In vitro and clinical characterisation of a Newcastle disease virus-modified autologous tumour cell vaccine for treatment of colorectal cancer patients.

A virus-modified autologous tumour cell vaccine prepared from human colorectal cancer cells is described. After dissociation an average of 5 x 10(7) cells/g tissue were obtained from primary tumours and 9 x 10(7)/g tissue from metastases with an average viability of 72% and 51%, respectively. Following irradiation (200 Gy), inactivation of the proliferative activity of the cells was demonstrated by their degeneration in tissue culture and the absence of incorporation of 3H-labelled thymidine. One third of the cells were still metabolically active, as shown by the incorporation of 3H-uridine and a mixture of 3H-aminoacids. The dissociated cells expressed MHC class I and II antigens in a qualitatively similar way to tissue sections. Epithelium-specific antigens (detected by MAb HEA125) were expressed on an average of more than 75% cells of the suspension, while leucocyte-specific antigens (detected by MAb CD53) were expressed on an average of less than 25% cells. The vaccine was prepared by admixing the nonlytic strain Ulster of Newcastle disease virus (NDV) with the tumour cell suspension. The NDV adsorption at tumour cells was shown by electron microscopy. Clinically, the treatment with the vaccine was associated with an increased sensibilisation against autologous tumour cells, measured by DTH skin reactivity. First results in 23 patients with colorectal liver metastases who underwent "curative" liver resection followed by vaccination show a clear correlation between the induced increase of DTH skin reaction against autologous tumour cells and the recurrence-free interval. No correlation was found for DTH reaction caused by standard antigens (Mérieux test), NDV alone or autologous normal liver tissue. The results demonstrate the possibility of preparing immunogenic virus-modified autologous tumour cell vaccine from colorectal cancer tissue, which could be used for cancer therapy.

HLA class I alleles and responsiveness of melanoma to immunotherapy with interferon-alpha (IFN-alpha) and interleukin-2 (IL-2).

MHC-restricted recognition of melanoma cells by cytotoxic T lymphocytes is an important mechanism in vitro. Several HLA alleles can function as restriction elements for melanoma cell recognition. In this study we investigated whether there is a correlation between responsiveness of patients with metastatic melanoma (MM) to immunotherapy and the HLA type of the patient. Seventy-two patients received immunotherapy with IFN-alpha and IL-2 according to two different schedules with an overall response rate of 24%. In 54 patients, including 16 responders and 16 patients with stable disease, in whom peripheral blood lymphocytes were available, we determined HLA haplotypes. As a control group we prospectively determined HLA types in 67 melanoma patients of all stages who underwent surgery or received chemotherapy and of 126 healthy donors from our blood bank. Two HLA alleles that have been shown to function as restriction elements in vitro were observed more frequently in responding patients, namely Cw7 (p = 0.014) and A1 (p = 0.19). No association was found for A2 and B44. These observations provide evidence that the responsiveness of MM to immunotherapy with IL-2 is associated with certain HLA types, suggesting an important role of HLA-restricted T lymphocytes for IL-2-induced tumour regression.

Postoperative active specific immunization in colorectal cancer patients with virus-modified autologous tumor-cell vaccine. First clinical results with tumor-cell vaccines modified with live but avirulent Newcastle disease virus.

Sixteen patients with colorectal carcinoma Dukes' Stage B2, C, or D were treated with an autologous virus-modified tumor-cell vaccine after potential curative tumor resection (R0-Resection). An inoculum of 1 X 10(7) cells incubated with 32 hemagglutination units of nonirradiated Newcastle disease virus (NDV) was given intracutaneously up to four times at 10-day intervals. The delayed-type hypersensitivity (DTH) skin reaction was measured. The vaccination was well tolerated. In 11 of 16 patients an increasing reactivity against the vaccine was observed during the vaccination procedure. A challenge test using autologous tumor cells without NDV after the vaccination cycle revealed a specific antitumor sensibilization in 12 patients. The DTH response was not due to bacterial contamination or sensibility to the virus. Histologic examination of the vaccination site showed a dense infiltration of predominantly helper T-lymphocytes. We conclude that in most of the patients treated active, specific immunization led to a specific antitumor sensitivity.

In vitro expansion and analysis of T lymphocyte microcultures obtained from the vaccination sites of cancer patients undergoing active specific immunization with autologous Newcastle-disease-virus-modified tumour cells.

In order to understand further the effects of Newcastle-disease-virus(NDV)-modified tumour vaccines we investigated the feasibility of isolating lymphocytes from the site of injection of patients undergoing postoperative active specific immunization (ASI) with autologous NDV-modified tumour cells. Delayed-type-hypersensitivity(DTH)-like reactions from five cancer patients were surgically removed, minced and the tissue particles were digested with collagenase and DNase. Lymphoid cells recovered were expanded in a highly efficient limiting-dilution analysis system optimized for T cell growth [Moretta et al. (1983) J Exp Med 157: 743] and lymphocyte microcultures (clonal probability > 0.8) could be grown for up to 1 year. Analysis of the microcultures for phenotype and function showed that the majority were positive for CD4 (92%) and TCR alpha beta (96%). Concanavalin-A-induced production of interleukin-2 (IL-2), IL-6, interferon gamma and tumour necrosis factor alpha was detected in more than 70% of the microcultures. Lectin-dependent cytotoxicity was only very rarely observed. The general characteristics of the microcultures obtained support the notion of a DTH-like reaction taking place at the site of tumour cell challenge. The possibility of in vitro expansion and cultivation of T lymphocytes from ASI vaccination sites should help to elucidate further the role of these cells in active specific immunization against autologous tumour cells.

Active specific immunotherapy with Newcastle-disease-virus-modified autologous tumor cells following resection of liver metastases in colorectal cancer. First evaluation of clinical response of a phase II-trial.

A group of 23 colorectal cancer patients were treated by a new type of active specific immunotherapy (ASI) following complete surgical resection of liver metastases (RO resection). For ASI treatment we used a vaccine consisting of 1 x 10(7) autologous, irradiated (200 Gy) metastases-derived tumor cells incubated with 32 hemagglutination units (HU) of Newcastle disease virus (NDV). The adjuvant vaccine therapy was started 2 weeks after surgery and was repeated five times at 14-days intervals followed by one boost 3 months later. The delayed-type hypersensitivity (DTH) skin reactions to the vaccine were measured as well as the DTH reactions to a challenge test of 1 x 10(7) non-virus-modified autologous tumor cells from liver metastases or 1 x 10(7) autologous normal liver cells. In addition 32 HU NDV alone and a standard antigen test (Merieux test) were applied pre- and post-vaccination. The vaccination was well tolerated. In 13 of 23 patients an increasing reactivity against the vaccine was observed during the vaccination procedure. Nine patients (40%) experienced an increased DTH reactivity against autologous tumor cells following vaccination, while 17% or fewer showed an increased reactivity to Merieux test antigens, NDV, or normal liver cells. The increased antitumor response was not correlated to responsiveness to NDV alone, autologous liver cells, enzymes and culture medium used for vaccine preparation or standard antigens (Merieux test). After a follow-up of at least 18 months 61% of the vaccinated patients developed tumor recurrence in comparison to 87% of a matched control groups from the same institution that had been only surgically treated. The results of this phase II trial are encouraging and should stimulate further prospective randomized studies.