Deuk-Su Kim

Glycomodification and characterization of anti-colorectal cancer immunotherapeutic monoclonal antibodies in transgenic tobacco

Anti-colorectal cancer mAb CO17-1A (IgG2a) recognizes the antigen GA733, which is highly expressed on the surface membrane of human colorectal carcinoma cells. In this study, a transgenic tobacco system for the production of mAb CO17-1A was developed. The mAb construct included a KDEL sequence, an endoplasmic reticulum (ER) retention signal attached to the C-terminus of the heavy chain, to target accumulation of mAb into ER. An immunoblot showed significantly enhanced levels of expression of the plant-derived mAbK (mAbPK) CO17-1A compared to mAbP CO17-1A mAb without the KDEL sequence. An ELISA assay using human colorectal carcinoma cells confirmed that expression of mAbPK was also significantly higher than that of mAbP. Glycosylation analysis revealed that mAbP had plant-specific glycans; whereas, mAbPK primarily had oligomannose glycans. FACS showed that the Fc domains of both mAbPK and mammalian-derived mAb (mAbM) had similar binding activity to the FcγRI receptor (CD64). However, the Fc domains of the mAbP had slightly lower binding activity to the FcγRI receptor than both mAbPK and mAbM. The antibody-dependent cell cytotoxicity of mAbPK, against human colorectal cancer cells, was as efficient as mAbM; whereas mAbP was very low. These results suggest that KDEL localized and accumulated mAbP in the ER and eventually enhanced the expression of mAbP with oligomannose glycan and similar anti-cancer biological activity to the parental mAbM.

Optimization of Ammonium Sulfate Concentration for Purification of Colorectal Cancer Vaccine Candidate Recombinant Protein GA733-FcK Isolated from Plants

A protein purification procedure is required to obtain high-value recombinant injectable vaccine proteins produced in plants as a bioreactor. However, existing purification procedures for plant-derived recombinant proteins are often not optimized and are inefficient, with low recovery rates. In our previous study, we used 25–30% ammonium sulfate to precipitate total soluble proteins (TSPs) in purification process for recombinant proteins from plant leaf biomass which has not been optimized. Thus, the objective in this study is to optimize the conditions for plant-derived protein purification procedures. Various ammonium sulfate concentrations (15–80%) were compared to determine their effects on TSPs yield. With 50% ammonium sulfate, the yield of precipitated TSP was the highest, and that of the plant-derived colorectal cancer-specific surface glycoprotein GA733 fused to the Fc fragment of human IgG tagged with endoplasmic reticulum retention signal KDEL (GA733P-FcK) protein significantly increased 1.8-fold. SDS-PAGE analysis showed that the purity of GA733P-FcK protein band appeared to be similar to that of an equal dose of mammalian-derived GA733-Fc (GA733M-Fc). The binding activity of purified GA733P-FcK to anti-GA733 mAb was as efficient as the native GA733M-Fc. Thus, the purification process was effectively optimized for obtaining a high yield of plant-derived antigenic protein with good quality. In conclusion, the purification recovery rate of large quantities of recombinant protein from plant expression systems can be enhanced via optimization of ammonium sulfate concentration during downstream processes, thereby offering a promising solution for production of recombinant GA733-Fc protein in plants.

Optimization of colorectal cancer vaccine candidate protein GA733‐Fc expression in a baculovirus–insect cell system

The baculovirus–insect cell expression system has been used to produce functional recombinant proteins. The antigen GA733 is a cell‐surface glycoprotein highly expressed on most human colorectal carcinoma cells. Conditions for the expression of GA733 fused to the human immunoglobulin IgG Fc fragment (GA733‐Fc) were optimized in the baculovirus expression system. Several variable factors were adjusted to optimize expression, including the cell line (Sf9 and High Five), multiplicity of infection (MOI) value (0.05, 0.1, 0.5, 1 and 3), post‐infection time (48, 72 and 96 h) and harvested sample (cell culture media (CM) or cell lysate (CL)). In addition, two pFastBac Dual vectors carrying the GA733‐Fc gene were constructed to express GA733‐Fc with or without an endoplasmic reticulum (ER) retention sequence KDEL and used to generate recombinant baculoviruses. Western blot showed that expression depended on the conditions used to express the recombinant proteins. The protein production level and secretion capability differed in each cell line. In Sf9 cells, the highest expression in the CM and CL was obtained with GA733‐Fc at 96 h post‐infection at 0.1 MOI and with GA733‐FcK at 96 h post‐infection at 3 MOI, respectively. In High Five cells, the highest expression in the CM and CL was obtained with GA733‐Fc at 48 h post‐infection at 1 MOI and with GA733‐FcK at 48 h post‐infection at 3 MOI, respectively. These results suggest that the MOI value, post‐infection time and subcellular localization affect expression, and that these conditions can be modified to optimize protein expression in the baculovirus–insect cell system.

