Part:BBa_K4961005
PEE6.4-33D9-L
The main task of this experiment is to combine the heavy chain variable region and light chain variable region sequences of 33D9 with the constant region sequences (mIgG2a) on pEE12.4 and pEE6.4, respectively, by molecular cloning technology to form an expression vector that can fully express the antibody. To construct our complete antibody expression plasmid, we asked the Gene Synthesis company to synthesise the DNA fragments, then, using the technique of molecular cloning(homologous recombination), we inserted the light chain DNA fragment of the antibody into the pEE6.4 vector. After obtaining the recombinant plasmid, we selected the PCR-correct monoclonal colonies and sent them to the company for sequencing. The validation results showed that we obtained the correct recombinant plasmid.
PEE6.4-33D9-L BBa_K4961005
Construction Design
The part consists of the gene sequence 33D9-L (encoding the antibody light chain/BBa_K4961002) and the vector backbone PEE6.4 (BBa_K4961001).
Figure 1. Profile of pee6.4
Engineering Principle
Antibody-drug conjugate (ADC) is an emerging class of highly effective drugs that is a perfect combination of chemotherapy and immunotherapy. As of September 2023, 16 ADC drugs have been marketed worldwide. With the development of ADCs, it is crucial to test the stability of ADCs in plasma[1]. Our product is an anti-paclitaxel (ptx) antibody (33D9) that binds Antibody-drug conjugate coupled to ptx with high specificity and affinity (eg:52B8hG1ptx). Thus, the stability of 52B8hG1ptx in plasma can be detected using the anti-paclitaxel antibody (33D9), and by analogy, any ADC with ptx as a small-molecule drug can be detected. in addition to this, the ability of 33D9 to bind not only ptx on an ADC, but also ptx alone, expands the scope of its application.
The most classical method of antibody production is the hybridoma technique, and through the preliminary research in the laboratory, we have obtained the heavy chain variable region and light chain variable region sequences of 33D9 respectively. The main task of this experiment is to combine the heavy chain variable region and light chain variable region sequences of 33D9 with the constant region sequences (mIgG2a) on pEE12.4 and pEE6.4, respectively, by molecular cloning technology, to form an expression vector that can fully express the antibody. Then, using the transfection technique, the heavy chain expression vector (pEE12.4-33D9H) and light chain expression vector (pEE6.4-33D9L) were packed with polyethyleneimine (PEI) transfection reagent, and the above complexes were transfected into human embryonic kidney epithelial cells (293F) for antibody expression. After 5 days of expression, we could purify the antibody 33D9 from the supernatant of the culture medium of 293F. finally, an enzyme-linked immunosorbent assay (ELISA) was applied to detect the ability of 33D9 to bind ADC (eg:52B8hG1ptx).
Experimental Procedure
For this experiment we designed two plasmids: antibody heavy chain (33D9H) protein expression plasmid, and antibody light chain (33D9L) protein expression plasmid. Among them, the heavy chain variable region sequence and light chain variable region sequence of the antibody were obtained from the genomic DNA of hybridoma cells.
In order to construct our complete antibody expression plasmid, we asked Gene Synthesis company synthesise the DNA fragments, then, using the technique of molecular cloning, we inserted the heavy chain DNA fragment of the antibody into the pEE12.4 vector and the light chain DNA fragment of the antibody into the pEE6.4 vector. We transformed the expression plasmid into E. coli competent cells, and then coated them on LB solid medium plates containing ampicillin and cultured them overnight at 37℃.
Figure 2 Purity verification and colony inoculation
A: Purity of the digested vector
B: Purity of amplified fragments
After constructing the antibody expression plasmid, it needs to be allowed to amplify inside E. coli, and after the amplified E. coli is lysed, a large amount of antibody expression plasmid will be released, which provides material for the subsequent transfection experiments. We can get the high purity and low endotoxin antibody expression plasmid by using the company's HiPure Plasmid EF Maxi Kit (Megan).
At present, we have obtained a large number of antibody light and heavy chain expression plasmids. The pEE12.4-33D9H and pEE6.4-33D9L were encapsulated with PEI transfection reagent, and then the complex was transfected inside a 293F cell line using a transfection method, and the cell supernatant was collected after 5-7 days. Subsequently, we used protein A affinity purification to obtain the 33D9 antibody from other proteins in the supernatant of the 293F cells.
The size of 33D9 antibody is 150kD, of which the size of light chain fragment is 25kD, and the size of heavy chain fragment is 50kD.The results of polyacrylamide gel electrophoresis (SDS-PAGE) showed that the purity of the protein that we purified from the supernatant of 293F was high. Therefore, the 33D9 antibody was expressed and purified with high quality, which meets the requirements of our subsequent experiments.
Figure 3 Purity of the 33D9 antibody(DetaiBio), the size of light chain fragment is 25kD
Function Testing
Now we have the product we want to develop, the 33D9 antibody, which binds specifically to paclitaxel. With the double antibody sandwich method, we can detect the concentration of ADC coupled to ptx in different media. Before applying it to a real scenario, we have to make a standard curve for our product to determine its upper and lower limits of detection.
The results of the double-antibody sandwich method showed that we successfully made a standard curve that could detect 52B8hG1ptx (Figure 4). By analogy, any drug coupled to paclitaxel, which itself can be directly coupled to fluorescence or indirectly coupled to fluorescence, can be detected in different solutions using our antibody. This provides strong experimental data to support the development of our ELISA kit.
Figure 4 ELISA results：Standard Curve of 52B8G1PTX
Reference:
[1] Kaur, S., Xu, K., Saad, O. M., Dere, R. C. & Carrasco-Triguero, M. Bioanalytical assay strategies for the development of antibody-drug conjugate biotherapeutics. Bioanalysis 5, 201-226, doi:10.4155/bio.12.299 (2013)
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal XbaI site found at 2523
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- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 6975
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Illegal XhoI site found at 2505 - 23INCOMPATIBLE WITH RFC[23]Illegal XbaI site found at 2523
- 25INCOMPATIBLE WITH RFC[25]Illegal XbaI site found at 2523
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Illegal AgeI site found at 2493 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 759
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