APPLICATION OF PLANT AS HOST IN EXPRESSING PD-1 ANTIBODY AND/OR PD-L1 ANTIBODY

Abstract

Provided is an application of a plant as a host in expressing a PD-1 antibody and/or a PD-L1 antibody, wherein the plant, such as lettuce, is used as an effective expression platform for preparing recombinant proteins, and a simple and effective agrobacterium-mediated vacuum infiltration method is used for expressing the PD-1 monoclonal antibody (Keytruda, pembrolizumab) and the PD-L1 monoclonal antibody (Atezolizumab).

Claims

1. A method for expressing a PD-1 antibody and/or a PD-L1 antibody, comprising using a plant as a host.

2. The method according to claim 1, wherein the plant is selected from the group consisting of lettuce, tobacco, Chinese cabbage, rice, corn, soybean and wheat, and the organ of the plant is selected from the group consisting of a leaf, a seed, a rhizome and a whole plant.

3. An expression vector comprising a vector and i. a heavy chain sequence or a light chain sequence of PD-1; or ii. a heavy chain sequence or a light chain sequence of PD-L1.

4. The expression vector according to claim 3, wherein the codons of the heavy chain sequence or the light chain sequence of PD-1 are optimized to plant-preferred codons to obtain an optimized PD-1 heavy chain sequence or an optimized PD-1 light chain sequence; and the codons of the heavy chain sequence or the light chain sequence of PD-L1 are optimized to plant-preferred codons to obtain an optimized PD-L1 heavy chain sequence or an optimized PD-L1 light chain sequence.

5. The expression vector according to claim 4, wherein the optimized PD-1 heavy chain sequence is set forth in SEQ ID No: 1, the nucleotide sequence of the optimized PD-1 heavy chain is set forth in SEQ ID No: 2; the optimized PD-1 light chain sequence is set forth in SEQ ID No: 3, the nucleotide sequence of the optimized PD-1 light chain sequence is set forth in SEQ ID No: 4; the optimized PD-L1 heavy chain sequence is set forth in SEQ ID No: 5, the nucleotide sequence of the optimized PD-L1 heavy chain is set forth in SEQ ID No: 6; and the optimized PD-L1 light chain sequence is set forth in SEQ ID No: 7, the nucleotide sequence of the optimized PD-L1 light chain is set forth in SEQ ID No: 8.

6. The expression vector according to claim 3, wherein the vector is a binary plant vector.

7. The expression vector according to claim 3, which is constructed by the steps of: Step 1: respectively optimizing the codons of the PD-1 heavy chain, the PD-1 light chain, the PD-L1 heavy chain and the PD-L1 light chain to plant-preferred codons to obtain i. optimized PD-1 heavy chain sequence, ii. optimized PD-1 light chain sequence, iii. optimized PD-L1 heavy chain sequence, and iv. optimized PD-L1 light chain sequence; Step 2: respectively adding XbaI restriction site at the 5 end and XhoI site at the 3 end of the optimized PD-1 heavy chain sequence, the optimized PD-L1 heavy chain sequence and the optimized PD-L1 light chain sequence; adding XmaI restriction site at the 5 end and XhoI site at the 3 end of the optimized PD-1 light chain sequence; respectively cloning the sequences into pUC57 vector to obtain pPD-1H, pPD-1L, pPD-L1H and pPD-L1L cloning vectors; and Step 3: producing gene fragments from the cloning vectors obtained in Step 2 by KpnI/SacI, and cloning the gene fragments into the binary plant vector pCam35S to obtain expression vectors p35S-PD-1H, p35S-PD-1L, p35S-PD-L1H and p35S-PD-L1L.

8. A method for expressing a PD-1 antibody and/or a PD-L1 antibody comprising using the expression vector according to claim 3.

9. The according to claim 8, comprising transforming the expression vector into agrobacterium, performing agrobacterium-mediated vacuum infiltration on a plant tissue, and extracting and isolating protein to obtain the PD-1 antibody and/or the PD-L1 antibody.

