A nano antibody against PD-L1 protein and its preparation method and application

Abstract

The present invention provides a nano antibody against PD-L1 protein and a preparation method and application thereof. The amino acid sequence of the nano antibody is shown in SEQ ID NO: 1. The nano antibody can bind to PD-L1 with specificity and high affinity. Since the nano antibody has a small molecular weight, it can be better taken up into cells. Therefore, an ADC drug against PD-L1 can be prepared, further expanding the application potential of the antibody.

Description

Technical Field

[0001] The present invention belongs to the technical field of nano antibodies and relates to a nano antibody against PD-L1 protein and a preparation method and application thereof. Background Art

[0002] PD-L1 (Programmed Death Ligand 1) is a protein expressed on the surface of tumor cells and is a member of the B7 family. It is a single transmembrane protein that contains an extracellular cytoplasmic region, a transmembrane region, and an intracellular cytoplasmic region. The extracellular cytoplasmic region contains an N-terminal immunoglobulin-like region (IgV) for binding to PD-1. The intracellular cytoplasmic region contains multiple sites, including a C-terminal PDZ binding domain, which can interact with intracellular signal transduction pathways and participate in the regulation of immune cells. Studies have found that when PD-L1 on the tumor cell membrane binds to PD-1 on immune cells such as T cells, tumor cells send inhibitory signals, causing T cells to be unable to recognize tumor cells, and thus unable to kill tumor cells, the body's immune function is suppressed, and tumor cells grow wantonly. PD-L1 is expressed in many tumors, including melanoma, breast cancer, colorectal cancer, non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, cutaneous squamous cell carcinoma, and hematological tumors. Based on the importance of PD-L1 in tumors, exhausted T cells in the tumor immune microenvironment can be reactivated by blocking PD-1 / PD-L1 signals, restoring the activity of T cells, and enhancing the recognition and killing of tumor cells, thereby exerting anti-tumor effects and restoring the body's anti-tumor immune function. However, due to the heterogeneity of tumors and the complexity of the regulatory mechanism of PD-L1 expression, the clinical application of anti-PD-L1 monoclonal antibodies is greatly limited, and the overall objective response rate of patients is around 15%-30%. Given that PD-L1 is expressed in multiple tumor types and its expression in normal tissues is limited, combining anti-PD-L1 antibodies with cytotoxins to make ADCs targeting PD-L1 will be a very promising research and development direction. However, the PD-L1 antibodies currently on the market are all large molecule antibodies, which have poor permeability to tumors, so small molecule antibodies such as anti-PD-L1 nanoantibodies are needed to develop ADC drugs.

[0003] Nanobodies are the variable regions of heavy chain antibodies that naturally lack light chains and exist in camelids and sharks, discovered by Belgian scientists in 1993. They are also called single-domain antibodies (sdAb), or VHH antibodies. VHH crystals are 2.5nm, 4nm long, and have a molecular weight of only 15KDa, which is about 1 / 10 of the molecular weight of monoclonal antibodies. Therefore, they are called nanobodies (Nb). They are the smallest units known to bind to target antigens. Compared with ordinary four-chain antibodies and single-chain antibodies, nanobodies have the advantages of small molecular weight, high solubility, high stability, easy expression, ability to bind to gap epitopes, and low immunogenicity. Therefore, nanobodies have broad application prospects in the field of new drug development. Summary of the invention

[0004] The object of the present invention is to provide a nano antibody against PD-L1 protein and a preparation method and application thereof. The antibody has a molecular weight of about 14KDa, can specifically bind to PD-L1, and can be used to prepare an anti-PD-L1 ADC drug.

[0005] To achieve the above object, the technical solution adopted by the present invention is:

[0006] The present invention provides a nanobody against PD-L1 protein, the amino acid sequence of the nanobody is shown in SEQ ID NO: 1.

[0007] The present invention also provides a pharmaceutical composition comprising the above-mentioned nanobody.

[0008] The present invention also provides a method for preparing the above-mentioned nanobody, comprising:

[0009] 1) Immunize alpacas with PD-L1 protein three times, collect peripheral blood from alpacas, and separate lymphocytes;

[0010] 2) extracting total RNA from the lymphocytes, amplifying cDNA by reverse transcription, and then amplifying the cDNA by nested PCR to obtain the target gene VHH;

[0011] 3) connecting the target gene VHH to the vector and transforming it into competent cells to construct an anti-PD-L1 nanobody gene library;

[0012] 4) inoculating and culturing the constructed anti-PD-L1 nanobody gene library, adding helper phage for cultivation, purifying the phage, and obtaining the anti-PD-L1 nanobody display library;

[0013] 5) Screening the anti-PD-L1 nanobody display library obtained in step 4) to obtain positive clones and prepare nanobodies against PD-L1 protein.

