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Publications and source records attributed to S Man.
The first generation of human cancer vaccines has been tested in phase III clinical trials, but only a few of these have demonstrated sufficient efficacy to be licensed for clinical use. This article reviews some of the mechanisms that could contribute to these limited clinical responses, and highlights the challenges faced for development of future vaccines.
The development of effective strategies against cervical cancer in Africa requires accurate type specific data on human papillomavirus (HPV) prevalence, including determination of DNA sequences in order to maximise local vaccine efficacy. We have investigated cervical HPV infection and squamous intraepithelial lesions (SIL) in an unselected cohort of 1061 women in a rural Gambian community. Squamous intraepithelial lesions was diagnosed using cytology and histology, HPV was typed by PCR-ELISA of DNA extracts, which were also DNA sequenced. The prevalence of cervical HPV infection was 13% and SIL were observed in 7% of subjects. Human papillomavirus-16 was most prevalent and most strongly associated with SIL. Also common were HPV-18, -33, -58 and, notably, -35. Human papillomavirus DNA sequencing revealed HPV-16 samples to be exclusively African type 1 (Af1). Subjects of the Wolof ethnic group had a lower prevalence of HPV infection while subjects aged 25-44 years had a higher prevalence of cervical precancer than older or younger subjects. This first report of HPV prevalence in an unselected, unscreened rural population confirms high rates of SIL and HPV infection in West Africa. This study has implications for the vaccination of Gambian and other African populations in the prevention of cervical cancer.
Rothenberg et al called for caution in the palliative use of irinotecan, 5-fluorouracil (5-FU) bolus and leucovorin (IFL schedule), because of early treatment related deaths in C89803 and N9741 studies. The objective of our multicenter phase II study was to evaluate the efficacy and safety of the combination of 5-FU bolus, folinic acid (FA) and irinotecan as first-line chemotherapy for metastatic colorectal cancer. From December 1999 to June 2002 138 patients (pts) were treated. The chemotherapy regimen was as follows: irinotecan 125 mg/m2 i.v. over 90 min and 5-FU 500 mg/m2 preceded by FA 20 mg/m2, both given by bolus, weekly, for 4 weeks every 6 weeks. Treatment continued until disease progression or unacceptable toxicity. Total number of administered cycles was 404. Average dose intensity was 75%. 47 out of 131 evaluable pts achieved a complete (n = 6) or partial (n = 41) response, leading to an overall response rate (RR) of 36% [95% confidence interval (CI) 24% to 48%], stable disease was registered in 50 (38%). The estimated median time to progression and survival were 6.5 months (95% CI 5.2-9.4) and 16.6 months (95% CI 15.1-19.3) respectively. Two-year survival was 35%. Toxicity was well manageable. In 18 (13.8%) pts the chemotherapy was stopped because of toxicity. Treatment related death was not observed. Close clinical monitoring, early recognition of toxicity, immediate therapeutic intervention are recommended for pts receiving IFL. In our experience this regimen has manageable toxicity and appropriate level of dose intensity and seems to be a good option for first-line therapy in metastatic colorectal cancer.
Human papilloma virus (HPV) found in 99.7% of cervical cancers represents an attractive immunotherapeutic target for novel adjuvant dendritic cell (DC) immunotherapy. DC primed with HPV antigens have been shown to be capable of inducing CTL responses powerful enough to eradicate established murine tumours expressing HPV16 antigen. The use of tumour lysate has been found to be an effective means of priming DC with tumour associated antigens in animal models and in clinical trials leading to significant anti-tumour responses. Autologous DC primed with sonicated HPV expressing tumour lysate have been shown to be capable of inducing HPV specific classes I and II T-cell immunity in a pilot clinical study.Synthetic double stranded polyribonucleotides are effective in vitro activation/maturation agents capable of inducing a stable mature DC phenotype producing high levels of IL12. However, the prototype polymer poly [I]:poly [G] has proved to be clinically toxic. Preliminary in vitro data have demonstrated that a novel clinically non-toxic analogue polymer poly [I]:poly [C(12)U] (Ampligen R) can effectively induce in vitro maturation of human monocyte derived DC with sustained bioactive IL12 production. Human monocyte derived DC primed with tumour lysate and matured with synthetic dsRNA may therefore offer an effective way of optimising Th1 specific anti-cancer T-cell responses in cancer patients. This strategy is currently being tested in a clinical trial in patients with cervical cancer.