10. The method according to claim 9, wherein the agrobacterium-mediated vacuum infiltration is performed by: Step 1: vacuuming for 2545 s; Step 2: maintaining under a vacuum of 95 kPa for 3060 s; Step 3: releasing the pressure and allowing penetrating fluid to penetrate into the plant tissue; and repeating Step 1 to Step 3 for 2 to 3 times, and then keeping the plant tissue in the dark for 4 d.

Description

BRIEF DESCRIPTION OF THE FIGURE

[0051] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings to be used in the embodiments or the description of the prior art will be briefly described below.

[0052] FIG. 1 shows a map of the cloning vector pUC57.

[0053] FIG. 2 (A) shows the construction of the PD-1 plant binary expression vector p35S-PD-1H (heavy chain) and P35S-PD-1L (light chain). PD-1H heavy chain sequence is obtained by double digestion with restriction endonuclease (Xbal/XhoI) from the cloning vector of FIG. 1 and inserted into the Xbal/XhoI sites of pCam35S to generate the plant binary expression vector P35S-PD-1H; the PD-1H heavy chain sequence is obtained by double digestion with restriction endonuclease (Xma1/XhoI) from the cloning vector of FIG. 1 and inserted into the Xma1/XhoI sites of pCam35S to generate the plant binary expression vector P35S-PD-1L;

[0054] LB and RB: the left and right borders of the Ti plasmid; 35S, the CaMV 35S promoter with the tobacco mosaic virus (TMV) 5UTR; NPT II, the expression of the nptII gene for kanamycin resistance; Nos3, Terminator.

[0055] FIG. 2 (B) shows the construction of the PD-L1 plant binary expression vector p35S-PD-L1H (heavy chain) and P35S-PD-L1L (light chain). PD-L1H light chain and PD-L1L heavy chain sequences are obtained by double digestion with restriction endonuclease (Xbal/XhoI) from the cloning vectors of FIG. 1 and inserted into Xbal/XhoI sites of pCam35S to generate the plant binary expression vector P35S-PD-L1H and P35S-PD-L1L;

[0056] LB and RB: the left and right borders of the Ti plasmid; 35S, the CaMV 35S promoter with the tobacco mosaic virus (TMV) 5UTR; NPT II, the expression of the nptII gene for kanamycin resistance; Nos3, Terminator.

[0057] FIG. 3 (A) shows the results of SDS-PAGE gel electrophoresis; Lane 1: PD-1 recombinant antibody; Lane 2: PD-L1 recombinant antibody;

[0058] FIG. 3 (B) shows the results of non-denaturing gel electrophoresis; Lane 3: PD-1 recombinant antibody; Lane 4: PD-L1 recombinant antibody;

[0059] FIG. 4 shows the inhibition of human non-small cell lung cancer (NSCLC) cells by purified PD-1 and PD-L1 antibodies in cell experiments.

DETAILED DESCRIPTION OF THE INVENTION

[0060] The invention discloses use of a plant as a host for expressing a PD-1 antibody and/or a PD-L1 antibody. Those skilled in the art can learn from the contents of this document and appropriately improve the process parameters. It is specifically to be understood that all such alternatives and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described by the preferred embodiments, and it is obvious that the methods and applications described herein can be modified or appropriately changed and combined to implement and apply the techniques of the present invention without departing from the scope of the present invention.

[0061] The present inventor has found through experiments that a plant system, especially a lettuce system, is a more economical and efficient expression platform for transient expression of recombinant proteins. The vacuum agrobacterium infiltration method described in the present invention is a simple and rapid method which can increase recombinant protein production. Lettuce can increase protein production by withstanding vacuum pressure and allow for a more complete penetration of each leaf. Since lettuce is easy to grow and commercially mass-produced, it is easier to be obtained and cheaper than other transiently expressed plants, such as tobacco, and the cost can be significantly reduced since no complicated special production equipment is required. In summary, the present invention can utilize the lettuce system to mass produce PD-1 and PD-L1 monoclonal antibodies in a short period of time.

