Among them, four transgenic lines had a single copy transgene, six lines had two copies, and the rest of lines had three or more copies Supplementary Figure S1. Transgenic calli were regenerated to produce transgenic rice plants which showed normal phenotypes similar to the wild type rice Supplementary Figure S2.
Thus, further analysis of transgenic lines with this construct was not performed. Protein molecular weights are indicated on the left side of each panel. In contrast, BLC and BHC in most transgenic lines with the 2A peptide constructs were proportional, although variations in the expression levels were observed among different transgenic events.
The finding that transgenic rice callus could produce correctly assembled mAb tetramers indicated that this plant expressed mAb could be functional. Biological activity of rice-derived Bevacizumab was detected by ELISA, an assay based on the highly specific antigen—antibody interaction.
B Standard curve of Bevacizumab. C mAb expression levels in transgenic plants. The experiment was performed in triplicates, and the means and errors of mAb contents in different transgenic plants were presented in graph. The Bevacizumab contents in four selected transgenic lines were from The major kDa mAb band was indicated.
Each measurement was performed in triplicates. Mass spectrometric analysis of glycopeptides from purified mAb after tryptic digestion. In this report, we designed and expressed a biologically functional mAb Bevacizumab in transgenic rice callus. The plant expression system has many advantages including high-level productivity, easy management and scaling-up, safety and low cost.
To maximize the benefit of the plant expression system, many strategies have been developed to improve the expression and to increase the production.
For example, transgenes can be stably integrated into the nuclear genome or transiently expressed using viral or Agrobacterium vectors Vaquero et al. In addition, stable expression in cell cultures can produce uniform recombinant proteins in controlled environments by using bioreactors so that good manufacturing practice GMP can be applied Shih and Doran, ; Xu et al. In this study, we produced bioactive Bevacizumab in stably transformed rice callus and the rice-derived Bevacizumab content reached With the development of the rice suspension culture system, it is possible to scale up the production to reach large-scale production by using the bioreactor technology such as the continuous or semi-continuous production system to reduce the production time and manufacturing cost Tekoah et al.
Low-level expression of mAbs in previous reports was mostly affected by the expression strategies. Unbalanced expression of LC and HC is unfavorable to the yield and quality of mAb production because efficient assembly and modification of mAb seem to require an optimal ratio of LC and HC Schlatter et al.
However, contradictory results were reported on the optimal ratio of LC and HC in recombinant mAb production. Excessive expression of LC was considered to be beneficial for mAb production because excess LC would allow more efficient mAb folding, assembly, and the clearance of HC, which is prone to aggregation when expressed in excess Vanhove et al.
Ho et al. Similarly, Schlatter et al. On the contrary, separate groups also reported that excessive HC was beneficial to IgG expression Jiang et al. Unlike the other systems, using of 2A self-cleavage peptide can ensure equal molars expression of LC and HC peptides to avoid unbalanced expression, and thus produce high levels of mAbs Chng et al.
The 2A peptide also drives a translational recoding of the downstream protein. Unlike other systems, 2A peptide-mediated cleavage is not dependent on cellular factors in different cell types because the self-cleavage activity is only dependent on the ribosomes, which are structurally conserved among all eukaryotic organisms. Thus, the 2A peptide is active in all eukaryotic systems, and has been used widely as a co-expression tool in the expression of two or more proteins Donnelly et al.
Recently, 2A peptide was applied in stable and high levels expression of mAbs in CHO cells and mice, and was considered to have potential applications of in vivo delivery of mAbs in gene therapy Fang et al.
However, the application of 2A in the production of complete mAb in plants has not been reported so far. We reported the expression of a mAb Bevacizumab LC and HC as a polyprotein with the 2A linker from FMDV in one gene expression cassette driven by a strong constitutive ubiquitin promoter in transgenic rice callus. The high levels of expression could be attributed to the following factors. First, the codon optimization makes the mRNA stable and the protein translation efficient.
Second, a strong constitutive ubiquitin promoter increases the transgene transcription level. Third, single copy transgene by the Agrobacterium -mediated transformation reduces the chances of gene silencing. Last but not least, the expression of a polyprotein using 2A self-processing peptide favors the accumulation of functional mAb.
The using 2A peptide expression system may affect the mAb yield by the following ways. First, the self-cleavage of 2A linked polyprotein produces equal molecules of HC and LC, which might be an optimal ratio for mAb accumulation. Although the excess of either LC or HC might speed up the mAb assembly as shown in a mathematical modeling Gonzalez et al.
