• Attenuated recombinant vaccinia virus expressing oncofetal antigen (tumor-associated antigen) 5T4 induces active therapy of established tumors.

      Mulryan, Kate; Ryan, Matthew G; Myers, Kevin A; Shaw, David M; Wang, Who W; Kingsman, Susan M; Stern, Peter L; Carroll, Miles W; Cancer Research United Kingdom Immunology Group, Paterson Institute for Cancer Research, Christie Hospital, Manchester, M20 9BX, United Kingdom. (2002-10)
      The human oncofetal antigen 5T4 (h5T4) is a transmembrane glycoprotein overexpressed by a wide spectrum of cancers, including colorectal, ovarian, and gastric, but with a limited normal tissue expression. Such properties make 5T4 an excellent putative target for cancer immunotherapy. The murine homologue of 5T4 (m5T4) has been cloned and characterized, which allows for the evaluation of immune intervention strategies in "self-antigen" in vivo tumor models. We have constructed recombinant vaccinia viruses based on the highly attenuated and modified vaccinia virus ankara (MVA strain), expressing h5T4 (MVA-h5T4), m5T4 (MVA-m5T4), and Escherichia coli LacZ (MVA-LacZ). Immunization of BALB/c and C57BL/6 mice with MVA-h5T4 and MVA-m5T4 constructs induced antibody responses to human and mouse 5T4, respectively. C57BL/6 and BALB/c mice vaccinated with MVA-h5T4 were challenged with syngeneic tumor line transfectants, B16 melanoma, and CT26 colorectal cells that express h5T4. MVA-h5T4-vaccinated mice showed significant tumor retardation compared with mice vaccinated with MVA-LacZ or PBS. In active treatment studies, inoculation with MVA-h5T4 was able to treat established CT26-h5T4 lung tumor and to a lesser extent B16.h5T4 s.c. tumors. Additionally, when C57BL/6 mice vaccinated with MVA-m5T4 were challenged with B16 cells expressing m5T4, resulting growth of the tumors was significantly retarded compared with control animals. Furthermore, mice vaccinated with MVA-m5T4 showed no signs of autoimmune toxicity. These data support the use of MVA-5T4 for tumor immunotherapy.
    • Glycosylation and epitope mapping of the 5T4 glycoprotein oncofoetal antigen.

      Shaw, David M; Woods, Andrew M; Myers, Kevin A; Westwater, Caroline; Rahi-Saund, Veena; Davies, Michael J; Renouf, David V; Hounsell, Elizabeth F; Stern, Peter L; CRC Immunology Group, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Wilmslow Road, Manchester M20 4BX, UK. (2002-04-01)
      The human 5T4 oncofoetal antigen is a focus for development of several antibody-directed therapies on the basis of the murine monoclonal antibody against 5T4 (mAb5T4), which recognizes a conformational epitope. 5T4 molecules are highly N-glycosylated transmembrane glycoproteins whose extracellular domain contains two regions of leucine-rich repeats (LRRs) and associated flanking regions, separated by an intervening hydrophilic sequence. Using a series of deletion and mutated cDNA constructs as well as chimaeras with the murine homologue, we have mapped the mAb5T4 epitope to the more membrane-proximal LRR2 or its flanking region. Analysis of the glycosylation of the seven consensus Asp-Xaa-Ser/Thr sites was consistent with all of the sites being glycosylated. A combination of two high-mannose chains (predominantly octasaccharide) and five mostly sialylated bi-, tri- and tetra-antennary complex chains with minor quantities of core fucose were detected. The two glycosylation sites, which are the most likely to have predominantly high-mannose chains, are in the only two regions that show significant differences between the human and the 81% identical mouse sequence. A site-directed mutation, which abolished glycosylation at one of these sites (position 192), did not alter antigenicity. The other, which is nearest to the N-terminus in the human, has an Asn-Leu-Thr to Asn-Leu-Leu conversion in the mouse, so cannot be glycosylated in the latter species. The large complex glycosylation at the other sites is likely to influence the antigenicity and tertiary structure generating the 5T4 epitope.
    • Isolation of a cDNA encoding 5T4 oncofetal trophoblast glycoprotein. An antigen associated with metastasis contains leucine-rich repeats.