[0062] The materials and reagents used in the application of the plant as a host for expressing PD-1 antibody and/or a PD-L1 antibody according to the present invention are commercially available.

[0063] The present invention is further illustrated below in conjunction with the examples.

Example 1. Construction of Plant Transient Expression Vector

[0064] In order to provide high-efficiency expression of exogenous proteins in plants, the codons of the human PD-1 heavy chain (GenBank Accession number: 5DK3_B) and light chain, (GenBank Accession number: 5DK3_A), PD-L1 heavy strand (GenBank Accession No.: AAO 17823.1) and light chain (GenBank Accession number: 4DKE_L) are optimized into plant-preferred codons using the protein sequence reverse translation software (https://www.idtdna.com/CodonOpt) and synthesized by Genescript (Nanjing, China). XbaI restriction site was added at the 5 end and XhoI site was added at the 3 end of the optimized PD-1 heavy chain, as well as PD-L1 light chain and heavy chain sequences, respectively. XmaI restriction site was added at the 5 end and XhoI site was added at the 3 end of the PD-1 light chain sequence, respectively. The sequences were cloned into pUC57 vector by genecript, to obtain pPD-1H, pPD-1L, pPD-L1H and pPD-L1L cloning vectors, respectively. The gene fragments were cut from the cloning vector by KpnI/SacI digestion, and cloned into the binary plant vector pCam35S to obtain expression vectors p35S-PD-1H, p35S-PD-1L, p35S-PD-L1H and p35S-PD-L1L, respectively. The four plant expression vectors were transformed into Agrobacterium tumefaciens GV3101 by electroporation using Multiporator (Eppendorf, Hamburg, Germany), respectively. The resulting strains were spread evenly on selective LB plates containing kanamycin antibiotic (50 mg/L). After incubating at 28 C. in the dark for 2 days, single colonies were picked and inoculated into 0.5 L YEB liquid medium (yeast extract broth, 5 g/L sucrose, 5 g/L tryptone, 6 g/L yeast extract, 0.24 g/LMgSO.sub.4, pH 7.2) supplemented with antibiotic (50 mg/L kanamycin). The inoculated culture was incubated at 25 to 28 C. for 72 h in a shaker (220 rpm). The O.D.600 value was measured and adjusted to 3.5 to 4.5 by adding YEB medium. The culture medium was then collected and centrifuged (4500 rpm) for 10 min. The agrobacterium cells were resuspended in penetrating solution (10 mM MES, 10 mM MgSO4) until O.D.600 was 0.5.

[0065] The obtained pPD-1H, pPD-1L, pPD-L1H and pPD-L1L gene fragments were cloned, and four binary plant expression vectors p35S-PD-1H, p35S-PD-1L, P35S-PD-L1H and p35S-PD-L1L were constructed. After completion of the construction, digestion with specific restriction enzymes was performed to confirm that the gene fragment was intact. During vacuum infiltration, most of the lettuce tissue was submerged in the penetrating solution, and except for the solid midrib area, the remaining parts of lettuce turned yellowish brown 4 days after vacuum infiltration.

Example 2. Agrobacterium-Mediated Vacuum Infiltration

[0066] The prepared agrobacterium containing p35S-PD-1H and agrobacterium containing p35S-PD-1L were mixed in equal amounts to O.D.600 of 0.5; also, the prepared agrobacterium containing p35S-PD-L1H and agrobacterium containing p35S-PD-L1L were mixed in equal amounts to O.D.600 of 0.5. The culture suspension was added into a 2 L beaker and the beaker was placed in a desiccator. The lettuce was inverted (core up) and gently spun in the bacterial suspension, and the desiccator was then sealed. Vacuum was applied using a vacuum pump (Welch Vacuum, Niles, Ill., USA) and the penetrating solution in the leaf tissue was observed. After keeping under the pressure for 3060 s, the pressure was released quickly, allowing the penetrating solution to penetrate into the space inside the tissue. This procedure was repeated 2 to 3 times until the significant diffusion of penetrating solution in the lettuce tissue was clearly visible. The lettuce tissue was then gently removed from the penetrating solution and rinsed three times with distilled water and then transferred to a container covered with a plastic film. The treated samples were kept in the dark for 4 days.

