An anti-PD-1 shark single-domain antibody or its antigen-binding fragment and its preparation method and application

Abstract

The present invention discloses an anti-PD-1 shark single-domain antibody or its antigen-binding fragment, and a preparation method and application thereof, relating to an anti-PD-1 shark single-domain antibody and its amino acid and nucleotide coding sequences, and also relates to a recombinant plasmid and recombinant expression bacteria containing the nucleotide coding sequence and their uses. It has higher affinity and other pharmacological properties, contributes to the improvement of medical level, is suitable for the preparation of drugs, and has broad market prospects.

Description

Technical Field

[0001] The present invention relates to anti-PD-1 shark single-domain antibodies, and more specifically, to anti-PD-1 shark single-domain antibodies and nucleotides thereof. The present invention also relates to recombinant plasmids and recombinant expression bacteria containing the nucleotide coding sequence, belonging to the field of single-domain antibody technology development. Background Art

[0002] Antibodies are immunoglobulins produced by plasma cells that differentiate into B cells in response to antigenic stimulation and can specifically bind to the corresponding antigen. Traditional antibodies have two variable domains, called VH and VL, which provide stability and binding specificity to each other.

[0003] In 1995, researchers such as Flajnik MF first isolated a homodimer similar to immunoglobulin heavy chain from the serum of nurse sharks. This homodimer is called immunoglobulin new antigen receptor (IgNAR). IgNAR is structurally different from IgW and IgM commonly found in fish. It is similar to HCABs in structure and naturally lacks Ig light chain structure. It has only two heavy chain structures, each heavy chain consists of 5 constant regions, 1 hinge region and 1 variable region. This type of antibody is also called single-domain antibody or nanobody.

[0004] The structural differences between single-domain antibodies and traditional antibodies determine their distinct physicochemical properties. Compared to traditional antibodies, single-domain antibodies possess smaller molecular weight, greater solubility, higher stability, lower immunogenicity, and higher antigen-binding activity. Therefore, single-domain antibodies have a wider range of applications than traditional antibodies. The PD-1 protein is an immunosuppressive molecule of the B7 / CD28 superfamily and a type I transmembrane protein with a relative molecular weight of approximately 52,500. The protein's clathrate contains immunoreceptor tyrosine-based inhibitory motifs and switch motifs. Various immune cells, including CD4+ and CD8+ T cells, B cells, natural killer T cells (NK T cells), dendritic cells (DCs), and monocytes / macrophages, express high levels of the PD-1 protein in response to inflammation or tumor stimulation. The PD-1 / PD-L1 signaling pathway functions to suppress immune cell activity. In cancer patients, tumor cells develop PD-1 / PD-L1 receptors on their cell membranes to evade recognition by the immune system and avoid being killed by cytotoxic T lymphocytes (CTLs). Antibodies specifically bind to PD-1 / PD-L1, blocking the PD-1 / PD-L1 signaling pathway and thereby suppressing the immune system's negative feedback mechanism, restoring the immune system's ability to kill tumor cells.

[0005] A large number of cancer immunotherapy drugs targeting PD-1 molecules have been successfully developed, but the development of single-domain antibodies with higher affinity and other pharmacological properties for the PD-1 target is still necessary and has important medical value. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a single-domain antibody against PD-1, and at the same time provide the coding sequence of the single-domain antibody, a plasmid containing the coding sequence, and a recombinant IgNAR protein having binding activity to the human and mouse homologous PD-1 protein.

[0007] In order to achieve the above object, the present invention is implemented through the following technical solutions:

[0008] The present invention provides an anti-PD-1 shark single-domain antibody or an antigen-binding fragment thereof, wherein the anti-PD-1 shark single-domain antibody or the antigen-binding fragment thereof comprises the variable region VNAR of the IgNAR protein, and the amino acid sequence of the variable region VNAR comprises SEQ ID NO: 2; the sequence of SEQ ID NO: 2 is

[0009] MEIVLTQPEAESGHPGATLRLTCKTSGFDLTSYWMYWVRQFPGQGLEWLLEYKSSSSSNYAPAIKNRFTASKDTSNNIFALEMTNLKIEDTAIYYCARYIQ.

