Cancer, a disease that the world is concerned about. Hundreds of millions of people died of it, and tens of thousands of families were destroyed by it.
Human struggle against cancer has been going on for thousands of years, and early people used plants and minerals to treat cancer, but it didn't work until surgery opened the door to cancer treatment.Anesthesiology and sterilization have greatly contributed to the development of cancer surgical treatment, which has continued to be used and improved for decades, until radiation and chemotherapy led to the first revolution in cancer treatment. In 1943, Alfred Gilman and Louis Goodman, pharmacologists at Yale University in the United States, successfully conducted a clinical trial to treat lymphoma with nitrogen mustard, which opened the way for modern cancer chemotherapy.
However, since chemotherapy drugs or therapies are usually non-targeted, they can also attack normal tissues and cells while killing cancer cells, causing numerous side effects.Later on, the discovery of DNA's double-helix structure revolutionized our understanding of many diseases, pinpointing mutations at a genetic level, including in cancer. This breakthrough paved the way for targeted drug therapies. These drugs, purposefully designed, hone in on specific cancer-causing agents, obliterating tumor cells while sparing surrounding healthy tissues.
To optimize the application of these targeted drugs, it's imperative to ensure their stability and specificity. Our innovation lies in an ELISA-based detection method to assess both the stability and targeting capacity of an ADC drug named "52B8G1-PTX", which is conjugated with paclitaxel (abbreviated as PTX).
Our product features a small molecular antibody termed "anti-ptx-mAb". This antibody ensures precise detection of the stability and targeting abilities of "52B8G1-PTX" in plasma. The "anti-ptx-mAb" is composed of two main parts: a light chain, referred to as "pEE6.4", and a heavy chain, designated "pEE12.4". These chains are conjoined using the vector pcDNA3.1 and target fragments "33D9VH" and "33D9VL" through a process of homologous recombination
Our entire experimental process, in fact, revolves around the anti-ptx molecular antibody, which is the most important and unique part of the whole reactor box. Through it we can test the stability and targeting of 52B8G1-PTX.
To produce the desired antibodies, we began by amplifying the DNA fragments, 33D9VH and 33D9VL, using PCR. These fragments were then integrated into the pEE12.4 and pEE6.4 vectors through an enzyme-mediated digestion process. After undergoing homogenization and sequencing tests, we successfully generated the constructs pEE12.4-33D9H and pEE6.4-33D9L.
Subsequently, we introduced both constructs into 293F cells via co-transfection, prompting the cells to express the desired antibody. The expressed antibody was then purified using protein A beads. With our anti-ptx molecular antibody in hand, we conducted a final validation step using the enzyme-linked immunosorbent assay (ELISA). This concluded our experimental workflow.
Figure1. Work flow of our design
Since the anti-ptx molecular antibody are composed of heavy chain and light chain, so, we need to construct two expression plasmids pEE12.4-33D9H, pEE6.4-33D9L that can express antibody heavy chain and antibody light chain respectively. In order to construct pEE6.4-33D9L and pEE12.4-33D9H, we first carried out an enzyme cut to obtain a carrier – in the two PCR tubes, the corresponding enzymes, water, templates, and rapid enzym cut buffers were added, respectively. Finally, 50 microliters of solution were obtained in two tubes. Place them in the 37°C enzyme cut in the PCR and remove them for 3 hours.
The second step is to extend the target fragments 33D9VH and 33D9VL.Similarly, two PCR tubes are prepared, and the corresponding template, water, upstream and reverse downstream and prime STAR MAX premix are inserted. Configure into 50 microliters of solution, then put them in the PCR instrument for 1 hour and remove.
The third step is the purification of the carrier and target fragments, where we have chosen to configure the liposuction gel to purify, adding the GDP buffer, the DW2 buffer, the Elution buffer and the HiPure DNA Mini Columns.
When the experiment reached this stage, the phased testing was introduced.We're going to test the concentration of purified DNA to see if it's too much lost and determine the optimal input. After the test, we combined the target fragments with the carrier using the co-source reconfiguration method and mixed them together in a 37°C connection in the PCR for 30 minutes.
Finally, we'll sequence the pEE12.4-33D9H and pEE6.4-33D9L plasmids. We successfully obtained a monoclonal clone of the recombinant plasmid.
Figure 2. The monoclonal clone of the recombinant plasmid
Figure 3. Heavy chain sequencing results
Figure 4. Light chain sequencing results
We recovered HEK293F cells for 5 minutes in a 37°C bain-marie. Raise 293F cells to the appropriate density. Expression plasmids PEE 12.4-33D9H and pEE 6.4-33D9L were transfected into 293F cells and cultured in 25 ml shake flasks for 5 days. From the culture medium supernatant of 293F cells, we can purify our antibody using protein A affinity chromatography columns and test the purity of the antibody we obtained by SDS-PAGE.
The specific experimental steps were as follows: collect the cell supernatant, centrifuge at 4000 rpm for 45 minutes, and collect the clarified liquid. After the protein A chromatography column was equilibrated with phosphate buffer salt, the clarified liquid from the previous step was injected, and then equilibrated with phosphate buffer salt, and finally eluted with glycine at pH=3, to get the antibody we want and test its protein concentration. Its concentration is:1.754mg/ml。
Next, we verified its purity using SDS-PAGE, due to the fact that the antibody is composed of light and heavy chains. Therefore with the addition of the reducing agent, we can see two bands, a 25kD one for the light chain and a 50kD one for the heavy chain. Non-reduced, the bands of the antibody should be around 150kD.
Figure 5 SDS-PAGE gel
Then we draw the standard curve for our ELISA kit. We diluted the anti-PTX small molecule antibody with 1 × PBS solution at a concentration of 3 μg / mL, and then coated it on a 96-well plate, added 50 μL to each well, and incubated overnight at 4 ° C.The sample with a concentration of 10 μg / mL was prepared with 52B8G1-PTX and added to 12 wells with a gradient dilution of 5 times. After incubation, the pre-made enzyme-labeled secondary antibody working solution was added to continue incubation.Finally, 50 μL TMB color developing solution was added to each well to avoid light, and then 50 μL concentrated sulfuric acid was added to each well. The detection wavelength was set at 450 nm in the microplate reader. The detected curve is as follows, and the detection range is about 0.125-8 ng / ml.
Figure 6. Standard Curve of 52B8G1PTX of the ADC drug "52B8G1-PTX"
After completing the experiment, we found that we could not rule out the non-specific binding of ADC and antibody during the detection process, and could not determine whether it was only about PTX small molecule. Therefore, in order to eliminate the interference of ADC, we try to add 52B8G1-PTX antibody and 20 % ethanol dissolved PTX small molecule drug to compete with it, and use competitive ELISA to eliminate non-specific immunity. Under this method, the color depth of the sample was negatively correlated with the concentration of PTX small molecule, and the standard curve was also opposite to the first method, showing a downward trend. Finally, the curve of the following figure was obtained, and the detection range was 0.01 ng / ml-1μg / ml.
Figure 7. Competitive ELISA to eliminate non-specific immunity