Plant-derived EpCAM antigen induces protective anti-cancer response

Abstract

Immunotherapy holds great promise for treatment of infectious and malignant diseases and might help to prevent the occurrence and recurrence of cancer. We produced a plant-derived tumor-associated colorectal cancer antigen EpCAM (pGA733) at high yields using two modern plant expression systems. The full antigenic domain of EpCAM was efficiently purified to confirm its antigenic and immunogenic properties as compared to those of the antigen expressed in the baculovirus system (bGA733). Recombinant plant-derived antigen induced a humoral immune response in BALB/c mice. Sera from those mice efficiently inhibited the growth of SW948 colorectal carcinoma cells xenografted in nude mice, as compared to the EpCAM-specific mAb CO17-1A. Our results support the feasibility of producing anti-cancer recombinant vaccines using plant expression systems.

Fig. 1

Expression of EpCAM in plants. a Colorectal cancer-associated antigen EpCAM/GA733-2 was assembled in plant expression vectors pBIN-plus (ImpactVector) and pICH115999 (MagnICON) for stable and transient plant transformation, respectively. The final expression cassette contains: the rubisco small subunit promoter, RbcS1, driving the coding sequence of EpCAM antigen; the ER signal peptide (SP) and retention signal KDEL; a cassette (nptII) conferring resistance to the antibiotic kanamycin, and c-myc and H ₆ tags. All components are located within the left (LB) and right (RB) borders of T-DNA (plasmid pRB74) for stable transformation. b Six-week-old Swiss chard plants were used for MagnICON-based expression (left). Soluble pGA733 was detected at 7, 8 and 9 days post inoculation (dpi) by Western blotting with antigen-specific mAb in total soluble protein (TSP) extracts (right). c Stably transformed tobacco plants (left) and Western blotting (right) of total protein extract from this plants probed with murine mAb AB733. Positive control (+) is the bacteria-expressed GA733 antigen; protein extract from non-transgenic wild-type (wt) tobacco served as a negative control. d SDS-PAGE and Western blotting (at 1:1000 dilution) of soluble pGA733 affinity-purified from plant leaf tissues compared with purified bGA733. Numbers on the left indicate molecular size (kDa)

Fig. 2

Serum antibody response to EpCAM in BALB/c mice. a ELISA titers of sera from BALB/c mice immunized with pGA733 or bGA733 antigen. Results are presented as mean ± SD. b ELISA IgG subclass titers of the pooled pGA733 and bGA733 sera in panel a. c Serial dilutions of pGA733 and bGA733 protein preparations (1:3) probed with the corresponding pooled sera (1:10,000 dilution) obtained after the third immunization of mice in panel A

Fig. 3

Suppression of tumor growth in nude mice by pGA733-specific antibodies. a BALB/c nu/nu mice xenografted with 10⁶ SW948 colorectal cancer cells were injected with serum from BALB/c mice immunized with either pGA733 or bGA733 antigen, or received serum from mock-immunized (plant TSP) mice, or murine mAb CO17-1A. At days 2, 4 and 7, all mice were injected with three additional doses of sera or antibodies. Tumor volumes (mm³) were recorded at 10, 15, 17, 19, 22, 24, 26, 29, 31, 35 and 38 days after initial inoculation with cancer cells. Data are given as mean ± SD. b Pictures of mice treated with pGA733 sera (top) or control plant TSP sera (bottom) were taken on day 35 after injection of tumor cells. Tumor areas are circled; arrows indicate tumor cell injection sites