Second, the self-cleavage is co-translational, so that both the LC and HC are simultaneously produced and mobilized into ER, where mAb can be immediately assembled and folded.
Third, correct and complete assembly of mAb may reduce proteolytic degradation of either LC or HC monomers or dimmers when present in excess because incorrectly or incompletely folded antibodies are susceptible to protease attack Gardner et al.
Post-translational protein degradation has been a common phenomenon that significantly influences the yield of foreign protein production Doran, ; De Muynck et al.
But, the degradation of mAb was not observed in our studies based on the Western blot results. Glycosylation is an important property of recombinant mAb that affects its binding affinity and circular half-life, and causes immunogenicity Saint-Jore-Dupas et al. It has been shown that plant expressed proteins are differently modified as compared to mammalian systems.
To avoid plant-specific glycosylation, two strategies are generally used: to keep the recombinant protein in the ER with an ER retention signal peptide or to knockout plant genes coding the plant-specific glycosylation enzymes by glycoengineering Strasser et al.
The loss of KDEL signal in upstream protein can be solved by adding a furin protease recognition site upstream of 2A, which will remove the residues of the 2A peptide Chng et al. Although this strategy can solve the ER retention problem in future studies, the retention of recombinant mAb in ER with the KDEL signal will limit the protein secretion and invariably increase the downstream process cost.
Thus, an alternative approach by glycoengineering to silence or knockout the plant-specific glycosylation genes and to introduce mammalian glycosylation genes into plants could be a better solution Strasser et al. No significant difference was observed between the two rice expressed mAbs and between the rice expressed mAb and the commercial Bevacizumab control, indicating that the rice expressed Bevacizumab mAb is functional.
It remains unknown if the rice expressed Bevacizumab mAb have other comparable properties such as the neutralization of hVEGF in vivo , the circular half-life in clinical studies, and most importantly the adverse immunogenicity related to plant expressed recombinant proteins, which need to be characterized in future studies.
In conclusion, we reported an efficient recombinant mAb expression system in transgenic rice callus using the FMDV 2A self-cleavage peptide. The cleavage of 2A peptide linked polyprotein in one gene expression cassette produced equal molecules of HC and LC of a mAb, allowed efficient assembly and folding of functional antibody, and produced recombinant mAb at high levels.
WY and DL conceived, designed, and supervised the study, discussed the results, and wrote the manuscript. LC and XY performed the experiments, analyzed the data, and wrote the manuscript. All authors approved the final manuscript.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. All materials generated from this research will be available on request. National Center for Biotechnology Information , U.
Journal List Front Plant Sci v. Front Plant Sci. Published online Aug 9. Author information Article notes Copyright and License information Disclaimer.
Edited by: Manoj K. Sharma, Jawaharlal Nehru University, India. Reviewed by: Taras P. This article was submitted to Plant Biotechnology, a section of the journal Frontiers in Plant Science. Received May 18; Accepted Jul The use, distribution or reproduction in other forums is permitted, provided the original author s or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
This article has been cited by other articles in PMC. PDF K. Abstract Bevacizumab, a humanized monoclonal antibody mAb targeting to the vascular endothelial growth factor VEGF , has been widely used in clinical practice for the treatment of multiple cancers. Keywords: Bevacizumab, monoclonal antibody mAb , plant transformation, 2A self-cleavage peptide, plant made antibody, rice. Introduction Plants including the whole body, specific tissues or organs and plant-derived suspension cells are considered as attractive and competitive platforms for recombinant protein production.
Open in a separate window. Plant Transformation Agrobacterium -mediated transformation was performed based on the method of Nishimura et al. Western Blot Total protein was extracted from calli of wild type and selected transgenic rice lines. N-glycan Analysis The rice-derived Bevacizumab was purified as described previously, digested with trypsin, and extracted from gel pieces according to Kolarich et al. Biological Activity and Quantification of Rice-derived Bevacizumab mAb The finding that transgenic rice callus could produce correctly assembled mAb tetramers indicated that this plant expressed mAb could be functional.
Table 1 Recovery of Bevacizumab mAb expressed in transgenic rice callus. Then, using animal and human proteins, scientists work to create a special antibody that will attach to the target antigen. Antibodies will attach to matching antigens like a key fits a lock. This technology allows treatment to target specific cells, causing less toxicity to healthy cells.
