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The monoclonal antibody (mAb) CO17‐1A specifically binds to the tumor‐associated 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 (mAbI) 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 mAbI CO17‐1AK was effective as mammalian‐derived mAb CO17‐1A (mAbM CO17‐1A). These results suggest that the insect cell expression system can produce and properly assemble mAbs that inhibit tumor cell migration. [ABSTRACT FROM AUTHOR]

The baculovirus-insect cell expression system is widely used to produce recombinant proteins for various biomedical applications. Our previous study demonstrated that Ep CAM, a colorectal cancer vaccine candidate protein, can be expressed in the baculovirus-insect cell expression system. However, its functionality (the ability to elicit an immune response), which is important for its possible use as a colorectal cancer vaccine for immunotherapy, still needed to be confirmed. In this study, we examined the ability of recombinant Ep CAM to induce maturation of immature dendritic cells ( DCs) derived from CD34+ cells isolated from human umbilical cord blood. We demonstrated that Ep CAM induces the expression of four DC maturation markers: CD80, CD83, CD86 and MHC II. These results suggest that Ep CAM produced in the baculovirus-insect cell expression system is functional in terms of its ability to trigger maturation of human DCs. [ABSTRACT FROM AUTHOR]

Monoclonal antibody (m Ab) CO17- 1A binds to GA733, which is a tumor-associated glycoprotein antigen highly expressed on the colorectal cancer cell surface. Thus, m Ab CO17- 1A is considered a useful biomolecule for diagnosis and treatment against colorectal cancer. Previously, we established a baculovirus-insect cell expression system for the production of m Ab CO17- 1A. In order to use m Ab CO17- 1A as a diagnostic and therapeutic tool, however, the antibody must be properly purified from the insect cells. In this study, our aim was to investigate effective purification processes of m Ab CO17- 1A expressed in Spodoptera frugiperda ( Sf 9) insect cells, using a French press and sonication for cell disruption. SDS-PAGE confirmed that both m Ab CO17- 1A and m Ab CO17- 1A fused to the KDEL endoplasmic reticulum ( ER) retention signal (m Ab CO17- 1AK) were expressed clearly in Sf 9 insect cells. Western blot analysis showed that detection levels of m Ab CO17- 1A and CO17- 1AK were higher when the insect cells were disrupted two times by the French press and then sonicated, compared to only one French press disruption plus sonication. Optical microscopy confirmed that insect cells treated with both the French press and sonication were properly disrupted. Analysis of gene sequence information on m Ab CO17- 1A verified that a signal peptide is present but a transmembrane protein does not exist. These results suggest that cell disruption by the French press twice and sonication once is an effective method for improving purification efficiency. [ABSTRACT FROM AUTHOR]

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 Ig G 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 p Fast Bac 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- Fc K 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. [ABSTRACT FROM AUTHOR]

Seafoods and seaweeds represent some of the most important reservoirs of new therapeutic compounds for humans. Seaweed has been shown to have several biological activities, including anticancer activity. This review focuses on colorectal and breast cancers, which are major causes of cancer-related mortality in men and women. It also describes various compounds extracted from a range of seaweeds that have been shown to eradicate or slow the progression of cancer. Fucoidan extracted from the brown algae Fucus spp. has shown activity against both colorectal and breast cancers. Furthermore, we review the mechanisms through which these compounds can induce apoptosis in vitro and in vivo. By considering the ability of compounds present in seaweeds to act against colorectal and breast cancers, this review highlights the potential use of seaweeds as anticancer agents. [ABSTRACT FROM AUTHOR]

Colorectal cancer is the third most commonly diagnosed cancer in the world. Monoclonal antibody (m Ab) CO17-1 A recognizes the tumor-associated antigen GA733-2, a cell surface glycoprotein highly expressed in colorectal carcinoma cells which is applicable for preventing and curing colorectal cancer. In this study, we tried to produce a new recombinant anti-colorectal cancer large single chain (lsc) m Ab based on m Ab CO17-1 A in the baculovirus-insect cell protein expression system. Two kinds of recombinant lsc mAbs were generated where variable light chain ( VL) and heavy chain ( HC) of m Ab CO17-1 A were fused together by an interchain linker. The only difference between the two m Abs is based on fusion of an ER retention signal ( KDEL) at its C-terminus of HC. Polymerase chain reaction analysis verified the presence of both recombinant genes in the bacmid for generating viral expression vectors in insect cells. Western blot confirmed the expression of lsc mAbs in baculovirus-infected insect cells. Cell enzyme linked immunosorbent assay ( ELISA) showed that the m Abs from cell lysates bound to SW480 and SW620 human colorectal cancer cells. These results indicate that the baculovirus insect expression system can produce anti-colorectal lsc m Ab recognizing human colorectal cancer cells. [ABSTRACT FROM AUTHOR]

The epithelial cell adhesion molecule (EpCAM) is a tumor-associated antigen and a potential target for tumor vaccine. The EpCAM is a cell-surface glycoprotein highly expressed in colorectal carcinomas. The objective of the present study is to develop an edible vaccine system through Agrobacterium-mediated transformation in Chinese cabbage (Brassica rapa). For the transformation, two plant expression vectors containing genes encoding for the EpCAM recombinant protein along with the fragment crystallizable (Fc) region of immunoglobulin M (IgM) and Joining (J)-chain tagged with the KDEL endoplasmic reticulum retention motif (J-chain K) were constructed. The vectors were successfully transformed and expressed in the Chinese cabbage individually using Agrobacterium. The transgenic Chinese cabbages were screened using genomic polymerase chain reaction (PCR) in T0 transgenic plant lines generated from both transformants. Similarly, the immunoblot analysis revealed the expression of recombinant proteins in the transformants. Further, the T1 transgenic plants were generated by selfing the transgenic plants (T0) carrying EpCAM–IgM Fc and J-chain K proteins, respectively. Subsequently, the T1 plants generated from EpCAM–IgM Fc and J-chain K transformants were crossed to generate F1 plants carrying both transgenes. The presence of both transgenes was validated using PCR in the F1 plants. In addition, the expression of Chinese cabbage-derived EpCAM–IgM Fc × J-chain K was evaluated using immunoblot and ELISA analyses in the F1 plants. The outcomes of the present study can be utilized for the development of a potential anti-cancer vaccine candidate using Chinese cabbage. [ABSTRACT FROM AUTHOR]

The antigen–antibody complex (AAC) has novel functions for immunomodulation, encouraging the application of diverse quaternary protein structures for vaccination. In this study, GA733 antigen and anti-GA733 antibody proteins were both co-expressed to obtain the AAC protein structures in a F1 plant obtained by crossing the plants expressing each protein. In F1 plant, the antigen and antibody assembled to form a large quaternary circular ACC structure (~30 nm). The large quaternary protein structures induced immune response to produce anticancer immunoglobulins G (IgGs) that are specific to the corresponding antigens in mouse. The serum containing the anticancer IgGs inhibited the human colorectal cancer cell growth in the xenograft nude mouse. Taken together, antigens and antibodies can be assembled to form AAC protein structures in plants. Plant crossing represents an alternative strategy for the formation of AAC vaccines that efficiently increases anticancer antibody production. [ABSTRACT FROM AUTHOR]