Immunity to the alpha(1,3)galactosyl epitope provides protection in mice challenged with colon cancer cells expressing alpha(1,3)galactosyl-transferase: a novel suicide gene for cancer gene therapy

Human immunity to alpha(1,3)Galactosyl epitopes (alpha Gal) may provide the means for a successful cancer gene therapy that uses the immune system to identify and to destroy tumor cells expressing the suicide gene alpha(1,3)Galactosyltransferase (alpha GT). Innate antibody specific for cell surface alpha Gal constitutes a high percentage of circulating IgG and IgM immunoglobulins in humans and is the basis for complement-mediated hyperacute xenograft rejection and antibody-dependent cell-mediated cytotoxicity. In humans, the gene for alpha GT is mutated, and cells do not express the alpha Gal moiety. We hypothesized that human tumor cells induced to express the alpha Gal epitope would be killed by the hosts' innate immunity. Previous in vitro work by our group has demonstrated complement-mediated lysis of alpha Gal-transduced human tumor cells in culture by human serum. To induce antibodies to alpha Gal in this in vivo study, alpha GT knockout mice were used to determine whether immunization with alpha Gal could provide protection from challenge with alpha Gal-expressing murine MC38 colon cancer cells. Knockout mice were immunized either a single time, or twice, with rabbit RBC. Antibody titers to alpha Gal measured by indirect ELISA were significantly higher in mice immunized twice and approached the titers observed in human serum. Anti-alpha Gal antibodies were predominantly of the IgG1 and IgG3 subtype. Immunized knockout mice were challenged i.p. with varying doses of alpha Gal(+) MC38 colon carcinoma cells. Nonimmunized control groups consisting of alpha GT knockout mice, and wild-type C57BL/6 mice were challenged as well with MC38 cells. Immunized mice survived and exhibited slower tumor development in comparison to nonimmunized knockout and control mice. This study demonstrates, in vivo, the protective benefit of an immune response to the alpha Gal epitope. Our results provide a basis to pursue additional development of this cancer gene therapy strategy.     

Antibody-based vaccines for the treatment of melanoma

Malignant melanoma remains a difficult clinical problem. Chemotherapy is not effective and immunotherapy has long been contemplated as the preferred approach to this disease. Extensive passive and active immunotherapy trials have been conducted. Active vaccination with whole cells or defined antigens, administered with a panoply of techniques to increase immunogenicity, has yielded inconsistent results. The development of antibody-based vaccines has allowed vaccination without the need for tumor tissue material or purified antigens. The idiotype network theory originally proposed by Lindemann and by Jerne provided the basis for the development of anti-idiotype (anti-Id) antibody vaccines, which mimic the internal image of the epitope targeted for immunization. Preclinical and phase I clinical data are available for various malignancies. In melanoma, some of the anti-Id vaccines have targeted gangliosides. One of these vaccines, TriGem, has been successful in generating a robust and specific humoral immunity in melanoma patients. Phase II data suggest this anti-Id vaccine has clinical activity.     

The serologic response of patients with stage II melanoma to allogeneic melanoma cell vaccines

Seventeen patients with Stage II malignant melanoma were treated with vaccines prepared from three allogeneic melanoma cell lines in an attempt to induce a humoral immune response against melanoma cell surface antigens. The patients were free of detectable melanoma at the time of vaccination. Vaccines were prepared from three melanoma cell lines that expressed highly restricted melanocyte differentiation antigens. One of these cell lines also expressed an antigen found only on this particular line. The antigens were initially identified by antibodies in autologous serum; they were thus known to be recognized by the human immune system. In addition, two of the cell lines expressed HLA-A, -B, -C, and -DR antigens; no HLA antigens were detectable on the third line. The vaccines were administered sequentially by subcutaneous injection, mixed with bacillus Calmette-Guerin (BCG) or Corynebacterium parvum. The patients' sera were tested for antibodies against cell surface antigens of the vaccine cells in protein A assays and immune adherence assays, and the specificity of observed reactions was defined by absorption tests. Antibodies against alloantigens of the vaccine cells developed in 16 patients and in 15 patients, against antigens related to fetal calf serum in the culture medium. The magnitude of the antibody response to alloantigens varied considerably, with no difference between patients who received BCG or C. parvum with their vaccines. Antibodies against the restricted melanocyte differentiation antigens or the unique melanoma antigen expressed by the vaccine cells were not detected.     