[0014] Preferably, in step 2), the nested PCR has two rounds.

[0015] First round primers:

[0016] F1:GGTACGTGCTGTTGAACTGTTCC,

[0017] R1: CTTGGTGGTCCTGGCTGC;

[0018] The first round of PCR system is as follows:

[0019]

[0020] The reaction conditions were: 98°C for 2 min, 98°C for 10 s, 48°C for 20 s, 72°C for 1 min, 25 cycles;

[0021] Second round primers:

[0022] F2: ACTGTGGCCCAGGCGGCCCAGKTGCAGCTCGTGGAGTC,

[0023] R2-1: CTCGCGGCCGGCCTGGCCATGGGGGTCTTCGCTGTGGTGCG, R2-2: CTCGCGGCCGGCCTGGCCGTCTTGTGGTTTTGGTGTCTTGGG;

[0024] The second round of PCR system is as follows:

[0025]

[0026] The reaction conditions were: 98°C for 2 min, 98°C for 10 s, 60°C for 20 s, 72°C for 1 min, and 30 cycles.

[0027] Preferably, in step 3), the vector is pComb3XSS plasmid, and the competent cell is TG1 Escherichia coli.

[0028] Preferably, step 4) further comprises: adding the anti-PD-L1 nanobody gene library constructed in step 3) to LB medium, culturing at 37°C for 2.5 hours, adding helper phage, and culturing overnight at 30°C and 220rpm; the next day, centrifuging at 10000g for 10 minutes, taking the supernatant, adding PEG-NaCl solution, placing at 4°C for 8 hours, and then centrifuging, resuspending the precipitate with PBS to obtain the anti-PD-L1 nanobody display library.

[0029] Preferably, step 5) further comprises:

[0030] ① Add the biotinylated PD-L1 protein and the anti-PD-L1 nanobody display library obtained in step 4) to the EP tube, incubate at 37°C for 1 hour, then add magnetic beads containing streptomycin and incubate again for 30 minutes;

[0031] ②Put the EP tube on the magnetic stand, remove the solution in the tube, take it off the magnetic stand, add PBST solution and blow the magnetic beads evenly with a pipette, repeat this step, and wash 3 times with PBST;

[0032] ③After washing three times with PBST, add the magnetic beads to TG1 E. coli, culture at 37°C for 2.5 hours, take part of the bacterial solution and apply it to the ampicillin-resistant plate to measure the first round of elution titer, and add the rest of the bacterial solution to LB medium and helper phage, culture at 30°C, 220rpm overnight;

[0033] ④ Centrifuge on the next day, take the supernatant, add PEG-NaCl solution, place at 4°C for 8 hours, then centrifuge, resuspend the precipitate with PBS to obtain the first round of screening display library of anti-PD-L1 nanoantibodies, and repeat three rounds of screening;

[0034] ⑤ Pick a single clone from the plate measuring the elution titer of the third round of screening and add it to LB medium. Culture at 37°C for 2.5 hours, then add helper phage and culture overnight at 30°C, 220rpm. Centrifuge on the next day, take the supernatant, and screen the nanoantibody against PD-L1 protein through Elisa affinity detection.

[0035] The present invention also provides the use of the above-mentioned nanobody in the preparation of an anti-PD-L1 ADC drug.

[0036] In the present invention, an anti-PD-L1 nanobody is prepared, which can bind to PD-L1 with specificity and high affinity. Because the nanobody has a small molecular weight, it can be better taken up into cells, so an anti-PD-L1 ADC drug can be prepared, further expanding the application potential of the antibody. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is a diagram of PD-L1 immune titer detection in an embodiment of the present invention.

[0038] Figure 2 The figure is an agarose gel electrophoresis diagram of RNA extracted in the embodiment of the present invention.

[0039] Figure 3 This is an agarose gel electrophoresis diagram of the gene obtained by two rounds of nested PCR in an embodiment of the present invention.

[0040] Figure 4 It is the ELISA result in the embodiment of the present invention.