Chimerism of multiple monoclonal antibodies expressed in a single plant

Transgenic plants offer a source for the sustainable, safe, and large-scale production of therapeutic recombinant proteins. In this study, both murine anti-colorectal cancer mAb (mAbMC) and human anti-rabies mAb 57 (mAbHR), expressed in a single plant were investigated for their cancer cell binding activity and rabies virus neutralization activity, respectively. Transgenic plants, expressing murine anti-colorectal cancer mAb CO17-1A (mAbMC) and human anti-rabies mAb 57 (mAbHR), respectively, were crossed to reproduce F1 transgenic plant, expressing both mAbs. PCR and immunoblot analyses demonstrated that heavy (HC) and light chain (LC) genes of mAbMC and mAbHR were present, and that both mAbs were expressed in F1 transgenic lines, respectively. Quantitative immunoblot for purified mAb also showed the presence of both mAbs in F1 transgenic lines. However, Cell ELISA analysis showed that in mAbPC and mAbPR purified from the F1 transgenic lines (mAbPC×R), the binding activity to SW948 human colorectal carcinoma cells was lower than mAbMC, and in vitro mAbMC, mixed with mAbHR (mAbMHC+R). The in vitro rabies virus neutralization assay demonstrated that the mAbPC×R, from the F1 transgenic plants, had lower bioactivity against rabies virus than mAbH57, and mAbMHC+R. N-glycan structure analysis revealed that mAbMC and mAbHR had Golgi type (94 and 14%) and ER type (6 and 86%), respectively, and the purified mAbs from the F1 transgenic plants had Golgi type (75%) and ER type (25%). These results indicate that the F1 transgenic plant produced both mAbPC and mAbPR; however, the HC and LC proteins of each anti-rabies virus and anti-colorectal cancer mAbs were assembled randomly, resulting in chimerism in HC and LC assembly for mAb.

Plant Recycling for Molecular Biofarming to Produce Recombinant Anti-Cancer mAb.

The expression and glycosylation patterns of anti-colorectal cancer therapeutic monoclonal antibody (mAb) CO17-1A recognizing the tumor-associated antigen GA733-2, expressed in human colorectal carcinoma cells, were observed in the leaf and stem tissues of primary (0 cycle), secondary (1 cycle), and tertiary (2 cycle) growths of seedlings obtained from the stem cut of T2 plants. The bottom portion of the stem of T2 seedlings was cut to induce the 1 cycle shoot growth, which was again cut to induce the 2 cycle shoot growth. In the 1 and 2 cycle growths, the periods for floral organ formation (35 days) was shorter than that (100 days) for the 0 cycle growth. The genes of heavy and light chains of mAb CO17-1A existed at the top, middle, and basal portions of the leaves and stem obtained from the 0, 1, and 2 cycle plants. The protein levels in the leaves and stem tissues from the 1 and 2 cycles were similar to those in the tissues from the 0 cycle. The glycosylation level and pattern in the leaf and stem did not alter dramatically over the different cycles. Surface plasmon resonance (SPR) confirmed that mAbs CO17-1A obtained from leaf and stem tissues of the 0, 1, and 2 cycles had similar binding affinity for the GA733-2 antigen. These data suggest that the shoot growth by bottom stem cutting is applicable to speed up the growth of plant biomass expressing anti-colorectal cancer mAb without variation of expression, glycosylation, and functionality.

Expression of a Human Prostatic Acid Phosphatase (PAP)-IgM Fc Fusion Protein in Plants Using In vitro Tissue Subculture

In this study, prostatic acid phosphatase (PAP), which is overexpressed in human prostate cancer cells, was cloned to be fused to the IgM constant fragment (Fc) for enhancing immunogenicity and expressed in transgenic tobacco plants. Then, the transgenic plants were propagated by in vitro tissue subculture. Gene insertion and expression of the recombinant PAP-IgM Fc fusion protein were confirmed in each tested the first, second, and third subculture generations (SG1, SG2, and SG3, respectively). Transcription levels were constantly maintained in the SG1, SG2, and SG3 leaf section (top, middle, and base). The presence of the PAP-IgM Fc gene was also confirmed in each leaf section in all tested subculture generations. RNA expression was confirmed in all subculture generations using real-time PCR and quantitative real-time PCR. PAP-IgM Fc protein expression was confirmed in all leaves of the SG1, SG2, and SG3 recombinant transgenic plants by using quantitative western blotting and chemiluminescence immunoassays. These results demonstrate that the recombinant protein was stably expressed for several generations of in vitro subculture. Therefore, transgenic plants can be propagated using in vitro tissue subculture for the production of recombinant proteins.

Enhanced activities of reproductive system in male rat treated with male silkworm pupae extract

A male silkworm pupae extract with herbal mixtures such as Rubus coreanus Miquel, Chinese matrimony vine Acanthopanax senticosus and tocopherol can be effectively used to recover or boost stamina. In this study, the effect of the male silkworm pupae extract on the reproductive system of Sprague–Dawley male rats was investigated. No clinical symptoms, and no dying or dead animals were found among experimental male silkworm larvae extract, male silkworm pupae extract and control rat groups during the study. No significant differences were found in body weights, feed or water consumption, or macroscopic examination among the three groups. No lesion was found upon necropsy. However, sperm in the group treated with male silkworm pupae extract were significantly more active than sperm in the control group. Sperm counts in both male groups treated with male silkworm larvae extract and male silkworm pupae extract were significantly higher than sperm counts in the control group. Analysis of phospholipid–hydroperoxide glutathione peroxidase (PHGPx) gene expression showed that the male silkworm pupae extract increased expression in testes by 26.7%. This study shows that the male silkworm pupae extract has potential to be used to enhance the function of the human reproductive system.