Overexpression of human epidermal growth factor receptor type 2 (HER2) is considered as a prognostic factor of breast cancer, which is positively associated with recurrence when cancer metastasizes to the lymph nodes. Here, we expressed the single variable domain on a heavy chain (VHH) form of anti-HER2 camelid single domain antibody in tobacco plants and compared its in vitro anticancer activities with the anti-HER2 full size antibody. The gene expression cassette containing anti-HER2 camelid single domain antibody VHH fused to human IgG Fc region with KDEL endoplasmic reticulum (ER) (VHH-FcK) was transferred into the tobacco plant via the Agrobacterium-mediated transformation. The transformants were screened with polymerase chain reaction and Western blot analyses. Enzyme-linked immunosorbent assay (ELISA) confirmed the binding of the purified anti-HER2 VHH-FcK to the HER2-positive breast cancer cell line, SK-BR-3. Migration assay results confirmed anticancer activity of the plant-derived anticancer camelid single chain antibody. Taken together, we confirmed the possibility of using anti-HER2 VHH-FcK as a therapeutic anticancer agent, which can be expressed and assembled and purified from a plant expression system as an alternative antibody production system.

A 53-year-old woman presented with left mandibular area pain, trismus, and facial numbness that had persisted for 4 years. Physical examination revealed a 3×5 cm, hard, non-tender, and round mass on the left mandibular area. Computed tomography and magnetic resonance imaging revealed an expansile tumor involving the left mandibular ramus and temporomandibular joint area with bone destruction, extending to the base of middle cranial fossa and left zygomatic bone. The mass at the segment of left mandible and zygomatic bone, and base of middle cranial fossa was removed. Pathological examination of the mass revealed a giant cell tumor. The defect was reconstructed with iliac bone for the mandible and temporal bone and fascia for the cranial bone and dura. The case is described along with a review of the literature.

Immunization with thetumor-associated antigen GA733 glycoprotein, which is highly expressed in colorectal cancer, is considered to be a promising strategy for cancer prevention and treatment. We cloned a fusion gene of GA733 and immunoglobulin Fc fragment (GA733-Fc), and that of GA733-Fc and an endoplasmic reticulum retention motif (GA733-FcK) into the Cowpea mosaic virus (CPMV)-based transient plant expression vector, pEAQ-HT. Agrobacterium tumefaciens (LBA4404) transformed with the vectors pEAQ-HT-GA733-Fc and pEAQ-HT-GA733-FcK was infiltrated into the leaves of Nicotiana benthamiana plants. To optimize harvesting of leaf to express therapeutic glycoproteins both spatially and temporally, protein expression levels at various leaf positions (top, middle, and base) and days post-infiltration (dpi) were investigated. The GA733-Fc and GA733-FcK genes were detected in leaves at 1–10 dpi using PCR. As assessed by western blot, GA733-Fc and GA733-FcK were expressed at the highest levels in the top leaf position at 5 dpi, and GA733-FcK was expressed more than GA733-Fc. The proteins were successfully purified from infiltrated N. benthamiana leaves using protein A affinity chromatography. ELISA verified that an anti-GA733 antibody recognized both purified proteins. Thus, a functional GA733-Fc colorectal cancer vaccine protein can be transiently expressed using a CPMV virus-based vector, with an optimized expression time and leaf position post-infiltration.

The monoclonal antibody (mAb) CO17-1A specifically binds to the tumorassociated cell surface glycoprotein GA733 in colorectal cancer cells. Thus, mAb CO17-1A has the potential to act as an immune therapeutic protein against colorectal cancer. Recently, it was shown that the baculovirus insect cell expression system produces anti-colorectal cancer mAb CO17-1A. In this study, the colorectal cancer antibody mAb CO17-1A fused to the endoplasmic reticulum (ER) retention signal sequence (KDEL), and the (mAb CO17-1AK) was expressed in Spodoptera frugiperda Sf9 insect cells. The yield, cell cytotoxicity, and in vitro anti-tumor activity of mAb CO17-1AK were verified. Western blotting was performed to confirm that both heavy and light chains of mAb CO17-1A were expressed in Sf9 insect cells. The insect-derived mAb (mAb I ) CO17-1A was purified using a protein G affinity column. An in vitro wound healing assay was conducted to determine the inhibition activity of mAb CO17-1A during tumor cell migration, showing that mAb I CO17-1AK was effective as mammalian-derived mAb CO17-1A (mAb M CO17-1A). These results suggest that the insect cell expression system can produce and properly assemble mAbs that inhibit tumor cell migration. Key words: baculovirus insect cell expression system, CO17-1A, colorectal cancer, insect-derived mAb, recombinant glycoprotein, wound healing Entomological Research •• (2020) •• -••

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