Targeted radionuclide therapies for pancreatic cancer.

Pancreatic malignancies, the fourth leading cause of cancer deaths, have an aggressive behavior with poor prognosis, resulting in a 5-year survival rate of only 4%. It is typically a silent malignancy until patients develop metastatic disease. Targeted radionuclide therapies of cancer such as radiolabeled peptides, which bind to the receptors overexpressed by cancer cells and radiolabeled antibodies to tumor-specific antigens provide a viable alternative to chemotherapy and external beam radiation of metastatic cancers. Multiple clinical trials of targeted radionuclide therapy of pancreatic cancer have been performed in the last decade and demonstrated safety and potential efficacy of radionuclide therapy for treatment of this formidable disease. Although a lot of progress has been made in treatment of pancreatic neuroendocrine tumors with radiolabeled (90)Y and (177)Lu somatostatin peptide analogs, pancreatic adenocarcinomas remain a major challenge. Novel approaches such as peptides and antibodies radiolabeled with alpha emitters, pre-targeting, bispecific antibodies and biological therapy based on the radioactive tumorlytic bacteria might offer a potential breakthrough in treatment of pancreatic adenocarcinomas.

Direct incorporation of the NKT-cell activator α-galactosylceramide into a recombinant Listeria monocytogenes improves breast cancer vaccine efficacy.

BACKGROUND: Immune suppression in the tumour microenvironment remains a major limitation to successful immunotherapy of cancer. In the current study, we analysed whether the natural killer T cell-activating glycolipid α-galactosylceramide could overcome immune suppression and improve vaccination against metastatic breast cancer. METHODS: Mice with metastatic breast cancer (4T1 model) were therapeutically treated with a Listeria monocytogenes-based vaccine expressing tumour-associated antigen Mage-b followed by α-galactosylceramide as separate agents, or as a complex of α-galactosylceramide stably incorporated into Listeria-Mage-b. Effects on metastases, tumour weight, toxicity and immune responses were determined. RESULTS: Sequential treatments of mice with established 4T1 breast carcinomas using Listeria-Mage-b followed by α-galactosylceramide as a separate agent was highly effective at reducing metastases, but was accompanied by severe liver toxicity. In contrast, combined therapy using Listeria-Mage-b modified by incorporation of α-galactosylceramide resulted in nearly complete elimination of metastases without toxicity. This was associated with a significant increase in the percentage of natural killer T cells in the spleen, and an increase in natural killer cell activity and in T cell responses to Mage-b. CONCLUSIONS: Our results suggest that direct incorporation of α-galactosylceramide into a live bacterial vaccine vector is a promising non-toxic new approach for the treatment of metastatic breast cancer.

Myeloid-derived suppressor cells have a central role in attenuated Listeria monocytogenes-based immunotherapy against metastatic breast cancer in young and old mice.

BACKGROUND: Myeloid-derived suppressor cells (MDSCs) are present in large numbers in blood of mice and humans with cancer, and they strongly inhibit T-cell and natural killer (NK) cell responses, at young and old age. We found that a highly attenuated bacterium Listeria monocytogenes (Listeria(at))-infected MDSC and altered the immune-suppressing function of MDSC. METHODS: Young (3 months) and old (18 months) BALB/cByJ mice with metastatic breast cancer (4T1 model) were immunised with Listeria(at) semi-therapeutically (once before and twice after tumour development), and analysed for growth of metastases and primary tumour, in relation to MDSC-, CD8 T-cell and NK cell responses. RESULTS: We found that Listeria(at)-infected MDSC, which delivered Listeria(at) predominantly to the microenvironment of metastases and primary tumours, where they spread from MDSC into tumour cells (infected tumour cells will ultimately become a target for Listeria-activated immune cells). Immunotherapy with Listeria(at) significantly reduced the population of MDSC in blood and primary tumours, and converted a remaining subpopulation of MDSC into an immune-stimulating phenotype producing IL-12, in correlation with significantly improved T-cell and NK cell responses to Listeria(at) at both ages. This was accompanied with a dramatic reduction in the number of metastases and tumour growth at young and old age. CONCLUSIONS: Although preclinical studies show that immunotherapy is less effective at old than at young age, our study demonstrates that Listeria(at)-based immunotherapy can be equally effective against metastatic breast cancer at both young and old age by targeting MDSC.

