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Most immunotherapy studies in animal tumor models are performed in early stages of the disease. Reports on the studies of treatment in late stages of tumor growth and metastasis are much rarer. To guide future efforts for treatment in late-stage disease, a model of effective immune rejection of advanced metastasized cancer is reviewed and lessons therefrom are summarized. Already cachectic DBA/2 mice with a subcutaneously transplanted syngeneic tumor (ESb-MP lymphoma) of 1.5 cm diameter and with macroscopic liver and kidney metastases at 4 weeks could be successfully treated by a combination of sublethal (5 Gy) irradiation followed by a single transfer of 20 million anti-tumor immune spleen cells from tumor-resistant allogeneic MHC-B10.D2 mice. Following intravenous cell transfer, the primary tumors became encapsulated and were eventually rejected from the skin while visceral metastases gradually disappeared leaving behind only scar tissue. There was wound-healing at the site of the rejected primary tumor, and the animals survived long term without any tumor recurrence. The complete eradication of late-stage disease by adoptive cellular immunotherapy could be corroborated noninvasively by P-NMR spectroscopy of primary tumors and by H-NMR microimaging of liver metastases. Conclusions from functional mechanistic studies in this model are summarized and clinical implications discussed. [ABSTRACT FROM AUTHOR]

The article looks at a reproducible therapy model for advanced intracerebral B16 melanoma. It is noted that implanted tumors suppressed by a single radiosurgical dose of 100 kVp X-rays were further suppressed by a single ip injection of a Treg-depleting mAb given two days prior to the initiation of four weekly then eight bi-monthly sc injections of GMCSF-transfeced, mitotically disabled B16 cells. This treatment course reduced tumors in mice.

Previously, safety and immunogenicity of human papillomavirus type 16 (HPV16) or 18 E7-pulsed dendritic cells (DC) vaccinations were demonstrated in a dose-escalation Phase I clinical trial which enrolled ten patients diagnosed with stage IB or IIA cervical cancer (nine HPV 16-positive, one HPV 18-positive). The goal of the study was to define the T-cell epitopes of HPV 16 or 18 E7 protein in these patients in order to develop new strategies for treating HPV-associated malignancies. This was accomplished through establishing T-cell lines by stimulating peripheral blood mononuclear cells with autologous mature DC pulsed with the HPV 16 or 18 E7 protein, examining the T-cell responses using ELISPOT assays, and isolating E7-specific T-cell clones based on IFN-γ secretion. Then, the epitope was characterized in terms of its core sequence and the restriction element. Twelve T-cell lines from eight subjects (seven HPV 16-positive, one HPV 18-positive) were evaluated. Positive T-cell responses were demonstrated in four subjects (all HPV 16-positive). All four were positive for the HPV 16 E7 46-70 (EPDRAHYNIVTFCCKCDSTLRLCVQ) region. T-cell clones specific for the E7 47–70 region were isolated from one of the subjects. Further analyses revealed a novel, naturally processed, CD4 T-cell epitope, E7 58–68 (CCKCDSTLRLC), restricted by the HLA-DR17 molecule. [ABSTRACT FROM AUTHOR]

Metastatic disease is a major cause of mortality in colorectal cancer patients. Even after complete resection of isolated liver metastases, recurrence develops in the majority of patients. Therefore, development of strategies to prevent recurrent liver metastases is of major clinical importance. The present prospectively randomised phase III trial investigates the efficiency of active specific immunotherapy (ASI) after liver resection for hepatic metastases of colorectal cancer. Patients with histologically confirmed liver metastases from colorectal cancer were randomised to the vaccination or control group. After complete resection of liver metastases, patients randomised to the vaccination group received six doses of Newcastle disease virus (NDV) infected autologous tumour cell vaccine (ATV-NDV). The primary end-point was overall survival, secondary end-points were disease-free survival and metastases-free survival. Fifty-one patients were enrolled in the study with 50 patients available for analysis. The follow-up period was 116.1 ± 23.8 month in the vaccination arm and 112.4 ± 18.5 month in the control group. In the total patient group, no differences in the primary and secondary end-points were detected. Most interestingly, subgroup analysis revealed a significant advantage for vaccinated colon cancer patients with respect to overall survival [hazard ratio: 3.3; 95%, confidence interval (CI): 1.0–10.4; P = 0.042] and metastases-free survival (hazard ratio: 2.7; 95%, CI: 1.0–7.4; P = 0.047) in the intention-to-treat analysis. Active specific immunotherapy in unselected colorectal cancer patients was not effective for prevention of recurrent metastatic disease. However, in colon cancer patients, ASI with ATV-NDV appears to be beneficial prolonging overall and metastases-free survival. [ABSTRACT FROM AUTHOR]