[0041] Figure 5This is the SDS-PAGE gel electrophoresis diagram of the expressed P1 nanobody in the example of the present invention.

[0042] Figure 6 It is the ELSA result in the embodiment of the present invention.

[0043] Figure 7 This is a graph showing the binding force between PD-L1 and P1 nanobody in an embodiment of the present invention. DETAILED DESCRIPTION

[0044] In order to explain the present invention more clearly, the present invention is further described in detail below in conjunction with embodiments and with reference to the accompanying drawings. It should be understood by those skilled in the art that the content described below is illustrative rather than restrictive, and should not be used to limit the scope of protection of the present invention.

[0045] Example

[0046] 1. Alpaca immunization of PD-L1 protein and construction of antibody library

[0047] (1) Dissolve 500 μg of PD-L1 protein (Beijing Sino Biological Technology Co., Ltd., Catalog No.: 10084-H08H) in 500 μl of PBS and mix with 500 μl of Freund's complete adjuvant. Place the mixture on a mixer and shake vigorously to fully emulsify the antigen (the aqueous phase and the oil phase are not separated).

[0048] (2) Alpaca immunization: healthy adult alpacas were selected for immunization, mainly at the neck and back by subcutaneous injection for multiple immunizations, 8-10 points each time, each point was injected with about 0.1 mL of the antigen prepared in step (1), a total of three immunizations, each interval of 2 weeks, and peripheral blood of alpacas was collected before immunization and one week after each immunization to extract serum for immune titer detection. The results are as follows: Figure 1 As shown, one week after the third immunization, 30 ml of peripheral blood was collected to extract lymphocytes for antibody library construction.

[0049] (3) Lymphocytes were extracted from the peripheral blood of alpacas, and total RNA was extracted from the extracted lymphocytes. The extracted RNA was subjected to agarose gel electrophoresis to detect its integrity. The complete RNA should be three bands. Figure 2 Three bands can be clearly seen indicating that the extracted RNA is intact.

[0050] (4) The total RNA was reverse transcribed into cDNA, and the VHH gene was amplified through two rounds of PCR using the nested PCR method.

[0051] First round primers:

[0052] F1:GGTACGTGCTGTTGAACTGTTCC,

[0053] R1: CTTGGTGGTCCTGGCTGC.

[0054] Second round primers:

[0055] F2: ACTGTGGCCCAGGCGGCCCAGKTGCAGCTCGTGGAGTC

[0056] R2-1:CTCGGCGGCCGGCCTGGCCATGGGGGTCTTCGCTGTGGTGCG

[0057] R2-2:CTCGCGGCCGGCCTGGCCGTCTTGTGGTTTTGGTGTCTTGGG

[0058] High-fidelity enzyme (2×Phanta UniFi Master Mix) was purchased from vazyme with the product number P516.

[0059] The first round of PCR system is as follows:

[0060]

[0061] The reaction conditions were: 98°C for 2 min, 98°C for 10 s, 48°C for 20 s, 72°C for 1 min, for 25 cycles.

[0062] After the reaction, the product nucleic acid was recovered by electrophoresis and gel cutting. Figure 3 As shown, a smaller band was recovered from rubber tapping, with a quantitative value of 55 ng / μL, which was the first round PCR product. The second round of PCR was carried out using the first round PCR product as a template. The system is as follows:

[0063]

[0064] The reaction conditions were: 98°C for 2 min, 98°C for 10 s, 60°C for 20 s, 72°C for 1 min, and 30 cycles.

[0065] The second round of PCR products were recovered and the molecular weight was identified by nucleic acid electrophoresis. Figure 3 As shown, the size is about 500 bp.

[0066] (5) The second-round PCR product and pComb3XSS plasmid were digested with Sfi I, respectively, and the second-round product was 122 ng / μL, and the pComb3XSS plasmid was 186 ng / μL. 100 μL of the plasmid and 41 μL of the second-round product were mixed together, and 10 μL of ligase (Takara, catalog number 2011A), 20 μL of 10× Ligase buffer, and 29 μL of ultrapure water were added. The reaction was incubated at 16°C for 2 h.

[0067] (6) The ligation product in step (5) was recovered, and the final recovered product was eluted with 20 μL of ultrapure water and added to TG1 Escherichia coli electroporation competent cells (LGC, catalog number 60502-1), 5 μL was added to each tube for electroporation, and the transformed bacterial liquid was coated on an ampicillin-resistant plate. After incubation at 37°C for 12 h, the colonies were scraped and washed with 50 ml of LB medium to construct an anti-PD-L1 nanoantibody gene library.