Vaccination with Mage-b DNA induces CD8 T-cell responses at young but not old age in mice with metastatic breast cancer.

BACKGROUND: Elderly individuals react less efficiently to vaccines than do adults, mainly because of T-cell unresponsiveness. In this study, we analysed whether tumour-associated antigen (TAA)-specific CD8 T-cell responses could be induced by vaccination in old mice with metastatic breast cancer. METHODS: The effect of pcDNA-3.1- and Listeria-based vaccines, expressing TAA Mage-b, on Mage-b-specific immune responses was tested in spleens and draining lymph nodes (LNs) of mild (4TO7cg) and aggressive (4T1) syngeneic metastatic mouse breast tumour models at young (3 months) and old (20 months) age. RESULTS: Interferon gamma and interleukin-2 levels increased significantly in draining LNs and spleens of Mage-b-vaccinated mice compared with those in control groups at young but not old age in both mouse tumour models. A significant increase was observed in the number of IFNgamma-producing Mage-b-specific CD8 T cells after Mage-b vaccination in the 4T1 model at young but not old age. This correlated with a reduced protective effect of Mage-b vaccination against metastatic breast cancer at old compared with young age. CONCLUSIONS: The absence of CD8 T-cell responses after Mage-b vaccination and the accompanying reduced protection against metastatic breast cancer in old compared with young mice point towards the need for tailoring cancer vaccination to older age.

Mage-b vaccine delivered by recombinant Listeria monocytogenes is highly effective against breast cancer metastases.

New therapies are needed that target breast cancer metastases. In previous studies, we have shown that vaccination with pcDNA3.1-Mage-b DNA vaccine is effective against breast cancer metastases. In the study presented here, we have further enhanced the efficacy of Mage-b vaccination through the improved delivery of the vaccine using recombinant Listeria monocytogenes (LM). Three overlapping fragments of Mage-b as well as the complete protein-encoding region of Mage-b have been expressed as a fusion protein with a truncated non-cytolytic form of listeriolysin O (LLO) in recombinant LM. These different Mage-b vaccine strains were preventively tested for their efficacy against breast cancer metastases in a syngeneic mouse tumour model 4T1. The LM-LLO-Mage-b/2nd, expressing position 311-660 of the cDNA of Mage-b, was the most effective vaccine strain against metastases in the 4T1 mouse breast tumour model. Vaccination with LM-LLO-Mage-b/2nd dramatically reduced the number of metastases by 96% compared with the saline group and by 88% compared with the vector control group (LM-LLO), and this correlated with strong Mage-b-specific CD8 T-cell responses in the spleen, after restimulation with Mage-b. However, no effect of LM-LLO-Mage-b/2nd was observed on 4T1 primary tumours, which may be the result of a complete absence of Mage-b-specific immune responses in the draining lymph nodes. Vaccination with LM-LLO-Mage-b/2nd could be an excellent follow-up after removal of the primary tumour, to eliminate metastases and residual tumour cells.

The alpha C protein mediates internalization of group B Streptococcus within human cervical epithelial cells.

Group B Streptococcus (GBS) is the leading cause of bacterial chorioamnionitis and neonatal pneumonia, sepsis, and meningitis. Deletion of the alpha C protein gene (bca) attenuates the virulence of GBS in an animal model; significant survival differences in the first 24 h of infection suggest a pathogenic role for the alpha C protein early in the infection process. We examined the role of alpha C protein in the association between GBS and mucosal surfaces using a human cervical epithelial cell line, ME180. Fluorescent and confocal microscopy and flow cytometry demonstrated that 9-repeat alpha C protein binds to the surface of ME180 cells. Isolated N-terminal region of this protein also binds to these cells and competitively inhibits binding of the full protein. Wild-type GBS strain A909 and the bca-null isogenic mutant JL2053 bound similarly to the surface of ME180 cells. However, A909 entered these cells threefold more. Internalization of A909 was inhibited with 2- and 9-repeat alpha C and with N-terminal region alone but not by repeat region-specific peptide. Translocation across polarized ME180 membranes was fivefold greater for A909 than for JL2053. These findings suggest a role for the alpha C protein in interaction with epithelial surfaces and initiation of infection.

