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The natural history of a patient's cancer is often characterised by genetic diversity and sequential sweeps of clonal dominance. It is therefore not surprising that identifying the most appropriate tumour-associated antigen for targeted intervention is challenging. The 5T4 oncofoetal antigen was identified by searching for surface molecules shared between human trophoblast and cancer cells with the rationale that they may function to allow survival of the foetus as a semi-allograft in the mother or a tumour in its host. The 5T4 protein is expressed by many different cancers but rarely in normal adult tissues. 5T4 molecules are 72 kD, heavily N-glycosylated proteins with several leucine-rich repeats which are often associated with protein-protein interactions. 5T4 expression is associated with the directional movement of cells through epithelial mesenchymal transition, potentiation of CXCL12/CXCR4 chemotaxis and inhibition of canonical Wnt/beta-catenin while favouring non-canonical pathway signalling; all processes which help drive the spread of cancer cells. The selective pattern of 5T4 tumour expression, association with a tumour-initiating phenotype plus a mechanistic involvement with cancer spread have underwritten the clinical development of different immunotherapeutic strategies including a vaccine, a tumour-targeted superantigen and an antibody drug conjugate. In addition, a chimeric antigen receptor T cell approach targeting 5T4 expressing tumour cells is in pre-clinical development. A key challenge will include how best to combine each 5T4 targeted immunotherapy with the most appropriate standard of care treatment (or adjunct therapy) to maximise the recovery of immune control and ultimately eliminate the tumour.

The attenuated vaccinia virus, modified vaccinia Ankara, has been engineered to deliver the tumor antigen 5T4 (TroVax®). Here, we report results from a randomized open-label phase II trial in castration-resistant prostate cancer patients in which TroVax was administered in combination with docetaxel and compared against docetaxel alone. The aim was to recruit 80 patients (40 per arm), but the study was terminated early due to recruitment challenges. Therefore, this paper reports the comparative safety and immunological and clinical efficacy in 25 patients, 12 of whom were treated with TroVax plus docetaxel and 13 with docetaxel alone. 5T4-specific immune responses were monitored throughout the study. Clinical responses were assessed by measuring changes in tumor burden by CT and bone scan and by quantifying PSA concentrations. TroVax was well tolerated in all patients. Of 10 immunologically evaluable patients, 6 mounted 5T4-specific antibody responses. Patients treated with TroVax plus docetaxel showed a greater median progression-free survival of 9.67 months compared with 5.10 months for patients on the docetaxel alone arm (P = 0.097; HR = 0.31; 95% CI 0.08-1.24). Importantly, a pre-treatment biomarker previously demonstrated to predict 5T4 immune response and treatment benefit showed a strong association with 5T4 antibody response and a statistically significant association with progression-free survival in patients treated with TroVax plus docetaxel, but not docetaxel alone.

Cancer vaccines such as MVA-5T4 (TroVax(®)) must induce an efficacious immune response to deliver therapeutic benefit. The identification of biomarkers that impact on the clinical and/or immunological efficacy of cancer vaccines is required in order to select patients who are most likely to benefit from this treatment modality. Here, we sought to identify a predictor of treatment benefit for renal cancer patients treated with MVA-5T4. Statistical modeling was undertaken using data from a phase III trial in which patients requiring first-line treatment for metastatic renal cell carcinoma were randomized 1:1 to receive MVA-5T4 or placebo alongside sunitinib, IL-2 or IFN-α. Numerous pre-treatment factors associated with inflammatory anemia (e.g., CRP, hemoglobin, hematocrit, IL-6, ferritin, platelets) demonstrated a significant relationship with tumor burden and patient survival. From these prognostic factors, the pre-treatment mean corpuscular hemoglobin concentration (MCHC) was found to be the best predictor of treatment benefit (P < 0.01) for MVA-5T4 treated patients and also correlated positively with tumor shrinkage (P < 0.001). Furthermore, MCHC levels showed a significant positive association with 5T4 antibody response (P = 0.01). The latter result was confirmed using an independent data set comprising phase II trials of MVA-5T4 in patients with colorectal, renal and prostate cancers. Retrospective analyses demonstrated that RCC patients who had very large tumor burdens and low MCHC levels received little or no benefit from treatment with MVA-5T4; however, patients with smaller tumor burdens and normal MCHC levels received substantial benefit from treatment with MVA-5T4.