Treatment of patients with progressive unresectable metastatic melanoma with a heterologous polyvalent melanoma whole cell vaccine

Unresectable metastatic melanoma has no elective treatment. Neither chemotherapy, intravenous IL-2 nor biochemotherapy clearly improves the overall survival. Recent assays with therapeutic vaccines have been recently yielded promising results. Here, we describe the application, clinical tolerance and antitumoural activity of a heterologous polyvalent melanoma whole cell vaccine in patients with metastatic melanoma. Twenty-eight AJCC stage III/IV melanoma patients with progressive unresectable metastatic disease were treated with our heterologous polyvalent melanoma whole cell vaccine between July 1, 1998 and July 1, 2002. All patients had already been unsuccessfully treated with high doses of IFN-alpha2 and/or polychemotherapy and/or biochemotherapy and/or perfusion of extremities, or could not receive other treatments due to their age or underlying illness. Twenty-three were assessable. The vaccine was constituted by 10 melanoma cell lines, derived from primary, lymph node and metastatic melanomas. Prior to intradermal inoculation, the cells were irradiated and mixed with BCG, and 50% were treated with DNFB. After a median follow-up of 19 months, 26% of patients responded: 3 CR (18, 16+, and 26+ months), 2 PR (8 and 22 months) and 1 MR (36+ months). The median survival of the whole group was 20.2 months. None of the 28 patients initially included in the study presented significant toxicity. This vaccination program had specific antitumoural activity in advanced metastatic melanoma patients and was well tolerated. The clinical responses and the median survival of our group of patients, together with the low toxicity of our polyvalent vaccine, suggest that this approach could be applied to earlier metastatic melanoma patients.     

Effective chemotherapy for melanoma after treatment with interleukin-2.

Twenty patients with biopsy-proven metastatic malignant melanoma, previously treated with interleukin-2 (IL-2), received combination chemotherapy for progressive disease. Treatment included carmustine, cisplatin, dacarbazine, and tamoxifen (BCDT). Nausea was the most common toxicity (100%) and usually was mild. Persistent thrombocytopenia was the most frequent toxicity limiting further treatment. Eleven patients (55%) had an objective partial response, three patients (15%) had a minor response, and six patients (30%) had no change or progressive disease in response to this treatment. These results were comparable to the high response rates (21 of 40, 53%) achieved with BCDT in previously untreated patients with melanoma. It was concluded that prior therapy using IL-2 does not significantly alter the response rate of metastatic melanoma to BCDT, thus suggesting that immunomodulators (e.g., IL-2) and chemotherapeutic agents are not cross-resistant treatments.     

Features associated with survival in metastatic melanoma patients treated with patient-specific dendritic cell vaccines

Previously, a 54% 5-year survival was reported for metastatic melanoma patients treated with patient-specific vaccines consisting of autologous dendritic cells loaded with antigens from autologous proliferating tumor cells. This study attempted to determine which clinical and laboratory factors best explained long-term survival in this group of patients. Univariate analyses were used to identify factors associated with continuous survival after initiating vaccine therapy. Multivariate logistic regression was used to identify independent factors to classify survival at 3.5 years. Survivors were followed a minimum of 3.7 years (median: 5.7). Univariate analyses identified eight features associated with improved survival: Eastern Cooperative Oncology Group (ECOG) performance status (PS) of 0, no measurable disease at study entry, receiving 8 vaccinations, age <50 years, normal baseline lactate dehydrogenase, no history of visceral metastases, anergy to standard skin tests, and failure of interferon-gamma (IFN-γ) to induce apoptosis in autologous tumor cells. After examining 54 variables for which complete information was available over all patients, the best multivariate regression for survival at 3.5 years utilized six features: prior radiation therapy, younger age, male gender, ECOG PS 0, higher numbers of cells administered during the first 3 injections, and lower numbers of viable cells administered during the first 3 injections. This model correctly classified survival for 28 of 32 patients (87%) and death for 20 of 22 (91%). When features with incomplete information were included in the analysis, addition of IFN-γ-induced apoptosis (n=49) improved predictive accuracy to 27 of 29 (93%) for survival and 19 of 20 (95%) for death. Dependencies between variables were common, but these multivariate linear models yielded high classification accuracy for survival at 3.5 years and identified two features of the vaccine itself as being of independent significance.     