Tailoring cancer vaccines to the elderly: the importance of suitable mouse models.

The incidence of cancer has increased over the last decade, mainly due to an increase in the elderly population. Vaccine therapy for cancer is potentially less toxic than chemotherapy or radiation and could, therefore, be especially effective in older, more frail cancer patients. However, it has been shown that older individuals do not respond to vaccine therapy as well as younger adults. This has been attributed to T cell unresponsiveness, a phenomenon also observed in cancer patients per se. Activation of tumor-specific T cells by cancer vaccines might be an approach, especially suitable for elderly patients, to eradicate or to prevent recurrence of tumors after primary treatment. To tailor pre-clinical testing of vaccine therapies to the elderly, it is important to have mouse models in which tumors develop at equivalent time points in their life span, as in humans. Such models are currently not available. This progress report first summarizes the current knowledge of tumor-immunological parameters potentially involved in T cell unresponsiveness in relation to aging in mice and humans. Secondly, it reviews those cancer vaccines that are known for their potential to induce tumor-specific T cell responses. Thirdly, it discusses the usefulness of currently available mouse models for pre-clinical testing of cancer vaccines applicable to the elderly population. Finally, experimental approaches are proposed, as to how to develop mouse models that allow the induction of specific tumors at will at different ages, expressing tumor-specific antigens in an 'immune competent' environment. These mouse models may teach us how to overcome immune deficits in the elderly, thereby facilitating the development of effective and safe cancer vaccines.

Characterization of two distinct opsonic and protective epitopes within the alpha C protein of the group B Streptococcus.

Group B Streptococcus (GBS) is a major cause of neonatal sepsis, meningitis in early infancy, postpartum endometritis, and serious invasive infections in adults in the United States. We previously cloned, sequenced, and characterized the alpha antigen gene, bca, and showed that the alpha C protein of GBS is a trypsin-resistant, surface-associated polypeptide that contains a signal sequence, a unique N terminus, nine identical tandem repeats, and a C-terminal membrane anchor structure. Polyclonal antiserum raised to the recombinant alpha C protein and an opsonic monoclonal antibody, 4G8, raised to the native protein from GBS have been shown to be protective in a mouse model. The binding site of 4G8 has now been localized to the tandem repeat region of the alpha C protein. To determine whether the N terminus of the alpha C protein contains additional opsonic and/or protective epitopes, the sequence corresponding to the alpha C protein N terminus was subcloned into a pET vector, the expressed peptide from Escherichia coli was purified by Ni2+ affinity chromatography, and rabbit polyclonal antibodies were raised to the purified recombinant peptide. Antibodies to the alpha C protein N terminus were shown to be opsonic by an in vitro opsonophagocytosis assay. In addition, 69% of newborn mouse pups from mothers passively immunized with the antiserum to the recombinant N-terminal polypeptide of the alpha C protein were protected against lethal challenge with GBS A909. These data indicate that at least two distinct regions of the alpha C protein, the N terminus and the tandem repeat region, contain opsonic and protective epitopes.

OK-432 and IL-2-augmented cytotoxicity of human natural killer cells and cytotoxic T lymphocytes at the clonal level.

The biological response modifiers OK-432 and interleukin-2 (IL-2) were found to enhance the lytic capacity of cloned CD3- natural killer (NK) cells and CD3+ T cells. With respect to NK cells, only those clones with a high proliferative capacity and cultured without phytohaemagglutinin (PHA) responded with enhanced lytic capacity to OK-432. OK-432, but not IL-2, was found to augment the antibody-dependent cellular cytotoxicity of cloned NK cells. With T-cell clones, OK-432 augmented the cellular cytotoxicity of CD3+8+ but not that of CD3+4+ cytotoxic T lymphocytes, while IL-2 augmented the cytotoxicity of both types of clone. Taken together, these results indicate that OK-432-augmented lytic capacity is not restricted to NK cells and its pathway of action may be independent of IL-2.

Efforts to produce human cytotoxic T-cell hybridomas by electrofusion and PEG fusion.