[0068] (7) Take 10 ml of the anti-PD-L1 nanobody gene library constructed in step (6) and add it to 40 ml of LB medium. Incubate at 37°C for 2.5 h, then add 50 μl of helper phage and incubate at 30°C, 220 rpm overnight.

[0069] (8) The next day, centrifuge at 10,000 g for 10 min, take the supernatant, add 10 ml of PEG-NaCl solution, incubate at 4 °C for 8 h, then centrifuge, resuspend the precipitate with 1 ml of PBS to obtain the anti-PD-L1 nanobody display library.

[0070] 2. Screening of PD-L1 protein display library

[0071] (1) Add 2 μg of biotinylated PD-L1 protein and 0.5 ml of PD-L1 nanobody display library (from the aforementioned "I. Alpaca immunization of PD-L1 protein and construction of antibody library" step (8)) into a 1.5 ml EP tube, incubate at 37°C for 1 h, then add magnetic beads containing streptomycin and incubate again for 30 min.

[0072] (2) Place the EP tube on the magnetic rack. You can see that the magnetic beads are enriched and adsorbed to the tube wall. After removing the solution in the tube, take it off the magnetic rack and add 1 ml of PBST solution. Use a pipette to blow the magnetic beads evenly.

[0073] (3) Place the EP tube on the magnetic rack. You can see that the magnetic beads are enriched and adsorbed to the tube wall. After removing the solution in the tube, take the EP tube off the magnetic rack and add 1 ml of PBST solution. Use a pipette to blow the magnetic beads evenly. Repeat this operation and wash with PBST three times in total.

[0074] (4) After washing three times with PBST, the magnetic beads were added to 5 ml of TG1 Escherichia coli and cultured at 37°C for 2.5 h. 10 μl of the bacterial solution was then applied to an ampicillin-resistant plate to measure the first round of elution titer. The remaining bacterial solution was added to 45 ml of LB medium and 10 μl of helper phage and cultured overnight at 30°C, 220 rpm.

[0075] (5) The next day, centrifuge at 10,000 g for 10 min, take the supernatant, add 5 ml of PEG-NaCl solution at 4 °C, incubate for 8 h, and then centrifuge. Resuspend the precipitate with 1 ml of PBS to obtain the first round of screening display library of anti-PD-L1 nanoantibodies.

[0076] (6) The anti-PD-L1 nanobody first round screening display library obtained was repeated with the above screening steps to form the anti-PD-L1 nanobody second round screening display library, and the third round of screening was repeated again.

[0077] (7) Twenty-four single clones were selected from the plate used to measure the elution titer of the third round of screening and added to 1 ml of LB medium. The culture was incubated at 37°C for 2.5 h. After that, 1 μl of helper phage was added and the culture was incubated at 30°C, 220 rpm, overnight.

[0078] (8) On the next day, centrifuge at 10,000 g for 10 min, collect the supernatant, and number them 1-24.

[0079] (9) Dilute the PD-L1 protein to 5 μg / ml and coat the ELISA plate, with 100 μl coated per well.

[0080] (10) The next day, the PD-L1 protein solution on the ELISA plate was discarded and the plate was washed twice with PBST solution. 100 μl of skim milk was added to each well and the plate was incubated at 37°C for 2 h.

[0081] (11) Remove the skim milk and add 100 μl of the supernatant from step (8) to each well and incubate at 37°C for 1 h.

[0082] (12) After washing five times with PBST, add anti-M13 protein antibody conjugated with HRP (Beijing Sino Biological Technology Co., Ltd., catalog number 11973-MM05T-H) and incubate at 37°C for 1 h.

[0083] (13) After washing five times with PBST, TMB colorimetric solution was added and the cells were incubated at 37°C for 5 min before adding ELISA stop solution.

[0084] (14) Read the ELISA plate at OD450. Figure 4 As shown, the cloned bacterial solutions of No. 3, 5, 12, 19, and 22 were sent for sequencing to obtain a nanobody sequence, the sequence of which is

[0085] QVQLQESGGGLVQAGDSLRLSCTASELTFQDYNMGWFRQAPGKAREYVAGIS

[0086] RFGGVKDYAESVKGRFTVSRNNANNTVALRMSSLEPDDTAQYYCAGRRELRAHSIARASSDYDTWGQGTQVTVSS (SEQ ID NO: 1), named P1.

