Usage and Biology:

Anti-PD-L1 antibodies (Figure 1) are used in cancer immunotherapy to block the interaction between PD-L1 on cancer cells and PD-1 on immune cells. By inhibiting this interaction, Anti-PD-L1 antibodies help prevent cancer cells from evading immune detection and enhance the immune response against tumors. The PD-L1 pathway is a crucial mechanism used by cancer cells to evade immune surveillance by inhibiting T cell activity. By blocking PD-L1, Anti-PD-L1 antibodies restore the ability of immune cells to recognize and attack cancer cells, leading to an enhanced anti-tumor immune response [5-7].

Figure 1. The DNA sequence of Anti-PD-L1

Engineering Principle

pRED carries the light-sensing module (PSM) containing the key gene bacteriophytochrome (DrBphP), which perceives red and far-red light through the biliverdin (BV) chromophore generated by heme oxygenase (HO). The light-induced structural changes in PSM are transmitted to the effector Histidine Kinase domain (YF1), altering its kinase and phosphatase activities. Additionally, pRED includes FixJ, encoding an inverted box of genes for the λ phage repressor protein cI and the λ promoter pR (Figure 2). We constructed a novel red light-induced nanobody production platform and utilized this system to produce PD-L1 nanobodies, achieving enhanced antibody yield.

Figure 2. Principle of Red Light-Induced Anti-PD-L1 Nanobody Production

Construction of pRED-Anti-PD-L1 plasmid

The construction of the pRED-Anti-PD-L1 plasmid is based on the plasmid pRED. Specific primers were designed to linearize the pRED plasmid. The Anti-PD-L1 was synthesized by a biotech company. Through homologous recombination, the two fragments were ligated to form a plasmid, resulting in the construction of pRED-Anti-PD-L1(Figure 3).

Figure 3. The plasmid map of pRED-Anti-PD-L1.

Using PCR technology, we successfully obtained fragments for the construction of plasmid pRED-Anti-PD-L1, depicted in Figure 4a. The fragments obtained was approximately 453 bp in length, aligning with our expected size. Subsequent to seamless ligation of the target fragments, they were transformed into Escherichia coli DH5α, as illustrated in Figure 4b.

Moreover, approximately 5 single colonies were randomly selected from the agar plates, and the plasmids were sequenced, yielding sequencing results confirming the accuracy of the plasmid, as presented in Figure 4c. Following cultivation, the correct plasmid was extracted and introduced into Escherichia coli BL21, as shown in Figure 4d. This comprehensive process culminated in the successful acquisition of bacterial strains carrying the correct plasmid pRED.

Figure 4. Construction results of plasmid pRED-Anti-PD-L1. a: PCR results; b: Transformation into DH5α after ligation; c: Sequencing results of selected single colonies; d: Successful transformation of correct plasmid into BL21

Protein expression and verification

The strains of Escherichia coli BL21 carrying the correct plasmids with Anti-PD-L1 were separately subjected to SDS-PAGE to verify the protein expression of the target genes. The target proteins Anti-PD-L1 have sizes of 16.2 kDa, respectively. Protein expression was induced with 12W Red and performed at 25°C for 20 hours. Based on the results shown in Figure 5, we can clearly observe protein bands of the target genes Anti-PD-L1 in both crude and purified proteins, confirming the accuracy of our results. To further validate the expression and correct molecular weights of our target proteins Anti-PD-L1, we conducted Western blot experiments. The experimental results, as shown in Figure 5, clearly demonstrate the expression of our target proteins at lengths of 16.2 kDa, confirming the accuracy of our protein expression.

Figure 5. The SDS-PAGE and WB results for Anti-PD-L1

Characterization/Measurement

1. ELISA analysis of production of two nanobodies under different lighting times

The duration of red-light induction is a critical factor influencing the production of nanobodies by the strains. In order to determine the detection time for subsequent experiments, we first conducted experiments on two strains under different durations (8h,24h and 30h) of red-light induction at 25°C. Before testing the production of nanobodies in the fermentation broth, we tested the absorbance of our antibody when it completely reacted with different concentrations of PD-L1 antigen and drew the standard curve. Through the standard curve, we can quantitatively calculate the antibody yield.

Based on this, we conducted the quantification of nanobodies in the fermentation broth. We ensured the reliability of our experimental data by diluting the samples at different multiples. Through the experimental results (Figure 6), we explored the duration of light exposure at which the strain's antibody production was maximized and did not decompose. Figure 6 shows that Anti-PD-L1 nanobody production at both 24h and 30h was significantly higher than at 8h, but there was no significant difference between the 24h and 30h yields. Based on this experiment, we determined that in subsequent experiments, samples would only be taken at 24 hours to measure antibody production at 25°C.



Figure 6. The results of nanobody production under different light exposure durations.

2. ELISA analysis of production of two nanobodies under different lighting intensities

To investigate the impact of light intensity on nanobody production in the strains, we selected three intensities 3W, 6W, and 12W based on data from literature reports for simultaneous induction of the strains, aiming to assess the effect of different red-light intensities on nanobody production. From the experimental results (Figure 7), it can be observed that when the red-light irradiation intensity increased from 3W to 6W, the production of the two antibodies increased by 10.53% and 16.06%, respectively. However, upon further increase to 12W, there was minimal production enhancement, and the production of Anti-PD-L1 even decreased by 4.44%. Therefore, for subsequent steps, we will utilize a red-light intensity of 6W as our irradiation intensity.



Figure 7. The results of nanobody production under different lighting intensities.

2.3ELISA analysis of production of two nanobodies under different oxygen conditions

Because Escherichia coli is a facultative anaerobe, it means that it can produce under both aerobic and anaerobic conditions. However, it is not clear whether the production of nanobodies requires sufficient oxygen supply. We conducted experiments using two types of Erlenmeyer flasks, as shown in Figure 7: a regular Erlenmeyer flask and a baffled Erlenmeyer flask, with the latter providing more oxygen. This was done to provide different oxygen levels to the strains and assess the production of nanobodies. The experimental results, as shown in Figure 9, revealed that as the dissolved oxygen level in the shake flask increased, the production of antibodies PD-1 and PD-L1 increased by 17.30% and 6.56%, respectively.

We hypothesize that with the increase in dissolved oxygen, the metabolism of the bacterial strain becomes more active, leading to higher antibody production.



Figure 8. Different Erlenmeyer flasks - regular Erlenmeyer flask on the left, baffled Erlenmeyer flask on the right.



Figure 9. The results of nanobody production under different oxygen conditions.

Table 1 presents the nanobody production yields achieved under the established conditions (baffled flasks, 6W illumination, 24h induction period), with ELISA measurements showing 1.9485 ng/L for anti-PD-L1 nanobodies.

Table 1. Nanobody production yield of PD-L1