      Myers, Kevin A; Rahi-Saund, Veena; Davison, M D; Young, J A; Cheater, Amanada J; Stern, Peter L; Cancer Research Campaign Department of Immunology, Paterson Institute for Cancer Research, Christie Hospital, Manchester, United Kingdom. (1994-03-25)
      The monoclonal antibody 5T4 defines a human oncotrophoblast antigen expressed by a variety of carcinomas but with a restricted pattern of expression in normal adult tissues. The 5T4 antigen has been isolated from term placenta as a 72-kDa glycoprotein consisting of a 42-kDa core protein with extensive N-linked glycosylation. A cDNA has been isolated from a human placental library using pools of oligonucleotides based on amino acid sequence obtained from purified 5T4 molecules. The predicted open reading frame encodes a protein of 420 amino acids with a molecular mass of 46 kDa and 8 potential N-glycosylation sites. There are N- and C-terminal hydrophobic segments corresponding to putative signal and membrane anchorage sequences, respectively. Northern analysis has demonstrated a major 2.5-kilobase mRNA present in cell lines serologically reactive with the monoclonal antibody 5T4. Comparison of the 5T4 protein sequence with current sequence data bases has identified the presence of leucine-rich repeats, which are found in a variety of proteins from yeast, insects, and mammals. The 5T4 antigen expression is strongly associated with metastasis in colorectal and gastric cancer, and, hence, the possible functions of the gene product and its relationship to tumor growth and progression are discussed.
    • Isolation of a high affinity scFv from a monoclonal antibody recognising the oncofoetal antigen 5T4.

      Shaw, David M; Embleton, Jim; Westwater, C; Ryan, Matthew G; Myers, Kevin A; Kingsman, Susan M; Carroll, M W; Stern, Peter L; CRC Immunology Group, Paterson Institute for Cancer Research, Christie Hospital, Manchester, UK. (2000-12-15)
      The oncofoetal antigen 5T4 is a 72 kDa glycoprotein expressed at the cell surface. It is defined by a monoclonal antibody, mAb5T4, that recognises a conformational extracellular epitope in the molecule. Overexpression of 5T4 antigen by tumours of several types has been linked with disease progression and poor clinical outcome. Its restricted expression in non-malignant tissue makes 5T4 antigen a suitable target for the development of antibody directed therapies. The use of murine monoclonal antibodies for targeted therapy allows the tumour specific delivery of therapeutic agents. However, their use has several drawbacks, including a strong human anti-mouse immune (HAMA) response and limited tumour penetration due to the size of the molecules. The use of antibody fragments leads to improved targeting, pharmacokinetics and a reduced HAMA. A single chain antibody (scFv) comprising the variable regions of the mAb5T4 heavy and light chains has been expressed in Escherichia coli. The addition of a eukaryotic leader sequence allowed production in mammalian cells. The two 5T4 single chain antibodies, scFv5T4WT19 and LscFv5T4, described the same pattern of 5T4 antigen expression as mAb5T4 in normal human placenta and by FACS. Construction of a 5T4 extracellular domain-IgGFc fusion protein and its expression in COS-7 cells allowed the relative affinities of the antibodies to be compared by ELISA and measured in real time using a biosensor based assay. MAb5T4 has a high affinity, K(D)=1.8x10(-11) M, as did both single chain antibodies, scFv5T4WT19 K(D)=2.3x10(-9) M and LscFv5T4 K(D)=7.9x10(-10) M. The small size of this 5T4 specific scFv should allow construction of fusion proteins with a range of biological response modifiers to be prepared whilst retaining the improved pharmacokinetic properties of scFvs.
    • Metastasis-associated 5T4 antigen disrupts cell-cell contacts and induces cellular motility in epithelial cells.

