A synthetic peptide adhesion epitope as a novel antimicrobial agent.

The earliest step in microbial infection is adherence by specific microbial adhesins to the mucosa of the oro-intestinal, nasorespiratory, or genitourinary tract. We inhibited binding of a cell surface adhesin of Streptococcus mutans to salivary receptors in vitro, as measured by surface plasmon resonance, using a synthetic peptide (p1025) corresponding to residues 1025-1044 of the adhesin. Two residues within p1025 that contribute to binding (Q1025, E1037) were identified by site-directed mutagenesis. In an in vivo human streptococcal adhesion model, direct application of p1025 to the teeth prevented recolonization of S. mutans but not Actinomyces, as compared with a control peptide or saline. This novel antimicrobial strategy, applying competitive peptide inhibitors of adhesion, may be used against other microorganisms in which adhesins mediate colonization of mucosal surfaces.

CD40 ligation for immunotherapy of solid tumours.

Tumour vaccines provide an important focus of current cancer research and are often based on the premise that although T-cells do respond naturally to certain tumours, this is usually weak and therefore ineffective at controlling disease. An integral and necessary part of a T-cell immune response involves triggering of CD40 on antigen-presenting cells (APC) by its ligand, CD154, on responding T helper (Th) cells. Furthermore, cytotoxic responses to tumours may fail because the Th-cell response is inadequate and unable to provide CD40 stimulation of APC. Growing evidence shows that stimulating APC with soluble CD40L or an agonistic anti-CD40 mAb can, at least in part, replace the need for Th cells and generate APC that are capable of priming cytotoxic T lymphocytes (CTL). The aim of this study was to investigate whether a range of solid tumours (CD40(-)) could be treated with anti-CD40 mAb. It was found that this treatment was effective, and correlated with the intrinsic immunogenicity and aggressiveness of the tumours. The mAb could be delivered locally or at a distal site, but increased antigen load provided by irradiated tumour cells added little to the effectiveness of the treatment. T-cells were required since cytokine (interferon-gamma) and CTL activity were demonstrated following treatment and the therapeutic efficacy was lost in nude mice. In addition, depletion of CD8(+) cells abrogated protection whilst depletion of CD4(+) cells had no effect. This study demonstrates that solid CD40(-) tumours are sensitive to anti-CD40 mAb therapy and that the response bypasses the need for Th cells.

Coxsackie B and adenovirus receptor, integrin and major histocompatibility complex class I expression in human prostate cancer cell lines: implications for gene therapy strategies.

Gene therapy strategies based on modifying tumour cells using high efficiency adenoviral vectors have shown promise in the clinic. Recently the Coxsackie and adenovirus receptor (CAR) has been shown to mediate adenoviral entry into tumour cells, although previous studies also suggested a role for MHC class I heavy chain. Detailed evaluation of the expression of both CAR and MHC class I in prostate cancer cell lines would have important implications for therapeutic strategies. We have found that, unlike cell lines derived from other malignancies, in human and murine prostate cancer loss of CAR expression appears to be relatively infrequent and does not correlate with loss of MHC class I expression. These findings, together with the demonstration of appreciable levels of cell-surface expression of integrins, suggest that cancer vaccine strategies based on modifying whole prostate cancer cells should be feasible using the current generation of recombinant adenoviral vectors, without deleterious effects on either the virus vector or the target cell.

Effect of route of immunisation and adjuvant on T and B cell epitope recognition within a streptococcal antigen.

Oral immunisation may elicit both systemic and mucosal immunity. Antibodies directed to a portion (residues 816-1213) of a cell surface adhesin termed streptococcal antigen I/II (SA I/II) of Streptococcus mutans prevent colonisation of this bacterium in vivo. This polypeptide is highly immunogenic in mice and is immunodominant in naturally sensitised humans. In this study, the effects of immunisation by different routes and of adjuvant on T and B cell epitope recognition were investigated. The recombinant polypeptide comprising residues 816-1213 of SA I/II was administered to groups of SJL mice intraperitoneally, subcutaneously or orally. For systemic immunisation, incomplete Freund's adjuvant was used, whereas for oral immunisation the antigen was coupled to the cholera toxin B subunit. The hierarchy of T and B cell epitope recognition differed significantly following different routes of immunisation. These differences in T and B cell responses may be accounted for by extracellular protease activity and processing by antigen presenting cells at the sites of immunisation. Furthermore, epitope recognition may be critical if the immune response elicited by a vaccine must be directed specifically to functional determinants within an antigen. This study emphasises the importance of route of immunisation in vaccine development.

The role of herpes simplex virus thymidine kinase in the treatment of solid tumours.