Type I interferon (IFN) possesses antiviral and antitumor activities and also having an immune regulatory effect, activating cellular immune response and upregulating several cytokines. Recent study has shown that type I IFN upregurates the dendritic cell production of IL-15 capable of activating natural killer cells and CD8+ memory T lymphocytes. However, it is still unknown if type I IFN induces IL-15 production in non-immune cells and if type I IFN affects IL-15 production in vivo. The present study investigated the effect of type I IFNs on IL-15 expression in hepatocellular carcinoma (HCC) cell lines in vitro and in patients with chronic hepatitis C in vivo. When three HCC cell lines, Huh7, HepG2, and JHH4 were cultured in vitro, IFN upregulation of IL-15 expression was observed at both the mRNA and protein levels. In experiments using Huh7 cells, upregulation of IL-15 expression occurred within 24 h of the start of IFN stimulation, and both IFN-α and -β dose-dependently increased IL-15 production in the range from 100 U/ml to 10,000 U/ml of concentration. IFN-β showed stronger activity in IL-15 production induction in vitro than IFN-α. For in vivo examination, sera were obtained from 21 chronic hepatitis C patients treated with IFN and 29 healthy individuals, and the serum IL-15 level was quantified by ELISA. The serum IL-15 level of chronic hepatitis C patients before IFN treatment was similar to that of the healthy controls and significantly increased only during the IFN administration period. These results confirm that IFN-α/β induce IL-15 production and also suggest that IL-15 may be associated with type I IFN-induced immune response. [ABSTRACT FROM AUTHOR]

Prostate specific antigen (PSA) is a serum marker that is widely used in the detection and monitoring of prostate cancer. Though PSA is a self-antigen, T cell responses to PSA epitopes have been detected in healthy men and prostate cancer patients, suggesting it may be used as a target for active immunotherapy of prostate cancer. A PSA DNA vaccine (pPSA) was evaluated in mice and monkeys for its ability to induce antigen-specific immune responses. Mice immunized intradermally with pPSA demonstrated strong PSA-specific humoral and cellular immunity. The anti-PSA immune responses were skewed toward Th1, as shown by high IFNγ and IL-2 production. The immune response was sufficient to protect mice from challenge with PSA-expressing tumor cells. Tumor protection was durable in the absence of additional vaccination, as demonstrated by protection of vaccinated mice from tumor rechallenge. Furthermore, pPSA vaccination induced PSA-specific antibody titers in male cynomolgus monkeys, which express a closely related PSA gene. These results demonstrate that vaccination with pPSA may be able to break tolerance and can induce an immune response that mediates tumor protection. [ABSTRACT FROM AUTHOR]

For active specific immunotherapy of cancer patients, we designed the autologous virus-modified tumor cell vaccine ATV-NDV. The rationale of this vaccine is to link multiple tumor-associated antigens (TAAs) from individual patient-derived tumor cells with multiple danger signals (DS) derived from virus infection (dsRNA, HN, IFN-a). This allows activation of multiple innate immune responses (monocytes, dendritic cells, and NK cells) as well as adaptive immune responses (CD4 and CD8 memory T cells). Preexisting antitumor memory T cells from cancer patients could be activated by antitumor vaccination with ATV-NDV as seen by augmentation of antitumor memory delayed-type hypersensitivity (DTH) responses. In a variety of phase II vaccination studies, an optimal formulation of this vaccine could improve long-term survival beyond what is seen in conventional standard therapies. A new concept is presented which proposes that a certain threshold of antitumor immune memory plays an important role (1) in the control of residual tumor cells which remain after most therapies and (2) for long-term survival of treated cancer patients. This immune memory is T-cell based and most likely maintained by persisting TAAs from residual dormant tumor cells. Such immune memory was prominent in the bone marrow in animal tumor models as well as in cancer patients. Immunization with a tumor vaccine in which individual TAAs are combined with DS from virus infection appears to have a positive effect on antitumor immune memory and on patient survival. [ABSTRACT FROM AUTHOR]