Monoclonal antibody therapy can be done only for cancers in which antigens and the respective antibodies have been identified. Avastin works by interfering with the process of angiogenesis by targeting and inhibiting human vascular endothelial growth factor VEGF. VEGF is a cytokine a small protein released by cells that have specific effects on the behavior of cells which when it interacts with its receptors in the cell leads to new blood vessel formation or angiogenesis. Note: We strongly encourage you to talk with your health care professional about your specific medical condition and treatments.
The information contained in this website is meant to be helpful and educational, but is not a substitute for medical advice. For information about the 4th Angel Mentoring Program visit www. Toggle navigation. Spanish About Chemocare. What Avastin Is Used For: Treatment of metastatic colon or rectal cancer, used as part of a combination chemotherapy regimen.
Treatment for non-squamous, non-small cell lung cancer. Treatment of metastatic breast cancer used as part of a combination chemotherapy regimen. Treatment of glioblastoma GBM. Treatment of metastatic renal cell carcinoma. The first dose is given over 90 minutes. The infusion time can eventually be shortened to 30 minutes if well-tolerated.
The amount of avastin that you will receive depends on many factors, including your height and weight, your general health or other health problems, and the type of cancer or condition being treated. Your doctor will determine your dose and schedule. Side Effects of Avastin: Important things to remember about the side effects of avastin: Most people do not experience all of the side effects listed.
Side effects are often predictable in terms of their onset and duration. Side effects are almost always reversible and will go away after treatment is complete. There are many options to help minimize or prevent side effects.
There is no relationship between the presence or severity of side effects and the effectiveness of the medication. There is no data as to the frequency of adverse reactions that may be attributed to avastin alone.
In clinical studies avastin was used in combination with other chemotherapy medications. This can put you at increased risk for infection. This syndrome is caused by damage to the glomeruli tiny blood vessels in the kidney that filter waste and excess water from the blood and send them to the bladder as urine. Congestive heart failure in patients who have received prior treatment with anthracycline based chemotherapy, or radiation therapy to the chest wall.
However, sometimes, Avastin and Temodar are used in combination for glioblastoma treatment. Avastin contains the active drug ingredient bevacizumab. According to estimates on WellRx. Here we look at how Avastin and Eylea are alike and different.
Avastin contains the active drug bevacizumab. In addition, Avastin is sometimes used off-label to treat age-related macular degeneration AMD. Avastin is also sometimes used off-label to treat diabetic retinopathy. With this condition, blood vessels in your retina an area inside your eye become damaged from diabetes. Both drugs are used to treat certain eye disorders, including macular degeneration and diabetic retinopathy.
However, unlike Eylea, Avastin is used off-label for these conditions. Eylea is approved to treat these eye-related conditions. In addition to Eylea, other drugs are also approved to treat certain eye conditions. And drugs other than Avastin are used off-label for eye-related conditions. For example, ziv-aflibercept Zaltrap is used off-label to treat AMD and diabetic retinopathy. However, for off-label treatment of wet AMD, Avastin is given by healthcare providers as an intravitreal injection.
With an intravitreal injection, the drug is injected into your eye. Eylea comes only as a brand-name drug. But your cost may vary based on how often you receive each drug. However, hair loss is a common side effect of some of the chemotherapy drugs that are used with Avastin.
If you have concerns about hair loss during your cancer treatment, talk with your doctor. They can recommend ways to help manage this side effect. Avastin works on VEGF, which is a certain substance made by cells in your body. Normally, VEGF helps your body make new blood vessels, such as after an injury. But because cancer cells make much more VEGF than healthy cells make, Avastin works mostly on cancer cells.
Chemotherapy drugs, on the other hand, work by attacking rapidly multiplying cells in your body. Chemotherapy affects all cells that multiply quickly, which includes both healthy cells and cancer cells. Yes, Avastin is an immunotherapy drug. Immunotherapy drugs work by helping your immune system to fight cancer. The active drug in Avastin, called bevacizumab, is a monoclonal antibody.
Monoclonal antibody drugs are a type of immunotherapy. Avastin is designed to seek out specific traits on cancer cells. And the drug works with your immune system to fight the cancer cells based on these traits. It depends. However, if your cancer worsens during Avastin treatment, your doctor may recommend that you stop taking the drug. If you have questions about how long you should take Avastin for brain cancer, talk with your doctor. And before Avastin can be used, any wounds you have must be fully healed.