Suicide gene therapy is the approach whereby the genetic alteration of a cell renders it susceptible to an otherwise non-toxic prodrug. Suicide gene therapy for solid tumours has progressed rapidly since the concept was originally described: nearly all tumour types have been explored, with some, such as glioma, melanoma and colon cancer frequently used experimentally. The exciting aspect of suicide gene therapy is the bystander effect, the phenomenon whereby there is extended tumour death when only a small fraction is transfected with the suicide gene. This phenomenon implies that there is a reduced need to target specifically all tumour cells, as the effect mechanism itself carries out this function. The bystander effect mode of action has not yet been fully characterised, but the role of gap junctions and the immune system are implicated as the main instruments in its potentiation. This approach is also amenable to pharmacological intervention, which may help to optimise parameters prior to commencing suicide gene therapy. Clinical trails have already commenced using this form of treatment and results are eagerly awaited.

Induction of immune responses to functional determinants of a cell surface streptococcal antigen.

Antibodies directed against the cell surface adhesin, termed streptococcal antigen I/II of Streptococcus mutans can protect against dental caries. Streptococcal antigen I/II (SA I/II) interacts with salivary glycoproteins and promotes adhesion to the tooth surface. Topical application of monoclonal antibodies which recognize a domain within residues 816-1213 (fragment 3) prevents colonization by S. mutans in primates. In this study the immunogenicity and antigenicity of fragment 3 was investigated in five strains of mice. Fragment 3 induced an immune response following immunization with whole cells of S. mutans in all strains of mice. Immunization with recombinant fragment 3 also induced T-cell proliferative and antibody responses both to fragment 3 and to the SA I/II. Antibody responses to the previously defined adhesion determinants (residues 1005-1044) were weak or undetectable. Immunization of three representative strains of mice with a recombinant polypeptide (residues 975-1044) comprising this adhesion epitope and an adjacent T-cell epitope (residues 975-1004) elicited both T- and B-cell responses to the polypeptide and to native SA I/II. The B-cell epitopes overlapped with the adhesion determinant. These findings provide a means of directing immune responses to functional determinants of SA I/II.

Can immunotherapy by gene transfer tip the balance against colorectal cancer?

Gene therapy, in particular the transfer of genes encoding immunostimulatory molecules (cytokines and costimulatory molecules) as well as selectively cytotoxic enzymes and DNA vaccination, has the potential of enhancing cell mediated immune responses against tumours including those of colorectal origin. Genes can be transferred using viral vectors either to cultured tumour cells in vitro that can be returned to the patient as a "cancer vaccine", or directly to tumour cells in vivo. Vaccination with DNA constructs expressing specific tumour antigens characteristic of colorectal neoplasia can trigger immune recognition and destruction of tumour cells. The aim is to tip the balance from protumour to antitumour mechanisms by generating a local immune response and systemic antitumour immune memory to destroy metastases. Studies in murine models, combined with human studies, show that such approaches could become an adjunct to current treatments for human colorectal cancer in the near future.

The immunotherapy of prostate cancer.

Advanced prostate cancer remains incurable with standard treatment options. Immunotherapy may be a realistic alternative given the growing evidence that the immune response can affect the growth of other solid tumours and the regulation of both specific and shared prostate cancer antigens. Early studies suggest that both non-specific and specific vaccines can effect relevant animal models and clinical trials based on these observations are now in progress. A number of other approaches including gene therapy with HSVtk are already undergoing clinical studies (Herman et al. Hum Gene Ther 1999; 10: 1239-1249). Prostate Cancer and Prostatic Diseases (2000) 3, 303-307

Efficacy of cytokine gene transfection may differ for autologous and allogeneic tumour cell vaccines.

Whole tumour cells are a logical basis for generating immunity against the cancers they comprise or represent. A number of human trials have been initiated using cytokine-transfected whole tumour cells of autologous (patient-derived) or allogeneic [major histocompatibility complex (MHC)-disparate] origin as vaccines. Although precedent exists for the efficacy of autologous-transfected cell vaccines in animal models, little preclinical evidence confirms that these findings will extrapolate to allogeneic-transfected cell vaccines. In order to address this issue a murine melanoma cell line (K1735) was transfected to secrete interleukin (IL)-2, IL-4, IL-7 or granulocyte-macrophage colony-stimulating factor (GM-CSF); cytokines currently in use in trials. The efficacy of these cells as irradiated vaccines was tested head-to-head in syngeneic (C3H) mice and in MHC-disparate (C57BL/6) mice, the former being subsequently challenged with K1735 cells and the latter with naturally cross-reactive B16-F10 melanoma cells. Whilst the GM-CSF-secreting vaccine was the most effective at generating protection in C3H mice, little enhancement in protection above the wild-type vaccine was seen with any of the transfections for the allogeneic vaccines, even though the wild-type vaccine was more effective than the autologous B16-F10 vaccine. Anti-tumour cytotoxic T-lymphocyte (CTL) activity was detected in both models but did not correlate well with protection, whilst in vitro anti-tumour interferon-gamma (IFN-gamma) secretion tended to be higher following the GM-CSF-secreting vaccine. Cytokine transfection of vaccines generally increased anti-tumour CTL activity and IFN-gamma secretion (T helper type 1 response). Further studies in other model systems are required to confirm this apparent lack of benefit of cytokine transduction over wild-type allogeneic vaccines, and to determine which in vitro assays will correlate best with protection in vivo.