Increased immunogenicity of tumor vaccines complexed with anti-Gal: studies in knockout mice for alpha1,3galactosyltransferase.

A major prerequisite for the success of tumor vaccines is their effective uptake by antigen-presenting cells (APCs) and transport of these APCs to the draining lymph nodes, where the processed and presented tumor-associated antigens activate tumor-specific naive T cells. We previously suggested that the immunogenicity of autologus tumor vaccines in humans may be augmented by engineering vaccinating tumor cell membranes to express alpha-galactosyl (alpha-gal) epitopes (i.e., Galalpha1,3Galbeta1,4GlcNAc-R). Subsequent in situ binding of natural anti-Gal IgG molecules to these epitopes would result in the formation of immune complexes that target tumor vaccines for uptake by APCs, via the interaction of the Fc portion of anti-Gal with Fcgamma receptors on APCs. This hypothesis was tested in a unique experimental animal model of knockout mice for alpha1,3galactosyltransferase (alpha1,3GT) and the mouse melanoma B16-BL6 (referred to here as BL6). Like humans, these mice lack alpha-gal epitopes and produce anti-GaL BL6 melanoma cels are highly tumorigenic, and like human tumor cells, they lack alpha-gal epitopes. Expression of alpha-gal epitopes on these melanoma cells was achieved by stable transfection with alpha,3GT cDNA. The transfected melanoma cells (termed BL6alphaGT) express approximately 2 x 10(6) alpha-gal epitopes per cell and readily form immune complexes with anti-Gal. Vaccination of the mice with 2 x 10(6) irradiated melanoma cells that express alpha-gal epitopes, followed by challenge with 0.5 x 10(6) live parental melanoma cells, resulted in protection for at least 2 months (i.e, no tumor growth) in one-third of the mice, whereas all mice immunized with irradiated parental melanoma cells developed tumors 21-26 days post-challenge. The proportion of protected mice doubled when the mice were immunized twice with irradiated melanoma cells expressing alpha-gal epitopes and challenged with 0.2 x 10(6) live BL6 cells. Histological studies on the developing tumors in challenged mice that were immunized with melanoma cells expressing alpha-gal epitopes demonstrated extensive infiltration of T lymphocytes and macrophages, whereas no mononuclear cell infiltrates were observed in tumors of mice immunized with parental tumor cells. Overall, these studies imply that immunization of alpha1,3GT knockout mice with BL6 melanoma cells that express alpha-gal epitopes elicits, in a proportion of the population, protective immune response against the same tumor lacking such epitopes. These studies further suggest that similar immunization of cancer patients with autologous tumor vaccines that are engineered to express alpha-gal epitopes may increase the immune response to autologous tumor-associated antigens and, thus, may elicit immune-mediated destruction of metastatic cells expressing these antigens.     

Effective introduction of T cell costimulatory molecules into virus modified tumor cell vaccines by modification with bispecific antibodies

This report describes the generation of bispecific antibodies which bind with one arm to virus modified tumor cell vaccines and introduce with the other arm anti-murine CD28 T cell costimulatory molecules. This is an effective alternative to somatic gene therapy strategies using genes coding for ligands of CD28 such as CD80 (B7-1) or CD86 (B7-2). While these B7 molecules interact not only with CD28 but also with CLTA-4, thereby generating a negative signal, agonistic anti CD28 antibodies only bind to CD28 and therefore deliver only positive costimulatory signals. The new bispecific antibody (bsAb) HN x CD28 allows the introduction of anti-CD28 antibodies into the tumor cell vaccine ATV-NDV, an autologous tumor cell vaccine already modified by infection with Newcastle Disease Virus (NDV). The bsAb HN x CD28 attaches with its anti-HN binding site to the NDV derived hemagglutinin-neuraminidase (HN) molecule which serves as a common foreign anchoring molecule in the vaccine. NDV infected tumor cells which were further modified with HN x CD28 on their cell surface (bs-vaccine), showed increased T cell stimulatory capacity in vitro. This was revealed by augmented proliferation as well as augmented CTL activity. When syngeneic mice were injected with aggressive murine ESb lymphoma cells which were infected with NDV and further modified with the bsAb HN x CD28, delayed tumor development and prolonged survival was observed in comparison to respective controls.     