These women received chemotherapy, either with or without Avastin. With early menopause, your ovaries stop ovulating like usual. They can discuss with you the risks and benefits of treatment. Typically, your doctor will start you on a low dosage. Your doctor will ultimately prescribe the smallest dosage that provides the desired effect. The following information describes dosages that are commonly used or recommended. However, be sure to take the dosage your doctor prescribes for you.
Your doctor will determine the best dosage to fit your needs. Avastin comes as a solution inside single-dose vials. Avastin is available in two vial sizes: 4 mL vials that hold mg of drug and 16 mL vials that hold mg of drug.
This dose is given once every 2 weeks. Your doctor will recommend how long treatment should be continued. This dose is given once every 3 weeks. This treatment regimen is continued for up to six 3-week cycles. After this period of time, Avastin may be used without chemotherapy.
Avastin can be given for up to a total of twenty-two 3-week cycles with or without chemotherapy. But it may be stopped sooner if your cancer worsens during treatment. Possible dosages are as follows:. These recommended dosages are as follows:. There is another Avastin dosage option for people with colon cancer that worsened during treatment with one of the regimens listed above.
The recommended dosage for this use is either:. And this dosage should be used in combination with a chemotherapy regimen that includes a group of drugs called fluoropyrimidines, with either irinotecan or oxaliplatin Eloxatin. Fluorouracil is an example of a fluoropyrimidine. The medical staff can help you reschedule your appointment to receive your dose of the drug. Avastin is meant to be used as a long-term treatment. Avastin is only given by healthcare providers.
During Avastin treatment, your first infusion will last about 90 minutes. Then your second infusion will last about 60 minutes.
For most people, Avastin is given every 2 to 3 weeks. But alcohol can interact with some chemotherapy drugs that may be used with Avastin. However, before taking Avastin, talk with your doctor and pharmacist. Tell them about all prescription, over-the-counter, and other drugs you take. Also tell them about any vitamins, herbs, and supplements you use.
Sharing this information can help you avoid potential interactions. If you have questions about drug interactions that may affect you, ask your doctor or pharmacist. This drug contains the medication bevacizumab. This substance stimulates new blood vessels to be made. In fact, VEGF is a major factor in helping cancer tumors to form new blood vessels.
And the new blood vessels that form help deliver nutrients and oxygen to tumor cells. This leads to rapid growth of tumors and helps them spread into other parts of your body. It also encourages the original tumor to keep growing. This stops the cancer from worsening inside your body. Avastin begins working as soon as you take your first dose.
After you stop taking Avastin, the drug will last in your body for about 3 to 4 months. But this length of time can vary from person to person. And it will depend on things like the dose of Avastin you used and how long you took the drug. As with all medications, the cost of Avastin can vary. To find current prices for Avastin in your area, check out WellRx. The cost you find on WellRx. This type of pharmacy is authorized to carry specialty medications. These are drugs that may be expensive or may require help from healthcare professionals to be used safely and effectively.
Your insurance plan may require you to get prior authorization before approving coverage for Avastin. This means that your doctor and insurance company will need to communicate about your prescription before the insurance company will cover the drug. The insurance company will review the request and let you and your doctor know if your plan will cover Avastin. If you need financial support to pay for Avastin, or if you need help understanding your insurance coverage, help is available.
Genentech, Inc. For example, if you have insurance coverage on Avastin, you may qualify for copay assistance or other ways to save on the drug. In animal studies , Avastin caused low birth weights in offspring who were exposed to the drug during pregnancy. And Avastin use also increased the risk of certain fetal complications, such as skeletal deformities. Post-marketing reports give information about a drug after the drug has been released onto the market.
They may recommend that you use a different cancer drug during pregnancy. Note: Sex and gender exist on spectrums. And you should continue to use birth control for at least 6 months after your last dose of Avastin. The manufacturer of Avastin recommends that women should not breastfeed during Avastin treatment. And women should continue to avoid breastfeeding for at least 6 months after their last dose of the drug.
They can recommend safe and healthy ways to feed your child. Before taking Avastin, talk with your doctor about your health history.
Avastin may not be right for you if you have certain medical conditions or other factors affecting your health. These include:. Avastin bevacizumab is an intravenous IV monoclonal antibody that is approved to treat the following conditions:.
This reduces endothelial cell proliferation, as well as new blood vessel formation. The approximate half-life of bevacizumab following IV administration is 20 days, with a range of 11 days to 50 days.
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