      Carsberg, Catherine J; Myers, Kevin A; Stern, Peter L; CRC Department of Immunology, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester, UK. (1996-09-27)
      The 5T4 antigen is defined by a monoclonal antibody (MAb) specific for human trophoblast. It is also expressed by many types of tumour cell and has been associated with metastasis and poor clinical outcome in a number of cancers. This pattern of expression is consistent with a mechanistic involvement of 5T4 molecules in the spread of cancer cells. The 5T4 antigen is a transmembrane glycoprotein with a 310 amino acid extracellular domain and a 44 amino acid cytoplasmic domain. Transfection of full-length 5T4 cDNA into epithelial cells alters cell-cell contacts and cellular motility. Thus, in 5T4-transfected CL-S1 murine mammary cells, 5T4 expression is associated with dendritic morphology, accompanied by abrogation of actin/cadherin-containing contacts and increased motility. In transfected MDCK canine kidney epithelial cells, 5T4 over-expression also results in increased motility, but disruption of cell-cell contacts, either by culturing cells in low calcium medium or by addition of HGF/SF, is needed. The effects of 5T4 expression on morphology and motility are separable since cells transfected with a truncated form of 5T4 cDNA in which the cytoplasmic domain is deleted reveal that the latter is necessary to abrogate actin/cadherin-containing contacts but does not influence the effects on motility. Thus, 5T4 molecules can deliver signals through both the extracellular and intracellular domains, and the resultant effects are consistent with a role for 5T4 molecules in invasion processes.
    • Metastasis-associated 5T4 oncofoetal antigen is concentrated at microvillus projections of the plasma membrane.

      Carsberg, Catherine J; Myers, Kevin A; Evans, Gareth S; Allen, Terence D; Stern, Peter L; CRC Department of Immunology, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester, UK. (1995-08)
      The 5T4 oncofoetal antigen is a 72 kDa glycoprotein defined by a monoclonal antibody raised against human placental trophoblast and is expressed in many different carcinomas but detected only at low levels in some normal epithelia. Immunohistochemical analysis of the patterns of expression in colorectal carcinomas has indicated a significant association between the presence of the antigen in tumour cells and metastatic spread. A cDNA encoding the 5T4 molecules has been isolated and the extracellular portion contains several leucine-rich repeats which have been implicated in cellular interactions. To study the cell biological role of 5T4 molecules, murine L cells (A9 derivative) were stably transfected with 5T4 cDNA under the control of the CMV immediate-early promoter. The 5T4-expressing cells exhibited a more spindle-shaped morphology compared to the vector alone transfected cells. Confocal immunofluorescence microscopy revealed a 'polkadot' pattern of 5T4 antigen expression, heterogeneous in intensity between cells, but distributed over the entire cell surface. Transmission and scanning electron microscopy showed that the 5T4 antigen is concentrated at microvillus projections of the plasma membrane both in the transfected A9 cells and in various carcinoma cell lines. Such projections express an array of surface molecules which function in cell adhesion and motility. This association of 5T4 antigen with microvillus projections was also observed in various carcinoma cell lines. 5T4 expression in A9 cells was also associated with an altered pattern of cell division, decreased cell-substratum adhesion and increased cellular motility. These results support the hypothesis that 5T4 molecules may have a direct function in trophoblast and tumour cell invasion processes.
    • Organisation of the mouse and human 5T4 oncofoetal leucine-rich glycoprotein genes and expression in foetal and adult murine tissues.

      King, Karen W; Sheppard, Freda C; Westwater, Caroline; Stern, Peter L; Myers, Kevin A; CRC Immunology Group, Cell and Tumour Biology Section, Paterson Institute for Cancer Research, Christie Hospital, Manchester M20 9BX, UK. (1999-06-09)
      The human 5T4 oncotrophoblast leucine-rich glycoprotein may contribute to the process of placentation or metastasis by modulating cell adhesion, shape and motility. To understand better the role of 5T4 in development and cancer, the gene structure has been elucidated from both human and mouse genomic clones and mRNA expression has been studied in foetal and adult mouse tissues. The protein coding region is located within the second of two exons, the first exon comprising solely of 5'-untranslated region. Upstream there are no TATA or CAAT boxes, but there are a number of potential Sp1 binding sites. The murine and human proteins show a homologous domain organisation of the leucine rich repeats (LRR) and associated N- and C-terminal flanking regions, although the hydrophilic sequence which intervenes between the two LRR domains contains six additional amino acids in the mouse. The signal peptide, transmembrane region and cytoplasmic tail sequences are identical as are 6 out of the 7 potential N-linked glycosylation sites. Mouse 5T4 transcripts are abundant in placenta and also highly expressed in embryos while in adult tissues transcripts are restricted to brain and ovary. These patterns of expression and the genomic organisation are discussed in relation to possible function and other recently described LRR containing proteins.
    • Prognostic significance of 5T4 oncofetal antigen expression in colorectal carcinoma.