[0010] Preferably, the sequence of the anti-PD-1 shark single domain antibody or its antigen-binding fragment is SEQ ID NO: 1, and the SEQ ID NO: 1 is

[0011] MIKSTIFLSLLLTFLSCVQSEIVLTQPEAESGHPGATLRLTCKTSGFDLTSYWMYWVRQFPGQGLEWLLEYKSSSSSNYAPAIKNRFTASKDTSNNIFALEMTNLKIEDTAIYYCARYIQ.

[0012] The present invention provides a nucleotide sequence encoding an anti-PD-1 shark single-domain antibody or an antigen-binding fragment thereof, comprising a nucleotide encoding a polypeptide, wherein the polypeptide comprises a shark single-domain antibody variable region VNAR; the variable region VNAR comprises SEQ ID NO: 1 or SEQ ID NO: 2, and the variable region VNAR can bind to human or mouse PD-1.

[0013] A preferred nucleotide sequence is SEQ ID NO: 3, wherein SEQ ID NO: 3 is

[0014] ATGGAGATAGTACTAACACAACCCGAGGCTGAATCAGGCCACCCGGGCGCTACCCTGCGTCTCACCTGTAAAACCAGCGGTTTTGATCTGACGAGCTACTGGATGTACTGGGTTCGTCAGTTCCCGGGTCAGGGTTTGGAGTGGCTGTTGGA GTATAAAAGCTCTAGCAGCAGCAATTACGCTCCGGCAATCAAGAACCGCTTCACCGCGAGCAAGGACACGTCCAACAACATTTTTGCGCTGGAAATGACCAATCTGAAAATCGAAGACACTGCGATTTATTACTGCGCCCGTTATATCCAA.

[0015] The invention discloses a recombinant plasmid containing a nucleotide sequence.

[0016] The invention discloses a recombinant expression bacterium containing a recombinant plasmid.

[0017] The present invention discloses an anti-PD-1 shark single-domain antibody and its use in the preparation of a PD-1 therapeutic antibody drug.

[0018] The present invention provides an application of a nucleotide sequence in the preparation of a PD-1 therapeutic antibody drug.

[0019] The present invention provides a method for preparing an anti-PD-1 shark single-domain antibody, characterized in that the preparation method comprises the following steps:

[0020] Step 1: preparing an expression vector containing a nucleotide sequence for expressing the anti-PD-1 shark single domain antibody or an antigen-binding fragment thereof;

[0021] Step 2: Construct a recombinant expression bacterium containing the expression vector of step 1;

[0022] Step 3: Cultivate the recombinant expression bacteria constructed in step 2;

[0023] Step 4: Isolate and purify to obtain the anti-PD-1 shark single domain antibody or its antigen-binding fragment.

[0024] Beneficial effects: Recombinant single-domain antibodies have a small molecular weight, so they have strong tissue penetration ability, can enter dense tissues, and can be quickly cleared; recombinant IgNAR protein is highly stable, and the unfolding of recombinant single-domain antibodies at high temperatures has been shown to be completely reversible. Unlike traditional antibody fragments, recombinant single-domain antibodies are also stable at extreme pH values and can survive in gastric juice; recombinant IgNAR protein has high antigen binding activity, and the CDR3 region of the single-domain antibody is relatively long, which can form an exposed convex loop structure, making its binding mode with the antigen more flexible. This structural feature allows the single-domain antibody to bind to the concave hidden epitope of the antigen; the antigen-binding fragment is a part of the antibody that retains the specific binding activity of the intact antibody, that is, any part of the antibody can specifically bind to the epitope on the target molecule of the intact antibody, which is suitable for the preparation of drugs and has broad market prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 SDS-PAGE electrophoresis of supernatant, precipitate and before disruption after ultrasonic disruption of bacterial sludge

[0026] Figure 2 Inclusion body SDS-PAGE electrophoresis

[0027] Figure 3 Nickel column purification SDS-PAGE electrophoresis

[0028] Figure 4 Purified protein samples were renatured by SDS-PAGE electrophoresis

[0029] Figure 5 After renaturation, protein was dialyzed and subjected to SDS-PAGE electrophoresis

[0030] Figure 6 Checkerboard titration double antibody sandwich ELISA to detect in vitro activity DETAILED DESCRIPTION

[0031] The effective amount of the present invention refers to the amount or dose of the product of the present invention (preferably an anti-PD-1 antibody or antigen-binding fragment thereof) that provides the desired treatment after single or multiple doses are administered to a patient, organ or individual.