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The production of cytotoxic T cells with specificity for cancer cells is a rapidly evolving branch of cancer therapeutics. A variety of approaches aim to amplify anti-tumour cytotoxic T cell responses using purified peptides, tumour cell lysates or recombinant HLA/peptide complexes in differing antigen presenting systems. Using a two-step biotin-streptavidin antibody targeting system, recombinant HLA-class I/peptide complexes were attached to the surface of B cells via the anti-CD20 B9E9-scFvSA antibody-streptavidin fusion protein. Flow cytometry with a conformation dependant monoclonal antibody to HLA class I indicated that targeted HLA-class I/peptide complexes remain on the surface of B cells in culture for periods in excess of 72 h. PBMCs were stimulated in vitro for 8-14 days using the autologous B cells as antigen presenting cells. Following a single cycle of stimulation specific cytotoxic T cell responses to targeted HLA-A2 complexes containing the M1, BMLF1 and Melan A peptides could be demonstrated by tetramer staining and Cr release assays. With the HLA-A2/BMLF1 complex up to 2.99% of CD8+ve cells were tetramer positive producing 20% lysis (E : T 10 : 1) of CIR-A2 target cells in an in vitro cytotoxicity assay compared to baseline levels of 0.09% tetramer +ve and 2% lysis in the unstimulated population. PBMCs from a healthy donor treated with two cycles of stimulations with targeted HLA-A2/Melan A complexes, demonstrated expansion of the melanA tetramer +ve population from 0.03% to 1.4% producing 15% lysis of Melan A pulsed target cells. With further consideration to the key variables of HLA/peptide complex density, the ratio of stimulator to effector cells and optimum cytokine support, this system should offer an easy and effective method for the in vitro amplification of specific cytotoxic T cell responses and warrants development for the in vivo induction of cytotoxic T cell responses in cancer therapy.
UNLABELLED: This study is a retrospective analysis of response, toxicity and freedom from progression of two single-agent chemotherapy regimens in patients with previously treated metastatic colorectal cancer. Thirty-five patients with histologically confirmed measurable metastatic colorectal cancer received chemotherapy after failure of first-line 5-fluorouracil (5-FU) and leucovorin treatment. The median age was 61 years. Twenty-seven patients had liver metastases, 6 had local recurrence, 1 had retroperitoneal lymph node metastases and 1 had lung metastases. Eighteen patients received weekly 2600 mg/m2 5-FU and 17 patients received weekly 125 mg/m2 irinotecan (CPT-11). Treatment was given until disease progression. Total number of cycles was 202 for 5-FU and 248 for CPT-11. The relative dose intensity was 1.0 for 5-FU and 0.84 for CPT-11. No grade 3-4 toxicity was registered in patients who received 5-FU. Grade 3-4 toxicity rates were as follows in those who received CPT-11: vomiting 1 (5.9%) patient in 1 cycle, diarrhea 3 (17.7%) patients in 3 cycles and neutropenia in 3 (17.7%) patients in 3 cycles. No patients manifested febrile neutropenia. Two patients (11.8%) needed hospital admission because of toxicity: 1 for vomiting and 1 for diarrhea. No objective responses were observed in the 5-FU group of patients. Three patients (17.6%) who received CPT-11, achieved partial response with a median duration of 8 months. Stable disease was registered in 3 (17.6%) and 9 (52.9%) patients in 5-FU and CPT-11 groups respectively (p=0.05). Median time to progression was 3.3 months for patients who received 5-FU and 4.2 months for those treated with CPT-11 (not significant). One-year survival was 22.2% and 54.3% respectively (p=0.05). CONCLUSION: Weekly chemotherapy with CPT-11 is tolerated with acceptable toxicity and leads to a better response rate than weekly high dose 5-FU. It also significantly improves survival but does not prolong freedom from progression.
