes (CTLs) in vitro. Both can be prevented by inhibitors of the NO synthase (NOS). To elucidate whether inhibition of the IL-2-induced NOS could boost efficacy of IL-2-stimulated CTLs in vivo, we assessed lung metastases in mice injected with IL-2, the NOS inhibitor aminoguanidine (AG), their combination and the diluent. No improvement was observed for IL-2 + AG compared to IL-2 while NO production was normalized. Since NO causes one of the two major side effects of IL-2 treatment, hypotension, we further studied whether capillary leak could be attributed to NO, too. While IL-2-inducible NO was reduced to control levels by AG, pulmonary edema was unaffected. Thus a decrease in NO does not improve antitumor effects of IL-2-stimulated CTLs nor does it attenuate IL-2-associated capillary leak.

Animals↗
PubMed1996

Detection of naturally processed and HLA-A1-presented melanoma T-cell epitopes defined by CD8(+) T-cells' release of granulocyte-macrophage colony-stimulating factor but not by cytolysis.

Several antigens, including the products encoded by the genes MAGE-1 and MAGE-3, are recognized on human melanoma cells by HLA-A1, HLA-A2, or HLA-Cw*1601*-restricted T cells on autologous or HLA-matched melanoma cell lines. T-cell recognition of naturally processed MHC class I-presented peptides, or alternatively synthetic peptides derived from MAGE-1 or MAGE-3, leads to cytokine release as well as to a cytotoxic T-cell response in these antimelanoma-directed polyclonal or clonal effector T-cell populations. Recent reports suggest that the activity of T lymphocytes infiltrating melanoma in vivo appears to be impaired. We report here the characterization of the in vitro (in the presence of 6000 IU interleukin 2) expanded tumor-infiltrating lymphocyte (TIL) T-cell line PM2-B2 derived from a patient with rapidly progressing and therapy-resistant head and neck melanoma. The TIL cell line PM2-B2 did not lyse, but instead released granulocyte-macrophage colony-stimulating factor in response to the autologous tumor or HLA-A1-matched allogeneic tumor cell lines. The TIL line PM2-B2 did not kill the MHC class I natural killer/lymphokine-activated killer target cell lines Daudi or K562. The fine specificity of the TIL line PM2-B2 restricted by HLA-A1 was further characterized by evaluating specific granulocyte-macrophage colony-stimulating factor release in response to MHC class I-eluted peptides derived from HLA-A1(+) melanoma cell lines. TIL PM2-B2 failed to recognize the recently described HLA-A1-presented peptides derived from the gene products encoded by MAGE-1 or MAGE-3. PCR-based analysis of the freshly harvested tumor from patient PM2-B2 revealed the presence of message for the melanoma-associated gene products MAGE-1 and MAGE-3, but not for tyrosinase or MART-1/MELAN-A. Acid elution and high performance liquid chromatography fractionation of MHC class I-presented peptides from HLA-A1-matched melanoma cell lines 397 or 888 revealed that TIL PM2-B2 recognized at least three distinct peptide epitopes eluting in high performance liquid chromatographic bioactive fractions 5/6, 36, and 51/52. These bioactive peaks appeared to be shared among HLA-A1(+) melanoma cell lines. We suggest, based on this report, that HLA-A1-presented melanoma-derived peptides (other than those previously reported peptides derived from MAGE-1 or MAGE-3) may represent targets for TIL recognition as defined by cytokine release, but not cytotoxicity. Such an immune response differentially defined by cytokine release, but absent cytotoxic functions, may either reflect the impaired cytolytic function of the TIL population or reflect the inherent nature of HLA-A1-presented melanoma T-cell epitopes leading to cytokine release, but not to a cytotoxic T-cell response. Additionally, this report suggests that the individual T-cell immune response to melanoma may be rather complex, involving diverse T-cell effector functions (e.g., cytotoxicity or cytokine release), each of which should be evaluated in studies of antitumor-specific T-cell reactivity.