Immunosuppressive effects of interleukin-12 coexpression in melanoma antigen gene-modified dendritic cell vaccines

Genetic immunotherapy with tumor antigen gene-modified dendritic cells (DC) generates robust immunity, although antitumor protection is not complete in all models. Previous experience in a model in which C57BL/6 mice immunized with DC transduced with adenoviral vectors expressing MART-1 demonstrated a 20-40% complete protection to a tumor challenge with B16 melanoma cells. Tumors that did develop in immunized mice had slower growth kinetics compared to tumors implanted in na?ve mice. In the present study, we wished to determine if the supraphysiological production of the Th1-skewing cytokine interleukin-12 (IL-12) could enhance immune activation and antitumor protection in this model. In a series of experiments immunizing mice with DC cotransduced with MART-1 and IL-12, antitumor protection and antigen-specific splenocyte cytotoxicity and interferon gamma production inversely correlated with the amount of IL-12 produced by DC. This adverse effect of IL-12 could not be explained by a direct cytotoxic effect of natural killer cells directed towards DC, nor the production of nitric oxide leading to down-regulation of the immune response - the two mechanisms previously recognized to explain immune-suppressive effects of IL-12-based vaccine therapy. In conclusion, in this animal model, IL-12 production by gene-modified DC leads to a cytokine-induced dose-dependent inhibition of antigen-specific antitumor protection.     

Effective inhibition of cell growth and invasion of melanoma by combined suppression of BRAF (V599E) and Skp2 with lentiviral RNAi

p27Kip1 that regulates the G1/S transition of cell cycle and inhibits Rho A signaling is frequently lost in several cancers leading to the deregulation of cell growth and cell motility. Mitogen-activated protein kinases (MAPK) regulate the export of p27Kip1 from nucleus to cytoplasm, followed by the degradation with proteases. Skp-2, a subunit of an SCF ubiquitin-protein ligase complex responsible for the ubiquitination of p27Kip1, is upregulated frequently in several cancers, leading to the decrease of p27Kip1. We applied human immunodeficiency virus (HIV) lentivirus-mediated RNA interference (RNAi) to melanoma cells with the BRAF mutation (V599E) and overexpressed Skp-2 and found that the simultaneous suppression of these activated oncogenes resulted in the effective inhibition of in vitro cell growth and invasive ability of melanoma cells accompanied by the additional increase of p27Kip1. Our results suggest that gene therapy against melanoma with the enhanced MAPK and ubiquitin-proteasomal pathways could be a specific and effective therapeutic strategy for cancers.     

Experience with interferon alpha 2b combined with dacarbazine in the treatment of metastatic malignant melanoma

A prospectively randomized trial was undertaken to compare dacarbazine (DTIC) alone with DTIC plus interferon in patients with metastatic malignant melanoma. Of the 73 patients who entered on the study, 36 were randomized to receive DTIC alone and 37 were randomized to receive the combination DTIC plus interferon. The two sections were well balanced. There was more toxicity on the combination section, but no life threatening toxicity. The overall response rate for patients on DTIC was 20% (two complete and five partial responses)(95% CI 7–39%) and for patients on DTIC plus interferon was 50% (13 complete and four partial responses)(95% CI 26–72%) (p=0.007). The median time to treatment failure, was significantly more in favour of the combination treatment (9versus 2.5 months;p < 0.01, Mantel-Cox). The median survival of 16.7versus 8 months was in favor of the combination treatment (p < 0.01). The reasons for the improved results with the combination treatment are discussed. The Eastern Cooperative Oncology Group is currently, based on the results of this study, investigating the role of interferon combinations in metastatic malignant melanoma     

Differential clinical significance of alpha(v)Beta(3) expression in primary lesions of acral lentiginous melanoma and of other melanoma histotypes