[0032] The term "diagnosis" as used herein refers to determining whether a patient has had a disease or condition in the past, at the time of diagnosis, or in the future, or to determining the progression or possible future progression of a disease, or to evaluating a patient's response to treatment.

[0033] "Treatment" as used herein means to slow down, interrupt, prevent, control, stop, alleviate, or reverse the progression or severity of a sign, symptom, disorder, condition, or disease, but does not necessarily involve the complete elimination of all disease-related signs, symptoms, conditions, or disorders, and refers to therapeutic intervention that improves the signs, symptoms, etc. of a disease or pathological state after the disease has begun to develop.

[0034] The terms "comprising" or "including" as used herein are open-ended descriptions encompassing the specified components or steps described, as well as other specified components or steps that do not materially affect the technical effect. When used in this application to describe a protein or nucleic acid sequence, the protein or nucleic acid may consist solely of the sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, while still exhibiting the activities described herein.

[0035] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0036] The present invention is described in detail below with reference to specific embodiments, but the present invention is not limited to these embodiments. Any improvement or substitution based on the basic spirit of the present embodiment still falls within the scope of protection required by the claims of the present invention.

[0037] Example 1

[0038] Obtaining PD-1 single-domain antibody sequences

[0039] Sequence screening:

[0040] Sharks are immunized with PD-1 antigen (preferably mouse antigen), and transcriptome sequencing is performed on the spleen and lymphocytes of the immunized sharks. Mass spectrometry is performed on the serum of the immunized sharks, and RNA from the spleen and lymphocytes of the immunized sharks is extracted for RT-PCR to obtain a high-throughput database containing only the shark VNAR region. The transcriptome high-throughput sequencing results obtained are compared with the high-throughput database of the VNAR region, and sequences that are present in both databases and are present in high abundance are screened. On the other hand, the mass spectrometry results are compared with the high-throughput database of the shark VNAR region, and sequences that are present in both databases and are present in high abundance are screened. The VNAR variable region sequence SEQ ID NO: 1 is obtained by combining the two screening results. The IgNAR protein is a dimer, with each chain containing one variable region (VNAR) and five constant regions (CNAR).

[0041] Gene sequence synthesis:

[0042] A fragment of the amino acid sequence SEQ ID NO: 1 of the variable region of the IgNAR protein VNAR was selected, namely the amino acid sequence SEQ ID NO: 2, and the nucleotide sequence encoding SEQ ID NO: 2 was codon-optimized for expression in E. coli, with the preferred sequence being SEQ ID NO: 3. Restriction sites (preferably NcoI and XhoI) were added to both ends of the optimized gene for artificial synthesis, and the synthesized nucleotide sequence was SEQ ID NO: 4: CCATGGAGATAGTACTAACACAACCCGAGGCTGAATCAGGCCACCCGGGCGCTACCCTGCGTCTCACCTGTAAAACCAGCGGTTTTGATCTGACGAGCTACTGGATGTACTGGGTTCGTCAGTTCCCGGGTCAGGGTTTGGAGTGGCTGTTGG AGTATAAAAGCTCTAGCAGCAGCAATTACGCTCCGGCAATCAAGAACCGCTTCACCGCGAGCAAGGACACGTCCAACAACATTTTTGCGCTGGAAATGACCAATCTGAAAATCGAAGACACTGCGATTTATTACTGCGCCCGTTATATCCAACTCGAG.