Antigen Presentation↗
PubMed1996

Growth stimulation of human head and neck squamous cell carcinoma cell lines by interleukin 4.

Interleukin 4 (IL-4) has been reported recently to inhibit growth of acute lymphoblastic lymphoma, non-Hodgkin's lymphoma, melanoma, sarcoma, breast, gastric, colon, and renal tumor cell lines, and treatment of murine tumors with IL-4 gene-transduced cells has been therapeutically successful. Therefore, we sought to determine the effect of IL-4 on the growth of human squamous cell carcinoma of the head and neck (SCCHN) cell lines. Growth of SCCHN cell lines incubated in the presence of various concentrations of IL-4 was measured in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide colorimetric assays and by cell counts. Specific binding of IL-4 to SCCHN cells was demonstrated by flow cytometry with phycoerythrin-labeled IL-4, blocking studies with antibodies to IL-4, and using the radiolabeled ligand 125I-labeled IL-4. Reverse transcription PCR for IL-4 and IL-4 receptor (IL-4R) mRNA was performed. SCCHN tissue biopsies were examined by immunohistology and in situ hybridization for the presence of IL-4 protein and IL-4 mRNA in the tumor, respectively. In contrast to earlier reports, we observed growth stimulatory effects of IL-4 consistently in 6 of 13 SCCHN cell lines tested. Growth stimulation by IL-4 ranged from 20 to 200% of control (P < 0.05) and was IL-4 dose dependent. The growth-promoting effect of IL-4 was inhibited completely by incubation of tumor cells in the presence of antibodies specific for IL-4. Reverse transcription PCR analysis of mRNA obtained from the SCCHN cell lines and ELISA performed with SCCHN cell supernatants respectively indicated that the tumor cells did not transcribe or secrete IL-4 actively. The SCCHN cell lines expressed 260-540 IL-4Rs/cell with a dissociation constant of 100 +/- 8 pM. SCCHN cell lines also contained IL-4R mRNA. Immunostaining of SCCHN tissue biopsies indicated that IL-4 may be produced and secreted within these tumors by tumor-infiltrating lymphocytes. In situ hybridization for IL-4 mRNA indicated the presence of positive cells in the tumor stroma. Our data suggest that IL-4 may regulate the growth of SCCHN cells by a paracrine mechanism. These data also indicate that immunotherapy with exogenous IL-4 or IL-4 gene therapy to treat head and neck cancer may not be effective, given the potential tumor growth-stimulatory effects of this cytokine.

Carcinoma, Squamous Cell↗
PubMed1996

Tumor escape from immune recognition: loss of HLA-A2 melanoma cell surface expression is associated with a complex rearrangement of the short arm of chromosome 6.

Specific CD8(+) CTL recognition of melanoma requires expression of MHC class I molecules as well as melanoma-associated peptide epitopes. Human melanoma cells may escape immune recognition by a variety of means, including global or allelic down-regulation of MHC class I molecules. Stable MHC class I cell surface expression requires delivery of cytosolic peptides into the endoplasmic reticulum by the peptide transporter molecules TAP1 and TAP2, with peptides subsequently transported to the cell surface in complexes containing MHC class I heavy chain and beta2-microglobulin. We have evaluated a series of mechanisms resulting in MHC class I down-regulation in a human melanoma cell line, Mz18, typed as HLA-A2(+), A3(+), B7(+), B57(+), Cw1(+), and Cw6(+) by genomic PCR analysis. The melanoma cell line Mz18 exhibits a global down-regulation of MHC class I heavy chain transcripts; beta2-microglobulin; the proteasome subunits LMP2/7, involved in generating cytosolic peptide fragments; and the peptide transporter molecules TAP1 and TAP2, involved in peptide transport from the cytosol into the endoplasmic reticulum. IFN-gamma treatment of Mz18 melanoma cells leads to up-regulation of LMP2/7 and TAP1/2, as well as to up-regulation of HLA-B and HLA-C MHC loci alleles, but not HLA-A2 or HLA-A3. Karyotypic analysis and fluorescence in situ hybridization with chromosome 6 and MHC class I-specific probes showed complex rearrangement of one chromosome 6 involving the MHC class I locus on 6p and translocation of 6q to the long arm of chromosome 19. To evaluate the capability of melanoma Mz18 to present tumor-specific peptides to HLA-A2-restricted, melanoma-specific CTLs, we restored HLA-A2 surface expression by retroviral-mediated transfer of functional HLA-A2 cDNA. Melanoma peptides could only be presented and recognized by CTLs if the HLA-A2-transfected Mz18 cell line was first treated with IFN-gamma, thereby restoring LMP2/7 and TAP1/2 expression and function. Because several melanoma antigens recognized by T cells have been reported to be presented by HLA-A2 (MART-1/Melan-A, tyrosinase, gp100, and MAGE-3), the loss of HLA-A2 molecules may represent an important mechanism by which many melanomas evade immune recognition. These findings suggest that patients entering clinical trials for immunotherapy with melanoma vaccines should be carefully examined for tumor cell allelic MHC class I loss and whether such MHC class I antigen down-regulation can be restored by cytokines.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1995