Despite its potential clinical relevance, alpha(v)beta(3) expression has been analyzed only in a limited number of melanoma lesions, mostly nodular melanoma (NM) and superficial spreading melanoma (SSM). Therefore, in the present study, we have correlated alpha(v)beta(3) expression in 33 acral lentiginous melanomas (ALMs), 6 lentigo maligna melanomas, 7 mucosal melanomas, 12 NMs and 9 SSMs with their antigenic profile, with their histo-pathological characteristics and with the clinical course of the disease. Furthermore, we have compared alpha(v)beta(3) expression in ALM lesions with that in NM and SSM lesions since this information helps to clarify the relationship of the latter 2 histotypes with ALM. Such a relationship is uncertain since ALM has a clinical course similar to that of NM and SSM despite different antigenic profiles and biological characteristics. The level of alpha(v)beta(3) expression in primary lesions was not correlated with that of high-m. w. melanoma-associated antigen and intercellular adhesion molecule-1, with lesion thickness and with disease recurrence in ALM but was significantly correlated with these 4 parameters in the other melanoma histotypes analyzed. Therefore, alpha(v)beta(3) expression appears to have a differential clinical significance in ALM and in the other histotypes of melanoma we have analyzed since it appears to play a significant role in the progression of the disease only in non-ALM histotypes.     

腺病毒转染黑色素瘤抗原基因3的树突状细胞疫苗抗胃癌的免疫效应

目的 研究趋化因子巨噬细胞炎症蛋白1α(MIP-1α)动员的树突状细胞(DC),经黑色素瘤抗原基因3(MAGE-3)腺病毒转染后对胃癌细胞的免疫效应.方法 615小鼠尾静脉注射MIP-1α,分选得到B220-CD11c+细胞,加入细胞因子连续培养,检测其细胞表面标志和混合淋巴细胞反应(MLR).收集培养后的B220-CD11c+细胞,加入编码MAGE-3的重组腺病毒进行转染,制备表达肿瘤抗原的DC疫苗,以荷载小鼠前胃癌(MFC)全肿瘤细胞抗原制备的DC疫苗作为阳性对照.采用二苯基溴化四氮唑蓝(MTT)法,榆测活化的T淋巴细胞在体外对MFC细胞的杀伤作用.采用酶联免疫吸附试验(ELISA),检测γ干扰素(INF-γ)的分泌情况.DC疫苗皮下注射MFC荷瘤小鼠,观察小鼠瘤体生长情况和存活时间.结果 MIP-1α注射后,外周血中B220-CD11c+细胞明显升高.新鲜分离的B220-CD11c+细胞在体外经过细胞因子诱导分化后具有典型的DC表面标志,并在MLR中具有极强的刺激T淋巴细胞增殖的能力.腺病毒转染MAGE-3的DC激活的T淋巴细胞表现出对MFC细胞的特异性杀伤作用,产牛高水平的INF-γ[(1460.00±16.82)ps/ml].实验组荷瘤小鼠接受DC疫苗治疗后,肿瘤牛长缓慢,观察至MFC细胞接种后的第27天,其肿瘤体积仅为(3.46±1.12)cm3;实验组小鼠的肿瘤体积与对照组之间的差异有统计学意义(P<0.01).实验组小鼠存活时间明显延长,与对照组之间的差异有统计学意义(P<0.01).结论 注射趋化因子MIP-1α可快速动员并诱导分化为成熟DC.肿瘤抗原基因MAGE-3经腺病毒转染制备的DC疫苗,在体外可以诱导出针对胃癌细胞的特异性杀伤作用,在体内对MFC荷瘤小鼠有明显的免疫治疗作用.     

Effective treatment of leukemic cell lines with wt1 siRNA.