Human papillomavirus (HPV) infection, particularly type 16, is causally associated with the development of cervical cancer. The E6 and E7 proteins of HPV are constitutively expressed in cervical carcinoma cells making them attractive targets for CTL-based immunotherapy. However, few studies have addressed whether cervical carcinomas can process and present HPV E6/E7-derived Ags for recognition by CTL. We generated HLA-A*0201-restricted CTL clones against HPV16 E6(29-38) that recognized HPV16 E6 Ags transfected into B lymphoblastoid cells. These CTL were unable to recognize HLA-A*0201(+) HPV16 E6(+) cervical carcinoma cell lines even when the level of endogenous HPV16 E6 in these cells was increased by transfection. This defect in presentation of HPV16 E6(29-38) correlated with low level expression of HLA class I, proteasome subunits low molecular mass protein 2 and 7, and the transporter proteins TAP1 and TAP2 in the cervical carcinoma cell lines. The expression of all of these proteins could be up-regulated by IFN-gamma, but this was insufficient for CTL recognition unless the level of HPV16 E6 Ag was also increased by transfection. CTL recognition of the HPV16 E6(29-38) epitope in 721.174 B cells was dependent on TAP expression but independent of immunoproteasome expression. Collectively, these findings suggest that presentation of the HPV16 E6(29-38) epitope in cervical carcinoma cell lines is limited both by the level of TAP expression and by the low level or availability of the source HPV E6 oncoprotein. These observations place constraints on the use of this, and potentially other, HPV-derived CTL epitopes for the immunotherapy of cervical cancer.
Loss of immunogenic epitopes by tumors has urged the development of vaccines against multiple epitopes. Recombinant DNA technologies have opened the possibility to develop multiepitope vaccines in a relatively rapid and efficient way. We have constructed four naked DNA-based multiepitope vaccines, containing CTL, Th cell, and B cell epitopes of the human papillomavirus type 16. Here we show that gene gun-mediated vaccination with an epitope-based DNA vaccine protects 100% of the vaccinated mice against a lethal tumor challenge. The addition of spacers between the epitopes was crucial for the epitope-induced tumor protection, as the same DNA construct without spacers was significantly less effective and only protected 50% of the mice. When tested for therapeutic potential, only the epitope construct with defined spacers significantly reduced the size of established tumors, but failed to induce tumor regression. Only after targeting the vaccine-encoded protein to the protein degradation pathway by linking it to ubiquitin, the vaccine-induced T cell-mediated eradication of 100% of 7-day established tumors in mice. The finding that defined flanking sequences around epitopes and protein targeting dramatically increased the efficacy of epitope string DNA vaccines against established tumors will be of importance for the further development of multiepitope DNA vaccines toward clinical application.
Cervical cancer is the second most common cause of cancer death in women worldwide. It is almost invariably associated with infection with human papilloma virus (HPV) particularly types 16 and 18. The ubiquitous expression of E6 and E7 oncogene products has been recognised as an attractive target for CTL-mediated immunotherapy. In-vivo expansion of an HPV oncogene product specific MHC class 1 restricted response has been demonstrated using intradermally administered live vaccinia virus HPV 16 and 18 E6/E7 gene construct (TA-HPV, Cantab Pharmaceuticals). Responses have been seen in 1/3 evaluable patients with advanced cervical cancer, and 3/12 CIN3 volunteers, and in 4/29 patients with early invasive cervical cancer (Rankin et al. Proceedings of 91st AACR Meeting, San Francisco, April 2000). In addition, the adoptive transfer of ex vivo HPV 16 or 18 positive autologous tumour lysate pulsed dendritic cells is currently being tested as an alternative means of expanding HPV specific CTL in advanced cervical cancer patients. So far an HLA-A*O201 restricted CD8 T cell response has been recorded in the single HLA-A*O201 patient whose tumour was shown to be HPV16 positive. It appears therefore feasible to induce HPV specific CTL responses in patients with cervical cancer using several vaccine strategies. However, further clinical trials are needed to determine the full anti-tumour potential of this vaccine based immunotherapy.
DNA co-condensates were formed by reacting [125I]DNA with an admixture of a high-mannose glycopeptide (Man9-CWK(18)) and either of two poly(ethylene glycol) peptides (PEG-VS-CWK(18) or PEG-SS-CWK(18)) followed by cross-linking with 6-50 mol equiv of glutaraldehyde. [125I]DNA co-condensates were administered intravenously in mice to determine the influence of peptide DNA formulation parameters on specific targeting to Kupffer cells. Optimal targeting to Kupffer cells required the combined use of 50 mol % Man9-CWK(18) and PEG-CWK(18) to mediate specific recognition by the mannose receptor to Kupffer cells. The cellular uptake of cross-linked Man9-CWK(18)/PEG-CWK(18) DNA co-condensates was receptor mediated since Kupffer cell targeting was inhibited by pre-administration of Man-bovine serum albumin (BSA) but not BSA. An optimized formulation targeted 60% of the dose to the liver, with 80% of the liver-targeted DNA localized to Kupffer cells. Cross-linking with either 6, 15, or 50 mol equiv of glutaraldehyde led to a corresponding decrease in the metabolism rate of DNA in liver as measured by half-live- of 4, 6, and 39 h, respectively. Tail vein dosing of 50 microg of DNA co-condensates cross-linked with 6 mol equiv of glutaraldehyde produced detectable levels of human alpha1-antitrypsin in blood after 12 h, which peaked at day six and persisted for 10 days. The level of human alpha1-antitrypsin was elevated two-fold each day when dosing with DNA co-condensates cross-linked with 15 mol equiv of glutaraldehyde, revealing a correlation between the metabolic stability of the DNA in liver and level of gene expression. In addition to possessing greater metabolic stability, DNA co-condensates cross-linked with 50 mol equiv of glutaraldehyde, but lacking a targeting ligand, avoided rapid liver uptake and possessed a prolonged pharmacokinetic half-life, providing insight into a means to target DNA condensates to peripheral tissues.