[0043] The nucleotide sequence of the recombinant IgNAR protein is artificially synthesized, with an accurate sequence. It can be obtained in large quantities through fermentation, has a low production cost, is environmentally friendly, and can be produced in large quantities. The recombinant IgNAR protein can effectively bind to antigen proteins, successfully replacing or partially replacing immunoglobulin IgG. The recombinant IgNAR protein has a small molecular weight. Compared with traditional antibodies, the V region of the recombinant single-domain antibody has a molecular weight of only about 12kDa due to the lack of CDR2. This feature enables it to carry drugs into cells or penetrate the blood-brain barrier.

[0044] Implementation 2

[0045] Construction of recombinant expression plasmid:

[0046] The gene sequence A was inserted into the expression vector pET-22b by double enzyme digestion to obtain the pET-22b-IgNAR recombinant plasmid.

[0047] Example 3

[0048] Construction of recombinant expression bacteria:

[0049] 1) Prepare competent E. coli cells. The strain can be E. coli BL21 (DE3), Rosetta (DE3) or other expression bacteria, preferably Rosetta (DE3).

[0050] 2) Slowly add 2 μL of recombinant plasmid pET-22b-IgNAR (no more than 50 ng) into the ultra-clean hood and slowly stir with a pipette tip to mix evenly. Incubate on ice for 30 minutes.

[0051] 3) Heat shock the cells in a 42°C water bath for 90 seconds. Do not touch the competent cells during the heat shock. After the heat shock, transfer the cells to ice and incubate for 5 minutes.

[0052] 4) Add 1 mL of LB liquid medium preheated at 37°C to the clean bench and incubate in a 37°C shaker at 220 rpm for 1 hour to revive the competent cells.

[0053] 5) After recovery, centrifuge at room temperature at 10,000 rpm for 5 minutes.

[0054] 6) Discard 1 mL of supernatant in a clean bench, mix the remaining supernatant and precipitate, and aspirate 50 μL to spread on an ampicillin-resistant plate.

[0055] 7) Place the plate in a 37°C biochemical incubator and incubate upside down for about 12 hours.

[0056] 8) Use an inoculating loop to pick a single colony and inoculate it into 5 mL of LB liquid medium. Then add 5 μL of Amp and incubate in a shaker at 37°C and 220 rpm for 12 h.

[0057] 9) Sequencing confirmed that the transformation was successful, and the recombinant expression strain Rosetta-pET-22b-IgNAR was obtained.

[0058] Example 4

[0059] Inducible expression of recombinant proteins:

[0060] 1) The recombinant expression strain Rosetta-pET-22b-IgNAR was inoculated into LB medium containing Amp resistance at a ratio of 1:100 and cultured at 37°C and 220 rpm / min for 4 h.

[0061] 2) IPTG is further added to make the final IPTG concentration 0.1-0.5 mM, preferably 0.5 mM, and the culture is continued for 4-6 h (preferably 5 h).

[0062] The recombinant expressed protein was obtained, whose amino acid sequence was shown in SEQ ID NO: 5: MEIVLTQPEAESGHPGATLRLTCKTSGFDLTSYWMYWVRQFPGQGLEWLLEYKSSSSSNYAPAIKNRFTASKDTSNNIFALEMTNLKIEDTAIYYCARYIQLEHHHHHH.

[0063] Example 5

[0064] 1. The bacterial sludge collected by centrifugation was added to Lysis Buffer: 20mM Tris, 500mM NaCl, 1% TritonX-100, pH 7.4

[0065] The cells were ultrasonically disrupted, and the disrupted bacterial solution was centrifuged and the precipitate and supernatant were collected for protein gel electrophoresis. Recombinant IgNAR was expressed in the precipitate, such as Figure 1 shown.

[0066] 2. Collect the precipitate after bacterial cell disruption and dissolve it with Solubilization buffer (20mM Tris, 6M Gua, 10mM DTT, pH 8.0). Figure 2 shown.

[0067] 3. Purification: Follow the nickel column purification instructions and elute with elution buffer containing 20 / 50 / 100 / 250mM Imidazole (Elution buffer: 20mM Tris, 8MUrea, 500mM NaCl, 1mM DTT, 0 / 20 / 50 / 100 / 250mM Imidazole, pH 8.0). 20 / 50 / 100mM Imidazole is preferred. The results are as follows: Figure 3 The eluate containing 20 / 50 / 100 mM imidazole had a better elution effect, and the eluate containing 20 / 50 / 100 mM imidazole was collected.