Cytotoxic human T cells from different sources were fused with different types of human T-lymphoma cells and mouse B-myeloma cells using variations of the polyethylene glycol (PEG) method and electrofusion. Both techniques yielded proliferating hybridomas. The frequency of wells with proliferating hybridomas depended on the tumor fusion partner used; the best results were obtained with HSB-1, whereas fusions with JURKAT-1 and HPB-1 did not yield any hybridomas. For one tumor cell line (HSB-1), considerably more hybridomas were obtained with electrofusion than with the PEG fusion (with or without heat shock). There was no consistent relationship between the presence or absence of cytotoxic activity of the T lymphocytes against the tumor fusion partner and the yield of hybridomas. In human-human as well as in human-mouse hybridomas most of the lymphocyte derived chromosomes were lost. Four of the more than 600 hybridomas tested showed transient cytotoxic activity, but in none of them this function could be immortalized. Two of the hybridomas obtained with CEM-1 as tumor fusion partner expressed low levels of lymphocyte-derived CD3 antigens. Two hybridomas obtained with HSB-1 were highly invasive in vitro in rat hepatocyte cultures, whereas HSB-1 tumor cells were not.

c-myc gene expression and interleukin-2 receptor levels in cloned human CD2+,CD3+ and CD2+,CD3- lymphocytes.

The levels of c-myc mRNA and interleukin-2 receptors (IL-2 Rec) were studied in human peripheral blood lymphocytes (PBL); mature CD2+,CD3+ T cell clones and CD2+,CD3- natural killer (NK) cell clones, and CD2+,CD3+ and CD2-,CD3- T lymphoma cell lines. A transient induction of the expression of c-myc and IL-2 Rec was observed in PBL after activation with phytohemagglutinin (PHA). Expression of c-myc and IL-2 Rec was also found in the CD2+,CD3+ and CD2+,CD3- clones. The CD2+,CD3+ showed higher levels of c-myc mRNA and IL-2 Rec than the CD2+,CD3- clones. In three T lymphoma cell lines constitutively high levels of c-myc mRNA but no IL-2 Rec were found. Only in JURKAT (CD2+,CD3+), c-myc mRNA levels could be further enhanced by PHA. These results suggest that in the presence of PHA, expression of c-myc and IL-2 Rec is induced via the CD3 receptor, and in the absence of PHA and/or the CD3 receptor alternative routes of induction are involved.

Interferon-beta and recombinant IL 2 can both enhance, but by different pathways, the nonspecific cytolytic potential of T3- natural killer cell-derived clones rather than that of T3+ clones.

We studied the enhancement of cytolytic activity of T3- natural killer cell-derived clones, of T3+ T cell activated killer (AK) clones, and of fresh peripheral blood lymphocytes (PBL) by various crude and recombinant interferon (r-IFN) as well as IL 2 preparations. It was found that IFN-beta had the highest cytotoxicity inducing potency as compared to crude or r-IFN-alpha or -gamma preparations. This enhancement was blocked by anti-IFN-beta antibodies but not by anti-IFN-gamma antibodies. IL 2 also strongly enhances cytolytic activity in cloned T3- killer cells that express the IL 2 receptors as determined with the anti-Tac monoclonal antibody (MAb) at concentrations of IL 2 (25 U/ml) which induced one-half of the maximal proliferation capacity in human T cells and murine CTLL cells. For enhancement of cytolytic activity in fresh NK cells, a much higher concentration of IL 2 is required. In addition, the enhancement of cytolytic activity by r-IL 2 but not that by IFN-beta can be reduced by anti-Tac MAb, suggesting that the IL 2 receptor is involved in the enhancement by IL 2, but not by IFN. Both IFN-beta and IL 2 were able to enhance (over threefold) the cytolytic activity of T3- cloned killer cells against a variety of tumor target cell types. Another remarkable observation was that K562 cells, the most commonly used target cell for determining NK cell cytolytic activity, are not the most suitable targets to assess enhancement of nonspecific lytic activity as compared to Daudi or lung tumor-derived cell lines. No enhancement of anti-body-dependent cellular cytotoxicity was observed. Finally, the effects of these biological response modifiers were much more pronounced on "fresh" and cloned T3- natural killer cell-derived than on T3+-activated killer mature T cell-derived clones.