Viral interleukin 10 (IL-10), the human herpes virus 4 cellular IL-10 homologue, induces local anergy to allogeneic and syngeneic tumors.

After the cloning of murine cytokine synthesis inhibitory factor, it was recognized that a homologous open reading frame was encoded within the Epstein-Barr virus (human herpes virus 4). This viral protein has now been termed viral interleukin 10 (vIL-10) to reflect its protein sequence homology to "cellular" IL-10 (cIL-10, either murine or human IL-10). It is now widely accepted that vIL-10 shares many functions with cIL-10, principally, the ability to enhance survival of newly infected B cells and to diminish the production of IFN-gamma and IL-2 during ongoing immune reactions. The immunomodulatory effect of locally secreted vIL-10 and murine IL-10 (mIL-10) was examined in tumor models using CL8-1 (a BL6 melanoma cell line transfected with the H-2Kb class I gene) in syngeneic animals. Although parental BL6 tumor cells grow in immunocompetent syngeneic hosts, CL8-1 are rejected. To achieve local secretion of vIL-10, we generated vIL-10 retroviral vectors. While nontransduced CL8-1 cells (1 x 10(4)) failed to grow when injected intradermally in C57BL/6 mice, CL8-1 cells (1 x 10(4)) transduced with vIL-10 formed palpable tumors and eventually killed 80% of injected animals. Suppression of tumor rejection was also noted when CL8-1 tumors with or without vIL-10 transfection were admixed with syngeneic vIL-10-transfected fibroblasts and inoculated. Since the in vitro proliferation of the tumor was not altered after transduction with the vIL-10 gene and injection of vIL-10-transduced CL8-1 does not affect the rejection of nontransduced CL8-1 inoculated at a distant site, local vIL-10 secretion appears to suppress the process of immune rejection of the target cells in a dose-dependent manner. Similar results were observed for the H-2b MCA105 sarcoma tumor model in allogeneic BALB/c mice (H-2d). Although all animals that received nontransfected MCA105 rapidly rejected these tumors, MCA105 sarcomas transfected with vIL-10 remained palpable for up to 37 d. The local immunosuppressive effect of gene-delivered vIL-10 could be neutralized by anti-human IL-10 monoclonal antibody or could be reversed by the systemic administration of IL-2 or IL-12. In marked contrast, mIL-10 transfection of CL8-1 significantly suppressed tumor growth and frequently led to the rejection of tumor. Similar results were obtained for the murine tumor cell lines MCA102.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1995

Cancer immunotherapy of established tumors with IL-12. Effective delivery by genetically engineered fibroblasts.