[0087] 3. Expression of P1 Nanobody

[0088] (1) The P1 nanobody sequence was synthesized into the pET28a plasmid, named P1-pET28a, and 2 ng of P1-pET28a was transformed into BL21 competent cells and cultured in an inverted kanamycin plate overnight.

[0089] (2) On the next day, a single clone was picked from the plate and placed in 5 mL of LB-kanana liquid medium. The culture was shaken at 37°C and 220 rpm for 8 h. Then, 1 L of LB-Amp liquid medium was added and the culture was shaken at 37°C and 220 rpm for 4 h.

[0090] (3) Add 1 mL of 0.1 M IPTG and culture overnight at 30°C and 180 rpm.

[0091] (4) The next day, the bacterial solution was collected by centrifugation and resuspended in 50 ml of PBS and then ultrasonically disrupted. The ultrasonic conditions were 150w, disruption for 10 seconds, and rest for 10 seconds.

[0092] (5) Collect the supernatant by centrifugation, pass it through a nickel column, then pass it through a nickel column with 20 ml of PBS solution containing 50 mM imidazole, then add 10 ml of PBS solution containing 300 mM imidazole and pass it through a nickel column to elute the nanoantibody, desalt the obtained nanoantibody solution, elute it with PBS, and obtain the P1 nanoantibody dissolved in PBS. Take 5 ug of the P1 nanoantibody and perform SDS-PAGE gel electrophoresis to detect its molecular weight. Figure 5 As shown, the molecular weight of P1 nanobody is around 14KDa.

[0093] 4. ELISA to identify the binding of P1 nanobody to PDL1

[0094] (1) Dilute BSA protein and PD-L1 protein to 2 μg and coat the plate with 100 μl per well, 20 wells per plate, and incubate at 4°C overnight.

[0095] (2) The next day, remove the protein solution from the ELISA plate, wash twice with PBST, add 100 μl of skim milk to each well, and incubate at 37°C for 2 h.

[0096] (3) Remove skim milk, add 20 μg / ml P1 nanobody solution to the first well of the coating, then dilute it in multiples and place it at 37°C for 1 h.

[0097] (4) After washing five times with PBST, add anti-His tag antibody with HRP (Abacm, catalog number ab1187) and incubate at 37°C for 1 h.

[0098] (5) After washing five times with PBST, TMB colorimetric solution was added and the cells were incubated at 37°C for 5 min before adding ELISA stop solution.

[0099] (6) OD 450 reading. The results are as follows Figure 6 The data showed that the data of the PD-L1 protein-coated wells formed a concentration-dependent curve with the added P1 nanoantibody, and the data of the BSA protein-coated wells showed that there was basically no binding with the P1 nanoantibody, proving that the P1 nano was specifically bound to PD-L1.

[0100] 5. Determination of the binding ability of P1 nanobody to PD-L1

[0101] (1) Prepare Fortebio Octet solution M (PBST solution containing 0.5% BSA).

[0102] (2) The PD-L1 protein was diluted to 10 μg / mL, and the P1 nanobody was diluted to 10 μg / mL, 5 μg / mL, 2.5 μg / mL, and 1.25 μg / mL.

[0103] (3) Place the biosensor in the corresponding clean well in the preparation box, and add 200 μL of Fortebio Octet special solution M to each well corresponding to the sensor.

[0104] (4) Set the program: the first stage is Baseline, 60 s, the second stage is Loading, 200 s, the third stage is Baseline2, 100 s, the fourth stage is Association, 300 s, and the fourth stage is Dissocition, 300 s. The reaction temperature of the whole process is 37 °C.

[0105] (5) After the reaction is completed, the data is analyzed using software. Figure 7 As shown, KD = 9.96 × 10 -10 M.

[0106] Obviously, the above embodiments of the present invention are merely examples to more clearly illustrate the present invention, and are not limitations on the implementation methods of the present invention. For ordinary technicians in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is impossible to list all the implementation methods here. All obvious changes or modifications derived from the technical solution of the present invention are still within the protection scope of the present invention.

Claims

1. A nanobody against PD-L1 protein, the amino acid sequence of the nanobody being as shown in SEQ ID NO:

1.

2. A pharmaceutical composition comprising the Nanobody according to claim 1.

3. Use of the nanobody according to claim 1 in the preparation of an anti-PD-L1 ADC drug.

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