[0068] 4. Renaturation: Add the collected urea and imidazole eluate to the renaturation solution at a volume ratio of 1:100. Centrifuge to collect the precipitate and supernatant, and perform protein electrophoresis. Figure 4 shown.

[0069] 5. Dialysis (dialysis conditions: molecular weight of dialysis bag 3kDa, 4℃, change the solution every 6 hours) Dialysis Buffer: 50mM Tris, 240mM NaCl, 0.4M L-Arginine, 0.5mM GSSG, 5mM GSH, 0.1% SKL, 1mM DTT, pH 8.0. The results are as follows Figure 5 shown.

[0070] Lyophilized to obtain recombinant IgNAR protein, i.e., anti-PD-1 single domain antibody

[0071] Example 6

[0072] In vitro activity assay:

[0073] Absorbance = OD450 - OD620

[0074] Absorbance of experimental group = experimental group (OD450-OD620)

[0075] Blank group absorbance = blank group (OD450-OD620)

[0076] Dilute the recombinant IgNAR protein to a concentration of 0.5 μg / mL using sodium carbonate-bicarbonate buffer (CBS, pH 9.6), and plate 100 μL per well. A sodium carbonate-bicarbonate buffer solution was used as a control. Incubate the plates at 4°C overnight. The next day, the plate was patted dry, washed once with TBST (pH 7.5), incubated with 5% BSA solution at 37°C for 1 hour, patted dry, and washed twice with TBST. For PD-1 protein binding, PD-1 protein was diluted to 0.2 μg / mL and 1 μg / mL with 5% BSA solution, respectively, and 100 μL was added to each well and bound at 37°C for 1 hour. Rabbit anti-PD-1 protein was used as the primary antibody, diluted 1:4000 with TBST, incubated at 37°C for 1 hour, patted dry, and washed three times with TBST. Goat anti-rabbit HRP-tagged antibody was used as the secondary antibody, diluted 1:10000 with TBST, incubated at 37°C for 1 hour, patted dry, washed three times with TBST, washed twice with ddH2O, and 100 μL of TMB colorimetric solution was added to each well. The reaction was carried out in the dark at room temperature for 15 minutes. After the reaction, 2 M H2SO4 was added to each well, and the absorbance of the sample at 450 nm and 620 nm was measured by a microplate reader.

[0077] Table 1

[0078]

[0079] The results are as follows Figure 6 As shown in the figure, when the concentration of single-domain antibody IgNAR was 0.5ug / mL and the concentration of antigen PD-1 protein was 0.2ug / mL and 1ug / mL, the absorbance of the experimental group (OD450-OD620) was greater than 1.0, the absorbance of the blank group (OD450-OD620) was less than 0.5, and the absorbance of the experimental group (OD450-OD620) / the absorbance of the blank group (OD450-OD620) was greater than 2.1. The test results were obvious and the test difference was significant, indicating that the single-domain antibody IgNAR and the antigen PD-1 protein had good binding.

[0080] Example 7

[0081] The present invention provides an anti-PD-1 single-domain antibody or an antigen-binding fragment thereof for use in antibody-drug conjugates and pharmaceutical carriers.

[0082] Example 8

[0083] The present invention provides an anti-PD-1 single domain antibody or an antigen-binding fragment thereof for use in a pharmaceutical composition.

[0084] Example 9

[0085] The present invention provides an anti-PD-1 single domain antibody or an antigen-binding fragment thereof for use in preparing a product for treating PD-1-related diseases.