IL-12 is a heterodimeric cytokine produced by macrophages, mitogen stimulated- or EBV infected-B lymphocytes, keratinocytes, and probably dendritic cells, with important immunoregulatory functions in vitro and in vivo. It directly stimulates activated NK and T cells to produce high levels of IFN-gamma, enhances their cytolytic activity, and promotes maturation of Th1 cells as well as IL-2-activated B cells. We have tested paracrine delivery of IL-12 using autologous or allogeneic fibroblasts engineered to secrete high levels of IL-12 to treat established tumors. Injection of IL-12-engineered fibroblasts at the site of an established (day 8) MCA207 sarcoma could efficiently eliminate or suppress tumor growth in a dose-dependent manner, requiring delivery of > 150 ng/kg/dose of bioactive IL-12. Weekly inoculations for 3 wk could also be used to effectively treat a day 4 sarcoma located intradermally in the opposite flank (80% protection using autologous fibroblasts), resulting in long-term protective antitumor immunity. In less immunogenic tumors (MCA102, MC38), 7-day established lung metastases could be significantly reduced (p = 0.001) following IL-12 delivery by fibroblasts and systemic administration of low doses of IL-2. Histologic findings included a mixed infiltrate of CD4+ and CD8+ T effectors and macrophages in the regressing sarcoma on day 21. In a day 41 MCA207 sarcoma locally injected in situ, similar findings were observed. No lymphoid hyperplasia or tissue necrosis were noted in liver, spleen, or lungs in mice receiving repeated inocula of IL-12-engineered fibroblasts. Tests of liver and renal function monitored during the repetitive weekly treatments were within the normal range. IL-12-engineered fibroblasts thus seem to serve as a safe and efficient means to deliver IL-12 in these three tumor models.

Animals↗
PubMed1995

Effective eradication of established murine tumors with IL-12 gene therapy using a polycistronic retroviral vector.

Our recent studies using IL-12 protein or fibroblasts genetically engineered to secrete IL-12 have demonstrated profound antitumor effects of IL-12 in murine models. The antitumor effects of local, high level IL-12 expression were examined using a retroviral vector, which can express both IL-12 subunits (p35 and p40) and the neomycin phosphotransferase (Neo)-marker gene from a polycistronic message utilizing internal ribosome entry site sequences. All animals intradermally (i.d.) receiving MCA207 murine sarcoma cell line nontransfected or Neo-transfected had progressively growing tumor, whereas all animals injected with MCA207 transfected with IL-12 were tumor free and were subsequently determined to be immune to a rechallenge of nontransfected MCA207 i.d. Similar results were obtained in experiments using the poorly immunogenic MCA102 murine sarcoma cell line. The inoculation of live MCA207-IL-12 tumor cells also caused the regression of contralateral nontransfected MCA207 inoculated either at the same time (80% protection) or up to 3 days before (33% protection) to the therapeutic tumor inoculation. In vivo depletion studies suggest that NK cells and IFN-gamma play important roles in the development of the early phase of the antitumor response, but that T cells (both CD4+ and CD8+) play the major role in the subsequent events, leading to long-term immunity. The potent antitumor effects observed for paracrine gene-delivered administration of IL-12 have thus been confirmed for multiple tumor cell types and in multiple murine strains. We believe that these results support the feasibility of IL-12 gene therapy for the treatment of human cancer.

Animals↗
PubMed1995

Mass spectrometric identification of a naturally processed melanoma peptide recognized by CD8+ cytotoxic T lymphocytes.

We and others have previously reported that melanoma-specific, cytotoxic T lymphocytes (CTL) define a minimum of six class I-presented peptide epitopes common to most HLA-A2+ melanomas. Here we show that three of these peptide epitopes are coordinately recognized by a CTL clone obtained by limiting dilution from the peripheral blood of an HLA-A2+ melanoma patient. Tandem mass spectrometry was used to characterize and sequence one of these three naturally processed melanoma peptides. One of the potential forms of the deduced peptide sequence (XXTVXXGVX, X = I or L) matches positions 32-40 of the recently identified melanoma gene MART-1/Melan-A. This peptide (p939; ILTVILGVL) binds to HLA-A2 with an intermediate-to-low affinity and is capable of sensitizing the HLA-A2+ T2 cell line to lysis by CTL lines and clones derived from five different melanoma patients. A relative high frequency of anti-p939-specific effector cells appear to be present in situ in HLA-A2+ melanoma patients, since p939 is also recognized by freshly isolated tumor infiltrating lymphocytes. p939 represents a good candidate for the development of peptide-based immunotherapies for the treatment of patients with melanoma.