Example 3 Protein Extraction and Isolation

[0067] The lettuce sample after agrobacterium vacuum infiltrated was stirred in a stirrer and homogenized at a high speed in the extraction buffer (100 mM KPi, pH 7.8; 5 mM EDTA; 10 mM -mercaptoethanol) at 1:1 ratio for 1 to 2 minutes. The homogenate was adjusted to pH 8.0, filtered through gauze, and the filtrate was centrifuged at 10,000 g for 15 min at 4 C. to remove cell debris. The supernatant was collected, mixed with ammonium sulfate (50%), and incubated on ice for 60 min with shaking, and then was again separated by a centrifuge (10,000 g) at 4 C. for 15 min. The resulting supernatant was subjected to a second round of ammonium citrate (70%) precipitation, suspended on ice for 60 min with shaking, and again centrifuged at 10,000 g for 15 min at 4 C. Then, the supernatant was discarded, and the precipitated protein from the treated sample was dissolved in 5 mL buffer (20 mM KPi, pH 7.8; 2 mM EDTA; 10 mM -mercaptoethanol) and stored at 4 C.

[0068] Downstream processing of plant-derived recombinant proteins is often difficult and expensive because cellulose cell walls are difficult to lyse and secondary plant metabolites are produced. In the present invention, a stirrer is used to perform homogenization, which greatly saves the cost and process of homogenization. After recombinant PD-1 antibody and PD-L1 antibody were separated by denaturing gel SDS-PAGE, bands with estimated molecular weights of approximately 23 kDa and 50 kDa in the lanes were observed (FIG. 3A), consistent with the protein sizes of the light and heavy chains of PD-1 and PD-L1 antibodies. A band of approximately 150 kDa (FIG. 3B) was observed in non-denaturing gel electrophoresis, consistent with the protein molecular weight of PD-1 and PD-L1 antibodies, demonstrating successful formation of the antibody structure by the light chain and heavy chain in lettuce. The protein content in the purified sample was determined to be approximately 0.72 mg/g based on the Bradford assay and the densitometric control group.

Example 4 SDS-PAGE Gel Electrophoresis

[0069] The purified protein extracted from the lettuce after agrobacterium vacuum infiltrated was collected, and the sample (5 L) was heat-denatured (95 C.), mixed with loading buffer (Biorad, Hercules, Calif., USA), and then electrophoresed on 4 to 12% Bolt Bis-Tris Plus SDS-gel (ThermoFisher Scientific, Waltham, Mass., USA). Also, the affinity of the antibody was detected in non-denaturing gel electrophoresis. After staining with Coomassie Blue G250 (Biorad), the gel was photographed again.

Example 5. Inhibition Experiment on Cancer Cells

[0070] Cells of human non-small cell lung cancer NSCLC cell line A549 were grown in RPMI-1640 medium (Gibco, USA) supplemented with 10% FBS (Gibco, USA), 100 U/mL penicillin and 100 g/mL streptomycin. All cells were cultured in a 5% CO.sub.2 humidified atmosphere at 37 C., with the medium changed daily, and cells were passaged every three days using 0.25% trypsin. A549 cells were collected by trypsinization and resuspended at a density of 110.sup.6 cells/mL. 10 g of purified PD-1 and PD-L1 antibodies were added, respectively, and double staining was performed with annexin V-luciferin isothiocyanate (FITC) and PI to evaluate the proportion of apoptotic cells.

