Earticle

Immunization with the highly expressed tumor-associated antigen GA733 glycoprotein in colorectal cancer is considered to 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-HTGA733- 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 protein expression was confirmed by western blot, and GA733-Fc and GA733-FcK proteins were successfully purified from infiltrated N. benthamiana leaves. The binding affinity of purified GA733-Fc and GA733-FcK with Recombinant Human Fcγ RI/CD64 protein (R&D Systems, MN) were measured by SPR spectrometry. Therefore, 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 after penetration. This work was carried out with the support of “Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ0162662021)” Rural Development Administration, Republic of Korea.

Overexpression of human epidermal growth factor receptor type 2 (HER2) protein is one of the prognostic factors for breast cancer, and HER2 is most likely to be recurrence when cancer metastasizes to lymph nodes. This study demonstrated the expression of the anti-HER2 camelid single domain antibody VHH in tobacco plant. And we confirmed the function of recombinant VHH for the treatment of breast cancer. The anti-HER2 VHH was fused to human IgG Fc domain along with KDEL endoplasmic reticulum (ER) (VHH-FcK) in a gene expression cassette. The plant gene expression cassette was transferred to tobacco plants using Agrobacterium-mediated transformation. The transformants were screened by PCR and western blots. ELISA confirmed the binding activity of purified anti-HER2 VHH-FcK to HER2 positive breast cancer cell line SK-BR-3. In conclusion, this study verified the possibility of anti-HER2 VHH-FcK as a therapeutic agent and suggests that the anti-breast cancer anti-HER2 VHH-FcK can be expressed, properly assembled and purified from plant expression system, which can be as an alternative system for antibody production. This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT)(2021R1F1A1063869).