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. 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).

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.

Figure 1 Principle of ClonExpress® Rapid Cloning Technology (Vazyme C115)

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℃.

As shown in Figure 2, we verified through DNA gel electrophoresis that we successfully obtained the pEE12.4 vector, pEE6.4 vector, the heavy chain DNA fragment of the antibody, and the light chain DNA fragment of the antibody. We also successfully obtained a monoclonal clone of the recombinant plasmid.

Figure 2 Purity verification and colony inoculation

A: Purity of the digested vector

B: Purity of amplified fragments

C: Overnight growth of Escherichia coli

We selected two monoclonal clones separatelyinto 15ml LB (Amp+) medium, incubated for 6h, took 500 µl of the bacterial solution and sent it to the company for sequencing, and then transferred the bacterial solution with the correct sequencing result into 1L of fresh LB (Amp+) medium and incubated it at 37℃ overnight. The sequencing results were as follows.The sequencing results in Figure 3 indicate that we successfully obtained two types of recombinant plasmids.

Figure 3A Heavy chain sequencing results

Figure 3B Light chain sequencing results

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.

Figure 4 Plasmid-vector complex formulation and protein expression

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(Figure 5). Therefore, the 33D9 antibody was expressed and purified with high quality, which meets the requirements of our subsequent experiments.

Figure 5 SDS-PAGE gel

Figure 6 Work flow of Purity of the 33D9 antibody(DetaiBio)

Now we have the product we want to develop, the 33D9 antibody, which binds specifically to paclitaxel. With the double antibody sandwich method (Figure 7)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.

Figure7 Double antibody sandwich method

The results of the double-antibody sandwich method showed that we successfully made a standard curve that could detect 52B8hG1ptx (Figure 8). 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 8 ELISA results：Standard Curve of 52B8G1PTX