      Starzynska, T; Marsh, P J; Schofield, Philip F; Roberts, Stephen A; Myers, Kevin A; Stern, Peter L; Department of Immunology, Paterson Institute of Cancer Research, Christie Hospital, NHS Trust, Manchester, UK. (1994-05)
      The 5T4 oncofetal antigen is a 72 kDa glycoprotein defined by a monoclonal antibody raised against human placental trophoblast and is expressed in many different carcinomas but detected only at low levels in some normal epithelia. Immunohistochemical analysis of the patterns of expression in colorectal carcinomas has indicated a significant association between the presence of the antigen in tumour cells and metastatic spread. The 5T4 antigen phenotype of 72 colorectal cancers has been compared with the clinical outcome of the patients in order to assess its relationship with prognosis. Forty per cent of tumours were 5T4 positive; the remainder were either unlabelled or exhibited stroma-associated labelling only. There was a significant correlation between 5T4 expression in the malignant cells and unfavourable course of disease (P < 0.001). The 5 year survival with 5T4-positive tumours was 22% compared with 75% for patients with 5T4-negative tumours; median survival was 24 versus > 90 months respectively. Stratified analysis showed that 5T4 antigen tumour positivity was acting independently of each of stage, site of tumour, age or sex. There were significant differences in survival for patients with Dukes' B and C stage carcinomas (P = 0.001 and P = 0.034). The results suggest that in colorectal cancer immunohistochemical assessment of 5T4 expression may be useful in identifying patients at high risk for tumour recurrence and for whom additional treatment strategies might be most appropriate.
    • Regional localization of a trophoblast antigen-related sequence and 16 other sequences to human chromosomes 6q using somatic cell hybrids.

      Boyle, John M; Hey, Yvonne; Myers, Kevin A; Stern, Peter L; Grzeschik, F H; Ikehara, Y; Misumi, Y; Fox, M; Department of Cancer Genetics, Paterson Institute for Cancer Research, Christie Hospital and Holt Radium Institute, Manchester, United Kingdom. (1992-04)
      Using a panel of 13 hybrid cell lines, we have regionally localized 22 markers to the long arm of chromosome 6. Revised or new locations are provided for 17 of the markers, and preliminary assignments to chromosome 6 of 11 loci are confirmed. The location of NT5, previously determined by antigen expression in hybrids, has been confirmed at 6q14-q15 by using a cDNA probe. Other DNA probes include one new anonymous sequence, designated D6S130, that maps to 6q12 and 4 VNTR probes that map to the proterminal band, 6q27. Probe CRI-L1065 also maps to 6q21, CRI-994 maps to 6q21-qter, and CRI-L322 maps to 6q14-15, information that may assist the merging of physical and genetic maps.
    • Repair of alkylated purines in the hepatic DNA of mitochondria and nuclei in the rat.

      Myers, Kevin A; Saffhill, Roy; O'Connor, Peter J; Paterson Institute for Cancer Research, Christie Hospital and Holt Radium Institute, Manchester, UK. (1988-02)
      Further evidence for the preferential interaction of carcinogens with mitochondrial DNA (mtDNA) has been obtained. In rats treated with high doses of N-nitrosodimethylamine (NDMA) or N-nitroso-N-butylurea (NBU), hepatic mtDNA contains 1.4 times more O6-methyl-2'-deoxyguanosine (O6-MedG) or 2.3 times more O6-butyl-2'-deoxyguanosine (O6-BudG) than does nuclear DNA (nDNA). The kinetics of removal of O6-MeG from mtDNA and nDNA are similar at both high (20 mg/kg) and low (2 mg/kg) doses of NDMA, and the removal of O6-MeG can be increased by pretreating the animals with 2-acetylaminofluorene (AAF), indicating that O6-MeG is repaired in the mitochondrion by a mechanism similar to that which functions in the nucleus. In contrast, O6-BudG is removed very slowly from mtDNA and rapidly from nDNA, an observation which is consistent with the absence of a nucleotide excision mechanism in the mitochondrion and the repair of O6-BudG, predominantly by an excision mechanism, in the nucleus of mammalian cells. A 23-kd methyltransferase (MT) protein, similar to the one found in the nucleus, has been isolated from hepatic mitochondria and is present in mitochondria from which the outer membrane has been removed. It is suggested that O6-MeG, but not O6-BuG can be repaired from mtDNA by a MT protein that is nuclear encoded and transported across the mitochondrial membrane.
    • Targeting immune effector molecules to human tumor cells through genetic delivery of 5T4-specific scFv fusion proteins.