Few immunotherapy compounds have demonstrated a direct link between the predicted mode of action of the product and benefit to the patient. Since cancer vaccines are thought to have a delayed therapeutic effect, identification of the active moiety may enable the development of an early marker of efficacy. Patients with renal cancer and requiring first-line treatment for metastatic disease were randomized 1:1 to receive MVA-5T4 (TroVax(®)) or placebo alongside Sunitinib, IL-2 or IFN-α in a multicentre phase III trial. Antibody responses were quantified following the 3rd and 4th vaccinations. A surrogate for 5T4 antibody response (the immune response surrogate; IRS) was constructed and then used in a survival analysis to evaluate treatment benefit. Seven hundred and thirty-three patients were randomized, and immune responses were assessed in 590 patients. A high 5T4 antibody response was associated with longer survival within the MVA-5T4-treated group. The IRS was constructed as a linear combination of pre-treatment 5T4 antibody levels, hemoglobin and hematocrit and was shown to be a significant predictor of treatment benefit in the phase III study. Importantly, the IRS was also associated with antibody response and survival in an independent dataset comprising renal, colorectal and prostate cancer patients treated with MVA-5T4 in phase I-II studies. The derivation of the IRS formed part of an exploratory, retrospective analysis; however, if confirmed in future studies, the results have important implications for the development and use of the MVA-5T4 vaccine and potentially for other similar vaccines.

We have recently reported the results of a phase II trial in which two TroVax [modified vaccinia ankara (MVA) encoding the tumour antigen 5T4] vaccinations were given to patients both pre- and post-surgical resection of liver metastases secondary to colorectal cancer (CRC). 5T4-specific cellular responses were assessed at the entry and 2 weeks after each vaccination by proliferation of fresh lymphocytes and ELISA for antibody responses; 18 from the 19 CRC patients mounted a 5T4-specific cellular and/or humoral response. Here, we present a comparison of individual and between patient responses over the course of the treatments using cryopreserved peripheral blood mononuclear cells (PBMC) samples from the baseline until after the fourth vaccination at 14 weeks. Assays used were proliferation assay with 5T4-Fc fusion protein, overlapping 32mer 5T4 peptides, MVA-LacZ and MVA-5T4 infected autologous monocytes. Responses to 5T4 protein or one or more peptide pools were pre-existing in 12/20 patients and subsequently 10 and 12 patients showed boosted and/or de novo responses, respectively. Cumulatively, 13/20 patients showed proliferative responses by week 14. We also assessed the levels of systemic T regulatory cells, plasma cytokine levels, phenotype of tumour-infiltrating lymphocytes including T regulatory cells and tumour HLA class I loss of expression. More than half of the patients showed phenotypes consistent with relative immune suppression and/or escape highlighting the complexity of positive and negative factors challenging any simple correlation with clinical outcome.

Modified vaccinia Ankara (MVA) encoding the tumor antigen 5T4 (TroVax) has been evaluated in an open label phase II study in metastatic colorectal cancer patients. The primary objective was to assess the safety and immunogenicity of TroVax injected before, during and after treatment with 5-fluorouracil, leukovorin and irinotecan. TroVax was administered to 19 patients with metastatic colorectal cancer. Twelve patients had blood samples taken following each of the six injections and were considered to be evaluable for assessment of immunological responses. Both antibody and cellular responses specific for the tumor antigen 5T4 and the viral vector MVA were monitored throughout the study. Administration of TroVax alongside chemotherapy was safe and well tolerated with no SAEs attributed to the vaccine and no enhancement of chemo-related toxicity. Of the 12 patients who were evaluable for assessment of immune responses, ten mounted 5T4-specific antibody responses with titers ranging from 10 to > 5,000. IFNgamma ELISPOT responses specific for 5T4 were detected in 11 patients with frequencies exceeding one in 1,000 PBMCs in five patients. Eight patients presented with elevated circulating CEA concentrations, six of whom showed decreases in excess of 50% during chemotherapy and four had CEA levels which remained stable for > 1 month following completion of chemotherapy. Of the 19 intention to treat (ITT) patients, one had a CR, six had PRs and five had SD. Potent 5T4-specific cellular and/or humoral immune responses were induced in all 12 evaluable patients and were detectable in most patients during the period in which chemotherapy was administered. These data demonstrate that TroVax can be layered on top of chemotherapy regimens without any evidence of enhanced toxicity or reduced immunological or therapeutic efficacy.

5T4 is a tumor associated antigen that is expressed on the surface of a wide spectrum of human adenocarcinomas. The highly attenuated virus, modified vaccinia Ankara, has been engineered to express human 5T4 (h5T4). In a pre-clinical murine model, the recombinant virus (TroVax) induces protection against challenge with CT26-h5T4 (a syngeneic tumor line expressing h5T4). Anti-tumor activity is long lived, with protection still evident 6 months after the final vaccination. In a therapeutic setting, injection of mice with TroVax results in a reduction in tumor burden of >90%. Depletion of CD8+ T cells has no effect upon therapy in the active treatment model, whereas depletion of CD4+ T cells completely abrogates anti-tumor activity. In a prophylactic setting, depletion of CD4+ and CD8+ T cells after the induction of a h5T4 immune response has no deleterious effect on protection following challenge with CT26-h5T4. In light of these studies, the role of antibodies in protection against tumor challenge was investigated. 5T4 specific polyclonal serum decreased tumor burden by approximately 70%. Thus, we conclude that CD4+ T cells are essential for the induction of a protective immune response and that antibodies are the likely effector moiety in this xenogeneic murine tumor model.