Allogeneic whole-tumour cell vaccination in the rat model of prostate cancer.

OBJECTIVE: To investigate cancer immunotherapy using whole allogeneic (differing tissue-type) tumour cells as vaccines in the rat prostate cancer model. Materials and methods Two rat models of prostate cancer were used; MAT-LyLu tumours which grow in Copenhagen rats and PAIII tumours which grow in Lobund-Wistar rats, with crossover of the cell lines to test allogeneic vaccination. The cell lines were immunologically characterized by flow cytometry. Irradiated tumour cells were administered as subcutaneous vaccines either before tumour challenge or after tumour establishment (both subcutaneous). A preparation of heat-killed Mycobacterium vaccae bacilli (SRL172) was used as an adjuvant to increase vaccine efficiency. RESULTS: Flow cytometry analysis of the cell lines showed that the PAIII cells had higher levels of major histocompatibility complex (MHC) class I and intercellular adhesion molecule (ICAM-1) expression than the MAT-LyLu cells. However, both tumour cell lines were rejected in their allogeneic hosts. Prophylactic vaccination with allogeneic MAT-LyLu cells protected against PAIII tumour challenge in Lobund-Wistar rats, with 80% of animals surviving for > 5 months, compared with 40% for animals receiving autologous cells. The immunity was prolonged, as rats were protected when rechallenged 5 months later. In Copenhagen rats allogeneic PAIII cells protected against the more aggressive MAT-LyLu tumour challenge only when the cells were combined with SRL172. Initial therapy experiments showed that vaccination with the cell lines mediated only limited tumour regression in the Lobund-Wistar rats. CONCLUSION: The allogeneic tumour cell vaccination model described is valuable for assessing the principle and efficacy of allogeneic prostate cancer cell vaccines for clinical use.

Genetic prodrug activation therapy (GPAT) in two rat prostate models generates an immune bystander effect and can be monitored by magnetic resonance techniques.

Treatment of hormone refractory prostate cancer requires new treatment strategies. Genetic prodrug activation therapy (GPAT) may provide a new therapeutic avenue. In this study the antitumour efficacy of the gene encoding herpes simplex virus thymidine kinase (HSVtk) activating the prodrug ganciclovir (GCV) was compared in two models of ectopic (subcutaneous) rat prostate cancer. Both models, which differ in their characteristics, were previously shown to be weakly immunogenic but susceptible to immunotherapy. Tumour cell lines were stably transfected with HSVtk and were rendered highly sensitive to GCV. Little or no bystander killing effect was observed by tk-transfected cells on wild-type cells in vitro. However, a significant in vivo bystander effect was observed suggesting an immune-mediated response. Indeed, such an immune response was capable of slowing the growth of distant wild-type tumours and increased overall animal survival. A T helper 1 immune response was generated as a result of GCV activation and cell kill, demonstrated by the secretion of IFNgamma by cultured splenocytes in response to tumour cells. BrDU staining of tk-transfected cells treated with GCV in vitro suggested apoptotic cell death, but Annexin V staining was less marked for one of the cell lines. Serial in vivo monitoring by non-invasive magnetic resonance spectroscopy (MRS) of the tk-transfected MATLyLu tumours demonstrated a decreased ATP/Pi ratio (a measure of cell energy status) during growth and an increase in the ATP/Pi ratio during regression initiated by treatment with GCV. Further, significant differences were found in the phosphomonester (PME) to total phosphate (SigmaP) ratios in treated compared with untreated tumours, a result rarely seen in animal models, but commonly observed in patients. This study showed that a Th1-biased immune response generated by killing prostate tumour cells with tk/GCV can kill distant as well as local wild-type tumour cells. These findings suggest that GPAT may have a potential application in patients with both confined and metastatic prostate cancer and MRS may provide a method of monitoring response to treatment.