DNA immunization targeting carcinoembryonic antigen in colorectal cancer patients

Active specific immunotherapy targeting carcinoembryonic antigen (CEA) may induce antigen-specific humoral and cellular responses in cancer patients. Plasmid DNA, encoding tumor antigens, represents a novel approach of delivering conformational antigens. Here we report immune data of an explorative study using CEA66-DNA (non-glycosylated cytoplasmic CEA) and tetwtCEA-DNA (wild type glycosylated and secreted CEA) for immunization in combination with cyclophosphamide and GM-CSF in the adjuvant setting of radically operated colorectal cancer (CRC). 10 patients received intradermal (id) or intramuscular (im) CEA66-DNA delivered by needle-free Biojector at weeks 0, 2, 6 (part 1). 10 patients received tetwtCEADNA id by needle injection and electroporation at weeks 0 and 12 (part 2). In part 3, 6 patients were primed with CEA66-DNA and boosted with tetwtCEA-DNA. A significant increase of CD4+ effector memory, CD8+ effector and CD8+ effector memory T cells was seen in part 1. An immune response against CEA atleast one time point was noted in 15/20 (75%) patients in parts 1 and 2 together. The frequency of patients mounting a CEA-specific cellular immune responses was significantly higher in part 1 (100%) than in part 2 (50%) (p=0.03). In part 3, 5/6 (83%) patients showed a CEA-specific immune response after a prime-boost protocol. The higher CEA-specific T cell responses seen in part 1, may indicate reduced immunological tolerance induced by the non-glycosylated intracellularly produced CEA66-DNA immunogen. Humoral responses determined by ELISA were low. Further studies are warranted to optimize vaccination schedules to induce both cellular and humoral anti-CEA responses of clinical significance. Clinical trials.gov. identification number (parts 2/3) is NCT01064375. Abbrevations CEA: Carcinoembryonic Antigen; CRC: Colorectal Carcinoma; DFS: Disease free survival; ELISA: Enzyme-linked immunosorbent assay; ELISPOT: Enzyme-linked immunospot; EP: Electroporation; GM-CSF: Granulocyte-macrophage colony-stimulating factor; HRP: Horseradish peroxidase; id: intradermal; IFN-γ: interferon γ; IL-4: Interleukin-4; IL-10: Interleukin-10; im: Intramuscular; OS: Overall survival; PBMC: Peripheral blood mononuclear cells; PBS: Phosphate buffered saline; PHA: Phytohemagglutinin; PPD: Purified protein derivative of tuberculin; sc: Subcutaneous; SI: Stimulation index; SIIR: Sustained induced immune response; STIIR: Single time point induced immune response; TAA: Tumor associated antigens; TCV: Therapeutic cancer vaccines; TNF-α: Tumor necrosis factor-α Introduction Colorectal carcinoma (CRC) is a major cause of cancer-related mortality. Despite introduction of new drugs, a large proportion of patients remain incurable. Adjuvant chemotherapy is standard for the treatment of stage III colon cancer and increases the 5-year survival rate to more than 70% and is routinely also used in stage II colon cancer patients with high-risk of relapse. Several chemotherapeutic agents approved for metastatic CRC have however failed to improve the prognosis for patients with stages II and III CRC [1]. New therapeutic approaches are needed and immunotherapy may offer a novel targeted therapeutic option [1]. The goal of therapeutic cancer vaccines (TCV) is to induce a robust long-lasting immune response with limited toxicity [2]. Most tumor cells express tumor-associated antigens (TAA), which might act as targets for the immune system [3]. A commonly expressed TAA in gastrointestinal cancer is the carcinoembryonic antigen (CEA) which has been explored in immunotherapy trials [4-11]. Vaccination targeting CEA in humans was shown to induce antigen-specific humoral, CD4+ helper as well as CD8+ cytotoxic T-cell (CTL) responses [8,11-14]. Immunisation with proteins comprising multiple CD4+ and CD8+ T cell epitopes, have failed to induce a robust CD8+ response which may partly be explained by defect peptide processing of exogenous proteins not entering the MHC class I antigen presentation pathway. DNA contains immunostimulatory CpG sequences that stimulate cytosolic DNA-sensing pathways, interferon regulatory factors and Fas-FasL Correspondence to: Håkan Mellstedt, MD, PhD, Prof, Department of Oncology and Pathology (Radiumhemmet), Cancer Centre Karolinska, Karolinska University Hospital Solna, SE-171 76 Stockholm, Sweden, Tel: +46851774641; E-mail: hakan.mellstedt@karolinska.se