[0071] The results show inhibition of lung cancer (NSCLC) cells. The purified recombinant PD-1 and PD-L1 antibodies were added to the cultured NSCLC cells, and the cell growth was checked at 72 h. The results showed that the NSCLC cells without treatment grew well, in contrast, cells cultured with purified recombinant PD-1 or PD-L1 antibodies mostly were destroyed (FIG. 4). These results indicate that exogenous PD-1 and PDL-1 antibodies transiently expressed by the lettuce system are biologically active and can kill NSCLC cells. The results indicate that plants, especially lettuce, are a suitable bioreactor for the production of PD-1 and PD-L1 antibodies.

Example 6

[0072] Control group: PD-1 and PD-L1 antibodies produced in animals;

[0073] Experimental group 1: PD-1 and PD-L1 antibodies produced in the plant provided by the present invention;

[0074] Experimental group 2: PD-1 and PD-L1 antibodies produced in leaves of tobacco.

TABLE-US-00001 TABLE 1 PD-1 and PD-L1 antibodies The degree of difficulty Production Protein Protein in the downstream Production Group cycle (d) content activity protein purification cost (yuan) Control 14 0.42 Binding assay It is difficult to remove About 16000 group mg/g 1.21 10.sup.6 animal cell impurities. In yuan per particular, animal cells gram of are often contaminated protein with human viruses and have low safety. Experimental 4**.sup.# 0.59**.sup.# Binding assay Relatively easy. The About group 1 mg/g 1.82 10.sup.6**.sup.# downstream homogenization 6500**.sup.## yuan is carried out with a stirrer, per gram of saving time and money, protein and eliminating the need to remove impurities such as nicotine and nicotine. Experimental 7* 0.51* Binding assay Relatively difficult. The About group 1 mg/g 1.27 10.sup.6* time-consuming, 13400* yuan laborious and expensive per gram of liquid nitrogen grinding protein was required, and special steps were required to remove nicotine, nicotine impurities. *indicates P 0.05 compared with the control group; **indicates P 0.01 compared with the control group; .sup.#indicates P 0.05 compared with the experimental group 2; .sup.##indicates P 0.01 compared with the experimental group 2.

[0075] As can be seen from Table 1, compared with the animal expression system of the control group, a lettuce transient expression system is provided by the present invention for the expression of PD-1 and PD-L1 antibodies, which reduces the production cycle very significantly (P0.01), improves the protein content very significantly (P0.01), improves the protein activity significantly (P0.05), simplifies the purification of the protein and reduces production costs very significantly (P0.01).

[0076] Compared with the tobacco expression system of experimental group 2, the lettuce transient expression system for PD-1 and PD-L1 antibodies reduces the production cycle significantly (P0.05), improves the protein content significantly (P0.05), improves the protein activity significantly (P0.05), simplifies the purification of the protein and reduces production costs very significantly (P0.01).

[0077] Compared with the control group, the tobacco leaf transient expression system of experimental group 2 for PD-1 and PD-L1 antibodies reduces the production cycle significantly (P0.05), improves the protein content significantly (P0.05), improves the protein activity significantly (P0.05), simplifies the purification of the protein and reduces production costs significantly (P0.05).

[0078] The above test results show that plant system, especially lettuce system, is a more economical and efficient expression platform for rapid and transient expression of recombinant proteins, and can produce large-scale PD-1 and PD-L1 monoclonal antibodies in a short period of time.

[0079] The above application of a plant as a host for expressing a PD-1 antibody and/or a PD-L1 antibody according to the present invention is described in detail. The principles and embodiments of the present invention are set forth herein in terms of specific examples, and the description of the above embodiments is only to aid in understanding the method of the present invention and its core concepts. It should be noted that those skilled in the art can make various modifications and changes to the present invention without departing from the spirit and scope of the invention.