Many murine tumor cells express not only individual haplotype-matched class I MHC molecules, but also species-specific CD1d molecules. The former class I MHC molecules generally present internally synthesized tumor-derived peptide antigens to highly specific CD8+ cytotoxic T lymphocytes (CTLs) in acquired immunity. In contrast, the latter CD1d molecules may present tumor-associated glycolipid antigens to broadly crossreactive natural killer T (NKT) cells, which might correlate with controlling tumor metastasis. Here, we showed that murine hepatoma cell line Hepa1-6-derived acid-eluted substances might contain both Db class I MHC-restricted antigens and CD1d-restriced substances, which could sensitize not only syngeneic bone marrow-derived DCs (BM-DCs), but also allogeneic BM-DCs expressing haplotype-mismatched class I MHC and species-specific CD1d molecules. To our surprise, intravenous (i.v.) immunization of C57BL/6 mice with the former syngeneic BM-DCs carrying acid-eluted materials primed both CD4–CD8– and CD8+ NKT cells in the spleen, whereas immunization with the latter allogeneic BM-DCs loaded the tumor-derived substances primed CD4-CD8-, but not CD8+ NKT cells. The findings shown in the present study will open a new area for cancer immunotherapy using allogeneic DCs and tumor-derived acid-eluted substances. [ABSTRACT FROM AUTHOR]

The role of β1 (CD29) integrins in natural killer (NK) cell–target cell conjugation and cytotoxicity has not been clearly established. Ligation of β1 integrins in NK cells can modulate the lytic capacity in both a positive and a negative manner; however, the contribution of the β1 integrins present on target cells remains to be evaluated. Here, we analyzed the effect of β1 integrins expressed by potential tumor target cells on conjugation and cytotoxicity. Using normalized flow cytometry binding assays, we demonstrated that the pretreatment of MOLT-4, K562, U-937 and HL-60 human leukemia target cell lines with selected anti-β1 monoclonal antibodies (mAb) increased conjugation to human NK cell line NKL as well as to purified NK cells. Only mAb recognizing residues 207–218 of the β1 subunit and functionally involved in the induction of homotypic adhesion (functional epitope A1) increased conjugation of all the target cells. Moreover, mAb to adhesion molecules different from β1 but also inducers of homotypic adhesion of the target cells, i.e. CD43 and CD50 (ICAM-3), failed to increase conjugation to NKL cells. Cytotoxicity assays demonstrated that lysis of NK-sensitive target cells (MOLT-4) also increased after pretreatment with anti-β1 epitope A1 mAb. Importantly, pretreatment of NK-resistant target cells (U-937 and HL-60) with anti-β1 mAb was not able to outweigh the cytotoxic inhibitory mechanisms controlled by HLA class I molecules. However, simultaneous masking of HLA class I molecules with mAb and pretreatment with anti-β1 mAb rendered NK-resistant cells susceptible to lysis, as predicted by the missing self hypothesis. Triggering of tumor target cells through β1 integrins may thus play a role in conjugation to NK cells as well as in co-stimulation of cell-mediated cytotoxicity. [ABSTRACT FROM AUTHOR]

Tumor necrosis factor α (TNFα) is a cytokine, produced by lymphocytes and monocytes, with cytotoxic activity against some but not all tumor cell lines. Resistance to the cytolytic effects of TNFα has been reported in cell lines with autocrine TNFα production. The purpose of this study was to investigate whether human primary malignant melanoma and tumor infiltrating lymphocytes produce TNFα in vivo. Optimal conditions for in situ hybridization for TNFα mRNA in paraffin-embedded tissue were established. Analysis of 13 primary malignant melanomas and 3 metastatic lesions with different degrees of immunohistochemical TNFα positivity demonstrated that, in some tumors, both melanoma cells and leukocytes contained TNFα mRNA and protein. These findings demonstrate variable production of TNFα in primary and metastatic melanoma in vivo.The previously described resistance to TNFα cytolytic activity may, therefore, be clinically important. [ABSTRACT FROM AUTHOR]

Tumour-specific cytotoxic T lymphocytes (CTL) are usually obtained after immunization in vivo and restimulation of immune cells in vitro. We here describe the generation of syngeneic tumour-specific CTL within no more than 9 days by priming and restimulation in vivo. This is achieved only if the correct sites are used both for primary immunization (ear pinna) and for restimulation (peritoneal cavity). The kinetics of immune T cell induction and of the secondary response in vivo will be reported. While a secondary CTL response could be generated in the peritoneal cavity, this was not possible in the spleen, no matter which routes of antigen restimulation were used. Upon transfer of immune spleen cells into the peritoneal cavity but not into the spleen, a secondary response could be generated upon in situ restimulation, indicating the importance of the correct microenvironment for this type of response. The peritoneal effector cells were true T cells and recognized a tumour-associated antigen in association with the K major histocompatibility (MHC class I) antigen. Finally the activated tumour-specific peritoneal exudate cells were able to transfer protective immunity without exogenous interleukin-2 into normal syngeneic mice. [ABSTRACT FROM AUTHOR]