[0086] Finally, it should be noted that the present invention is not limited to the above embodiments and may be subject to many variations. All variations that can be directly derived or imagined by a person skilled in the art from the disclosure of the present invention should be considered within the scope of protection of the present invention. Invention Name: An anti-PD-1 shark single-domain antibody or its antigen-binding fragment and its preparation method and application First applicant: Zhejiang Sci-Tech University Second applicant: Ningbo Yuchang Biotechnology Co., Ltd. SEQ ID NO: 1 is MIKSTIFLSLLLTFLSCVQSEIVLTQPEAESGHPGATLRLTCKTSGFDLTSYWMYWVRQFPGQGLEWLLEYKSSSSSNYAPAIKNRFTASKDTSNNIFALEMTNLKIEDTAIYYCARYIQ. SEQ ID NO: 2 is MEIVLTQPEAESGHPGATLRLTCKTSGFDLTSYWMYWVRQFPGQGLEWLLEYKSSSSSNYAPAIKNRFTASKDTSNNIFALEMTNLKIEDTAIYYCARYIQ SEQ ID NO: 3 is ATGGAGATAGTACTAACACAACCCGAGGCTGAATCAGGCCACCCGGGCGCTACCCTGCGTCTCACCTGTAAAACCAGCGGTTTTGATCTGACGAGCTACTGGATGTACTGGGTTCGTCAGTTCCCGGGTCAGGGTTTGGAGTGGCTGTTGGA GTATAAAAGCTCTAGCAGCAGCAATTACGCTCCGGCAATCAAGAACCGCTTCACCGCGAGCAAGGACACGTCCAACAACATTTTTGCGCTGGAAATGACCAATCTGAAAATCGAAGACACTGCGATTTATTACTGCGCCCGTTATATCCAA. SEQ ID NO: 4 is CCATGGAGATAGTACTAACACAACCCGAGGCTGAATCAGGCCACCCGGGCGCTACCCTGCGTCTCACCTGTAAAACCAGCGGTTTTGATCTGACGAGCTACTGGATGTACTGGGTTCGTCAGTTCCCGGGTCAGGGTTTGGAGTGGCTGTTGGAGTATAAAAGCTCTAGCAGCAGCAATTACGCTCCGGCAATCAAGAACCGCTTCACCGCGAGCAAGGACACGTCCAACAACATTTTTGCGCTGGAAATGACCAATCTGAAAATCGAAGACACTGCGATTTATTACTGCGCCCGTTATATCCAACTCGAG。 SEQ ID NO: 5 is MEIVLTQPEAESGHPGATLRLTCKTSGFDLTSYWMYWVRQFPGQGLEWLLEYKSSSSSNYAPAIKNRFTASKDTSNNIFALEMTNLKIEDTAIYYCARYIQLEHHHHHH。

Claims

1. An anti-PD-1 shark single domain antibody, characterized in that The anti-PD-1 shark single-domain antibody comprises the variable region VNAR of the IgNAR protein, and the amino acid sequence of the variable region VNAR is shown in SEQ ID NO: 2; the sequence of SEQ ID NO: 2 is MEIVLTQPEAESGHPGATLRLTCKTSGFDLTSYWMYWVRQFPGQGLEWLLEYKSSSSSNYAPAIKNRFTASKDTSNNIFALEMTNLKIEDTAIYYCARYIQ.

2. The anti-PD-1 shark single domain antibody according to claim 1, characterized in that: The sequence of the anti-PD-1 shark single domain antibody is SEQ ID NO: 1, and the SEQ ID NO: 1 is MIKSTIFLSLLLTFLSCVQSEIVLTQPEAESGHPGATLRLTCKTSGFDLTSYWMYWVRQFPGQGLEWLLEYKSSSSSNYAPAIKNRFTASKDTSNNIFALEMTNLKIEDTAIYYCARYIQ.

3. A nucleic acid encoding the anti-PD-1 shark single domain antibody according to claim 1 or 2. A recombinant plasmid comprising the nucleic acid of claim 3 .

5. A recombinant expression bacterium containing the recombinant plasmid according to claim 4.

6. A method for preparing an anti-PD-1 shark single domain antibody according to claim 1 or 2, characterized in that The preparation method comprises the following steps: Step 1: preparing an expression vector containing a nucleotide sequence for expressing the anti-PD-1 shark single domain antibody or an antigen-binding fragment thereof; Step 2: Construct a recombinant expression bacterium containing the expression vector of step 1; Step 3: Cultivate the recombinant expression bacteria constructed in step 2; Step 4: Isolate and purify to obtain the anti-PD-1 shark single domain antibody or its antigen-binding fragment.

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