The expression of wt1 and bcl-2 is considered to have a proliferating and survival supporting effect in leukemia blast cells. Here we describe the use of siRNA against wt1 and bcl-2 in leukemic cell lines for successful growth inhibition. We have used two different sequences designated as siRNA-A and siRNA-B corresponding to positions within the wt1 coding sequence to downregulate wt1 and a commercially available siRNA kit to downregulate bcl-2. WT1 and bcl-2 gene expression in transfected leukemic cell lines were evaluated with RT-PCR and western blot analyses. MTT assay was used to measure the cell viability and flow cytometry using annexin V/PI-staining for apoptosis. K562 and HL-60 cell lines transfected with siRNA-A targeted to wt1 had greatly decreased levels of both wt1 mRNA and protein expression. In contrast, siRNA-B and control siRNA led almost to no effect on wt1 mRNA and protein expression. siRNA-A-reduced wt1 mRNA expression was associated with a decreased cell proliferation and increased number of apoptotic cells in K562 and HL-60 cells by 24 and 48 h after transfection. Combined treatment with wt1 siRNA and bcl-2 siRNA simultaneously was not able to override the cell growth and apoptosis effects compared to single treatment with wt1 siRNA. siRNAs targeted against human wt1 might be a valuable tool as antiproliferative agent against wt1 expressing leukemic cells.     

A study on melanoma treatment using dendritic cells loaded with antigens purified from melanoma cell lines

Objective The aim of this study was to purify effective tumor peptide complexes from human melanoma cell lines to enhance the treatment effects on melanoma.Methods We purified heat shock protein 70(HSP70)-peptide complexes(PCs)from human melanoma cell lines A375,A875,M21,M14,WM-35,and SK-HEL-1.We named the purified product as M-HSP70-PCs and determined its immunological activities.Autologous HSP70-PCs purified from primary tumor cells of melanoma patients(9 cases)were used as controls.These two tumor antigenic complexes were loaded into dendritic cells(DCs)and used to stimulate an antitumor response against tumor cells in the corresponding patients.Results Mature DCs pulsed with M-HSP70-PCs stimulated autologous T cells to secrete the same levels of type I cytokines as the autologous HSP70-PCs.Moreover,DCs pulsed with M-HSP70-PCs endued CIK cells with an equal ability as autologous HSP70-PCs to kill melanoma cells in the patients.Conclusion M-HSP70-PCs may be used as an efficient and generalized tumor antigen in the treatment of DC-based malignant melanoma.     

Active immunotherapy of metastatic melanoma with allogeneic melanoma lysates and interferon alpha.

PURPOSE: A therapeutic lyophilized melanoma vaccine consisting of two mechanically disrupted allogeneic melanoma cell lines and the immunological adjuvant Detox-PC (Melacine) has demonstrated encouraging activity in metastatic malignant melanoma, often in regimens containing pretreatment with low-dose cyclophosphamide. In addition, IFN-alpha2b (INTRON A; Schering-Plough Corporation, Kenilworth, NJ) has shown efficacy in melanoma refractory to Melacine. In this Phase II trial, the combination of cyclophosphamide, Melacine, and IFNalpha was tested in metastatic malignant melanoma. EXPERIMENTAL DESIGN: Eligibility criteria included measurable disease, no prior systemic therapy for a minimum of 4 weeks, and adequate marrow, renal, and hepatic function. Cyclophosphamide was administered once at a dose of 300 mg/m(2) i.v. on day -3 before the first dose of Melacine. Melacine was administered at a dose of 2 x 10(7) tumor cell equivalents per dose admixed with 0.25 ml of Detox-PC s.c. once a week on weeks 1-4 and week 6. Melacine maintenance was then given monthly from the 8th week, until progression or intolerable toxicity. IFN was started in the evening after the fourth dose of Melacine at a dose of 5,000,000 units/m(2) 3 times a week, and continued until progression. RESULTS: Forty-seven patients were enrolled, of whom 39 completed the full course and were considered evaluable. The toxicity of the regimen was minimal and consisted mainly of pain at injection sites and granulomas caused by Detox-PC, and constitutional symptoms attributable to IFN. In 39 evaluable patients, the overall objective response rate was 10.2%, but 64% of patients had stabilization of their disease for at least 16 weeks. The median time to disease progression in evaluable patients was 8 months [95% confidence interval (CI), 6-13 months]. Median survival time for all of the 47 patients enrolled was 12.5 months (95% CI, 8-15 months) with a median time to disease progression of 4 months (95% CI, 3-7 months). CONCLUSION: Despite a low objective response rate, this combination holds great promise because of its tolerability and the high proportion of prolonged durations of remission or disease stabilization that it elicited.