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The ultimate target of anti-angiogenic drugs is the genetically stable, activated endothelial cell of a newly forming tumor blood vessel, rather than the genetically unstable tumor cell population per se. This led to the notion that acquired resistance to such drugs may not develop as readily, if at all. While there is some evidence that this lack of resistance development may be the case for some direct-acting angiogenesis inhibitors, it is becoming apparent that resistance can develop over time to many types of angiogenesis inhibitors including, possibly, some direct inhibitors, especially when used as monotherapies. Possible mechanisms for such acquired or induced resistance include: (i) redundancy of pro-angiogenic growth factors when the drug used targets a single such growth factor or its cognate endothelial cell-associated receptor tyrosine kinase; (ii) the anti-apoptotic/pro-survival function of growth factors such as VEGF, which, in high local concentrations, can antagonize the pro-apoptotic effects of various angiogenesis inhibitors; (iii) epigenetic, transient upregulation, or induction, of various anti-apoptotic effector molecules in host-endothelial cells; and (iv) heterogeneous vascular dependence of tumor cell populations. It is suggested that long-term disease control with anti-angiogenic drugs can be best achieved by judicious combination therapy. In this regard, the great molecular diversity of anti-angiogenic drug targets, in contrast to chemotherapy, makes this a particularly attractive therapeutic option, especially when approved, commercially available drugs considered to have anti-angiogenic effects are used in such combination treatment strategies.
Despite its being a relatively common virus, the study of human papillomavirus infection has lagged behind that of other viruses. Human papillomaviruses do not provoke strong systemic antibody or T-cell responses. Furthermore, the majority of those infected do not display clinical symptoms and are able to clear the virus by unknown mechanisms. In the last decade, however, research into human papillomavirus immunology has blossomed, for two main reasons. First, there is strong circumstantial evidence that the immune system can control papillomavirus infection, since the prevalence of human papillomavirus-associated neoplasia is increased in immunocompromised individuals. Second, the strong association between human papillomavirus infection and cervical cancer has led to attempts to develop prophylactic or therapeutic vaccines. In this chapter, our current knowledge of human papillomavirus immune responses will be reviewed, and how this relates to clinical practice will be discussed.
BACKGROUND & AIMS: Advanced gastric cancer has a poor prognosis and is largely unresponsive to currently available chemotherapeutic drugs. The development of more effective therapies would be aided by better preclinical models. METHODS: An in vitro multicellular gastric cancer spheroid model was established using the liquid overlay technique and compared with the corresponding xenografts in immunodeficient mice. RESULTS: Twelve of 17 (71%) gastric cancer cell lines reflected growth characteristics of their parental gastric carcinomas in three-dimensional culture. Thus, cell lines derived from peritoneal and pleural carcinomatosis grew as single cells (HSC-39, KATO-II, KATO-III) and cell aggregates (SNU-5, SNU-16). Cell lines representing adenosquamous (MKN-1) and tubular differentiation (MKN-28, MKN-74, N87) formed partly compact multicellular spheroids recapitulating the tumor architecture of the respective original tumor. The differentiated phenotype was lost after subcutaneous implantation of the in vitro spheroids in mice. The degree of morphologic differentiation was reflected by the levels of mucin and constitutive E-cadherin expression. Heterogeneous changes of other adhesion molecules (EpCAM, alpha2beta1, CD44s, Le(x), sLe(x)) were observed. In contrast, cell lines derived from poorly differentiated gastric carcinomas (Hs-746T, RF-1, RF-48) formed fully compact spheroids mimicking the poorly differentiated phenotype, were E-cadherin negative, and showed only CD44s up-regulation. CONCLUSIONS: Recapitulating some complexity of their in vivo counterparts, multicellular gastric cancer spheroids may represent a physiologically valid model for studying the biology of this cancer, and testing new therapeutic strategies.