Active specific immunotherapy was performed in a phase I study in 20 colorectal cancer patients after surgical resection of the tumor. An autologous tumor cell vaccine surface modified by Newcastle disease virus (NDV) was used, which showed the following characteristics. After mechanical and enzymatic dissociation of the tumor tissue an average of 5 × 10 cells/g tissue was obtained. According to trypan blue dye exclusion assay the average viability was 72%. Following irradiation (200 Gy) the inactivation of proliferative activity of the cells could be demonstrated by the absence of incorporation ofH-labelled thymidine. The cells were, however, still metabolically active as shown by the incorporation of [H]-uridine and a mixture ofH-labelled amino acids. Epithelium-specific antigens (detected by mAb HEA125) were expressed on more than 75% cells of the cell suspension indicating a high amount of (epithelium-derived) tumor cells. In order to increase the immunogenicity of the tumor cells the suspended cells were infected by the nonlytic, apathogenic Ulster strain of NDV. The successful modification of tumor cells with NDV could be shown by electron microscopy. Three weeks postoperatively cells were thawed, virus-modified, and inoculated intradermally in the upper thigh. Several cell and virus concentrations were tested in each patient. As control, tumor cells without NDV, NDV alone and normal colon mucosa were used. The number of tumor cells ranged from 2 × 10 up to 2 × 10 cells and NDV concentrations from 4 to 64 hemagglutination units (HU) were tested. Sixteen patients responded with a delayed-type hypersensitivity (DTH) skin reaction to the vaccine. The best DTH reaction, measured 24 h following vaccination, was obtained using a vaccine consisting of 1 × 10 tumor cells and 32 HU NDV (median induration of 8 mm). Response to NDV alone was seen in 2 patients only (median induration of 3 mm); 12 patients responded to tumor cells (1 × 10) alone (median induration of 4 mm). Of 10 patients tested with normal colorectal mucosa, 4 responded with a median induration of 3.5 mm. DTH responses to the vaccine of 1 × 10 tumor cells and 32 HU NDV increased throughout the repeated vaccinations to a median induration of 9.5 mm at the end of the therapy. No severe side-effects in the course of the immunotherapy, except for mild fever in 4/20 patients, were observed. The results of our phase I study show that this type of autologous colorectal tumor cell vaccine is ready for a large clinical trial to prove its efficacy. [ABSTRACT FROM AUTHOR]

To see whether cancer patients possess CD3 CD4 lymphocytes able to proliferate in response to autologous tumor cells (Auto-Tu), this lymphocyte subset was isolated either by positive or negative selection, both methods resulting in highly enriched CD4 populations. Unseparated and isolated CD3 CD4 lymphocytes were then assayed for proliferating activity in the presence or absence of various amounts of Auto-Tu, with or without recombinant interleukin-2 (IL-2) (1.5-15 U/ml) and DR adherent cells or E lymphocytes as autologous accessory cells (Auto-AC). Isolated CD3 CD4 lymphocytes were stimulated by Auto-Tu alone in only 1 out of 12 cases. CD3 CD4 cells failed to proliferate significantly in response to low doses of IL-2 alone but the addition of Auto-Tu caused stimulation in 8 out of 12 cases (67%). The further addition of Auto-AC to Auto-Tu + IL-2 resulted in enhanced response of isolated CD3 CD4 lymphocytes in 6 out of 8 cases tested. When reactivities to Auto-Tu in the presence of IL-2 and IL-2 + Auto-AC were considered together, positive responses of CD3 CD4 lymphocytes were seen in 11 out of 12 cases (92%). On the other hand, unseparated lymphocytes were stimulated by Auto-Tu alone in none out of 12 cases. Unseparated lymphocytes, however, responded to IL-2 in 11 out of 12 cases; such a response was increased by the addition of Auto-Tu in only 2 cases. Moreover, the IL-2 proliferation of unseparated lymphocytes was suppressed in 4 and in 3 out of 12 cases tested when Auto-Tu or Auto-Tu + Auto-AC were added respectively. These data indicate that lymphocytes of cancer patients contain CD3 CD4 cells that are usually unable to proliferate in response to Auto-Tu only. This proliferation, however, occurs when low doses of exogenous IL-2 are present and can be further amplified by the addition of Auto-AC. No response of CD4 cells is observed in the presence of DR Auto-AC + IL-2 except in 2 out of 7 cases tested (28%), suggesting an Auto-Tu-restricted reactivity of CD3 CD4 lymphocytes in the majority of cases. [ABSTRACT FROM AUTHOR]