Amino Acid Sequence↗
PubMed1995

Identification of Meth A sarcoma-derived class I major histocompatibility complex-associated peptides recognized by a specific CD8+ cytotoxic T lymphocyte.

The finding that class I major histocompatibility complex (MHC)-restricted cytotoxic T lymphocytes (CTL) recognize peptide antigens (epitopes) bound to class I MHC molecules has accelerated efforts to identify CTL-defined tumor peptides for the development of peptide-based cancer immunotherapy. The Meth A sarcoma is probably one of the best studied of all murine tumors. It is extremely lethal unless protective immunity is induced. We recently reported the characterization of a cloned H-2Kd-restricted, CD8+ anti-Meth A CTL line (CTLMA-9C; Frassanito et al., Cancer Res., 54: 4424-4429, 1994). The cytotoxic reactivity of this CTL was shown to be restricted to Meth A sarcoma, and the results of the analysis of the immunogenicity of the CTL-resistant variant of Meth A, designated Meth A4R, indicate that the CTL-defined epitope is functional in tumor rejection. Here we have isolated class I MHC-associated peptides from Meth A sarcoma by mild acid treatment and resolved them into sixty fractions by reverse phase-HPLC. These fractions were then tested for their ability to sensitize the DBA/2 mastocytoma P815 to cytolysis by the anti-Meth A CTL. A single fraction, fraction 27, has been repeatedly identified as containing the CTL-defined epitope. Peptides eluted from the CTL-resistant variant, Meth A4R, failed to sensitize P815 to cytolysis by the anti-Meth A CTL, while fraction 27 derived from Meth A sensitized Meth A4R to lysis by the CTL. These findings confirm the peptide nature of the epitope recognized by CTL on the surface of Meth A. Our future efforts will focus on the identification and sequence analysis of the tumor peptides and the development of a tumor peptide-based vaccine model for immunotherapy.

Animals↗
PubMed1995

Administration of systemic or local interleukin-2 enhances the anti-tumor effects of interleukin-12 gene therapy.

Interleukin-12 (IL-12) is a cytokine with a wide variety of immunoregulatory activities. These include stimulation of interferon-gamma production, cytolytic activity of natural killer (NK) cells and T-cell subsets, the development of cellular immunity, and induction of maturation of Th1 cells. IL-12 also has potent anti-tumor activity in vivo. In the present study the possibility of enhanced anti-tumor activity was examined using a combination of local IL-12 by cytokine gene therapy at the tumor site, combined with systemic or local IL-2 delivery. NIH 3T3 fibroblasts transfected with the genes for both subunits of IL-12, p35 and p40, were used as the source of IL-12 therapy producing 240 HLRU/10(6) cells/48 hr. In the first part of the study the effect of different regimens of systemic IL-2 delivery with local IL-12 administration on the size and growth rate of subcutaneous MCA-105 murine sarcoma was examined. Local IL-12 alone reduced the sizes of tumors after 32 days from 163 to 26.8 mm2 (P < 0.002). Adding the longer-acting polyethylene-glycol-modified IL-2 (PEG IL-2; 30,000 IU) for 5 days prevented the development of tumors in all treated mice compared to 1/3 mice treated with PEG IL-2 alone and 3/6 mice with IL-12, but this was a highly toxic therapy and most of the animals died. Administration of 60,000 IU of IL-2 on Days 1-5 postinoculation of tumor, delivered with IL-12 gene therapy, reduced the tumor growth rate compared to animals treated with IL-2 alone (P < 0.02) or IL-12 (0.1).(ABSTRACT TRUNCATED AT 250 WORDS)

3T3 Cells↗