During acute human viral infections, such as influenza A, specific cytotoxic T lymphocytes (CTL) are generated which aid virus clearance. We have observed that in HLA-A*0201+ subjects, CTL expressing Vbeta17+ TCR and recognizing a peptide from the influenza A matrix protein (M1(58-66)) dominate this response. In experimental models of infection such dominance can be due to inheritance of a restricted T cell repertoire or acquired consequent on expansion of CTL bearing an optimum TCR conformation against the MHC-peptide complex. To examine how influenza A infection might influence the development of TCR Vbeta17 expansion, we studied influenza A-specific CTL in a cross-sectional study of 82 HLA-A*0201+ individuals from birth (cord blood) to adulthood. Primary M1(58-66) -specific CTL were detected in cord blood, but their TCR were diverse and depletion of Vbeta17+ cells did not abrogate specific cytotoxicity. In contrast following natural influenza A infection, TCR Vbeta17+ CTL dominated to the extent that only one of nine adult CTL lines retained any functional activity after in vitro depletion of Vbeta17+ CTL. These results suggest that the dominance of Vbeta17+ TCR among adult M1(58-66)-specific CTL results from maturation and focussing of the response driven by exposure to influenza, and have implications for optimum immunization strategies.
We have shown that the dominance of CD8+ T cells expressing TCR Vbeta17 in the adult HLA-A*0201-restricted influenza A/M1(58-66)-specific response is acquired following first antigen exposure. Despite the acquired dominance of Vbeta17+ cells, subdominant M1(58-66)-specific clones expressing non-Vbeta17+ TCR persist in all individuals. To determine whether the affinity of the expressed TCR for the HLA-A*0201/M1(58-66) complex could influence functional properties, M1(58-66)-specific clones expressing subdominant (non-Vbeta17+) TCR were compared to cytotoxic T lymphocyte (CTL) clones expressing dominant (Vbeta17+) TCR. The Vbeta17+ CTL required up to 10,000-fold lower amounts of M1 peptide to mediate lysis compared to CTL clones expressing other Vbeta gene segments. All Vbeta17+ CTL clones tested bound HLA-A*0201/M1(58-66) tetramer, but two of three CTL clones expressing other TCR did not bind tetramer. The inability of non-Vbeta17+ CTL to bind tetramer did not correlate with phenotype, CD8 dependence or with cytokine production profiles. This suggests a limitation for the use of tetramers in examining subdominant T cell responses. Together these findings suggest that Vbeta17+ CTL which dominate the HLA-A*0201-restricted CTL response against influenza A are not functionally distinct from subdominant non-Vbeta17+ CTL. The dominance of Vbeta17+ CTL is likely to result from a competitive advantage due to superior CTL avidity for the HLA-A*0201/M1(58-66) complex.
Human Papillomaviruses (HPVs) are sexually transmitted pathogens, which are implicated in the etiology of cervical cancer. The early proteins E6 and E7 of HPV have transforming capacity and interfere with the cell cycle control of infected host cells and are essential for the maintenance of the transformed state. Identification of MHC class I-restricted, immunogenic peptides derived from either the E6 or the E7 protein is essential for the design of vaccines as well as the monitoring of clinical trials and immunotherapeutic approaches for the treatment of HPV-18-induced carcinomas. We have determined the binding affinities for all possible 9-mer peptides spanning the entire E6 and E7 amino acid sequence for the HLA-A*0101, HLA-A*0201, HLA-A*0302, HLA-A*1102, and HLA-A*2402101 molecules by a competition assay with reference peptides, thereby establishing the binding peptides as potential cytotoxic T-cell epitopes. From the HLA-A*0201 binding peptides, we selected five E6-derived and one E7-derived peptide with high affinities for HLA-A*0201. These six peptides were tested for their immunogenicity by in vitro immunization assays with purified human CD8+ T cells. We identified three HPV-18 E6-derived peptides (ELTEVFEFA, KTVLELTEV, and KLPDLCTEL) and the E7-derived peptide TLQDIVLHL to be highly immunogenic. Overall, these results will help to design vaccines for the prevention or treatment of HPV-18-induced cervical cancer.