      Myers, Kevin A; Ryan, Matthew G; Stern, Peter L; Shaw, David M; Embleton, Jim; Kingsman, Susan M; Carroll, Miles W; Oxford BioMedica (UK) Ltd., Medawar Centre, Oxford Science Park, UK. (2002-11)
      Although several clinical trials have shown beneficial effects by targeting tumor-associated antigens (TAAs) with monoclonal antibodies, a number of issues, including poor penetration of the tumor mass and human antimouse antibody responses, remain. The use of recombinant single-chain Fv (scFv) fragments has the potential to address these and other issues while allowing the addition of different effector functions. To develop therapeutic strategies that recruit both humoral and cellular arms of the immune response, we have constructed chimeric proteins linking either the human IgG1 Fc domain or the extracellular domain of murine B7.1 to a scFv specific for the oncofetal glycoprotein, 5T4. This TAA is expressed by a wide variety of carcinomas and is associated with metastasis and poorer clinical outcome. We have engineered retroviral constructs that produce fusion proteins able to interact simultaneously with both 5T4-positive cells and with the receptor/ligands of the immune effector moieties. Genetic delivery through a murine leukemia virus vector to 5T4-positive tumor cells results in the secreted scFv fusion protein binding to the cell surface. Furthermore, the scFv-HIgG1 fusion protein is able to direct lysis of 5T4-expressing human tumor cell lines through antibody-dependent cell cytotoxicity, indicating its potential as a gene therapy for human cancers.
    • Vaccination of colorectal cancer patients with modified vaccinia Ankara delivering the tumor antigen 5T4 (TroVax) induces immune responses which correlate with disease control: a phase I/II trial.

      Harrop, Richard; Connolly, Noel B; Redchenko, Irina; Valle, Juan W; Saunders, Mark P; Ryan, Matthew G; Myers, Kevin A; Drury, Noel L; Kingsman, Susan M; Hawkins, Robert E; et al. (2006-06-01)
      PURPOSE: The highly attenuated strain of vaccinia virus, modified vaccinia Ankara (MVA), encoding the tumor antigen 5T4 (termed TroVax), has been evaluated in an open-label phase I/II study in colorectal cancer patients. The primary objectives were to assess the safety and immunogenicity of ascending doses of TroVax and to determine the biodistribution of the vector. EXPERIMENTAL DESIGN: TroVax was given to 22 patients with metastatic colorectal cancer. Seventeen patients received doses of TroVax ranging from 5 x 10(7) up to 5 x 10(8) plaque-forming units at 0, 4, and 8 weeks and were considered to be evaluable for assessment of immunologic responses. Both antibody and cellular responses specific for the tumor antigen 5T4 and the viral vector were monitored throughout the study. RESULTS: TroVax was well tolerated in all patients with no serious adverse events attributed to vaccination. Of 17 evaluable patients, 16 showed 5T4-specific cellular responses whereas 14 had detectable antibody levels following vaccination. TroVax was able to boost 5T4-specific immune responses in the presence of MVA neutralizing antibodies. Periods of disease stabilization ranging from 3 to 18 months were observed in five patients, all of whom mounted 5T4-specific immune responses. Furthermore, statistical analysis showed a positive association between the development of a 5T4 (but not MVA) antibody response and patient survival or time to disease progression. CONCLUSION: These data indicate that vaccination with TroVax is safe and well tolerated and that immune responses to 5T4 can be induced without any evidence of autoimmune toxicity. Furthermore, 5T4-specific antibody responses correlate with evidence of disease control.