CN118085093B

CN118085093B - Agonist type anti-human PD-1 antigen binding polypeptide and application thereof

Info

Publication number: CN118085093B
Application number: CN202410512477.1A
Authority: CN
Inventors: 刘强; 常国强
Original assignee: Airport Hospital Of Tianjin Medical University General Hospital
Current assignee: Airport Hospital Of Tianjin Medical University General Hospital
Priority date: 2024-04-26
Filing date: 2024-04-26
Publication date: 2024-07-02
Anticipated expiration: 2044-04-26
Also published as: CN118085093A

Abstract

The invention discloses an excited anti-human PD-1 antigen binding polypeptide and application thereof, wherein the antigen binding polypeptide is an anti-human PD-1 nano antibody and comprises a CDR1, a CDR2 and a CDR3; the amino acid sequence of CDR1 is SEQ ID No:1, amino acid sequence of CDR2 is SEQ ID No:4, amino acid sequence of CDR3 is SEQ ID No:7, preparing a base material; or CDR1 has the amino acid sequence of SEQ ID No:2, amino acid sequence of CDR2 is SEQ ID No:5, amino acid sequence of CDR3 is SEQ ID No:8, 8; or CDR1 has the amino acid sequence of SEQ ID No:3, amino acid sequence of CDR2 is SEQ ID No:6, amino acid sequence of CDR3 is SEQ ID No:9. the antigen binding polypeptide has the advantages of simple humanization, high affinity, high stability, microbial expression, low immunogenicity, good solubility, strong penetrability, capability of recognizing hidden epitopes and the like, and has excellent application prospect in treating or preventing autoimmune diseases.

Description

Agonist type anti-human PD-1 antigen binding polypeptide and application thereof

Technical Field

The invention relates to the technical field of biological medicines, in particular to an excited anti-human PD-1 antigen binding polypeptide and application thereof.

Background

The PD-1 is called as programmed death receptor-1, the PD-1/PD-L1 pathway can regulate immunocompetence, effectively maintain T cell activation, realize balance between immune tolerance and tissue injury, and prevent autoimmune reaction and chronic infection. A variety of therapeutic antibodies targeting PD-1 and/or PD-L1 have been developed, with three of the most common drugs approved for sale at home and abroad: PD-1 mab opdivo, keystuda and PD-L1 mab TECENTRIQ are widely used in a variety of cancers such as Hodgkin's lymphoma, melanoma, lung cancer, head and neck squamous cell carcinoma. However, the antibodies are all inhibitors of PD-1/PD-L1, and through inhibiting the combination of PD1 and PD-L1 in a cancer patient, the identification of T cells in the patient to tumor cells is activated, so that the effect of killing tumors is achieved, and the treatment effect is required to be further improved.

The antibody of the present invention is an agonist antibody, and is completely different from the above antibody in action mechanism, and can specifically bind to and activate PD-1 receptors on T cells and B cells, activate PD-1/PD-L1 signaling pathway of T, B cells, inhibit T, B cell activation, induce apoptosis, disability and exhaustion of T cells and B cells, thereby regulating immune response of autoimmune diseases of nervous system, and treat autoimmune diseases of central nervous system typified by neuromyelitis optica. At present, the PD-1 excited antibody has no corresponding patent in China, the foreign patent has a PD-1 excited antibody, but the foreign patent antibody is a monoclonal antibody, the antibody is a nano antibody, and the antibody has the advantages of the nano antibody, such as small molecular weight, high humanization degree, excellent antigen binding specificity and the like, and does not occupy the binding site of PD-L1 and PD-1, does not influence the binding of PD-1 and PD-L1 in a physiological state, thus being easier to cooperate with PD-L1, enhancing the effect of inhibiting T, B cell activation and having better therapeutic effect.

Disclosure of Invention

In view of the above-mentioned drawbacks or shortcomings in the prior art, it is desirable to provide an agonist anti-human PD-1 antigen binding polypeptide and its use, which has the advantages of simple humanization, high affinity, high stability, microbial expression, low immunogenicity, good solubility, strong penetrability, recognizable hidden epitopes, and the like.

The invention provides an excited anti-human PD-1 antigen binding polypeptide, which is an anti-human PD-1 nanometer antibody and comprises a CDR1, a CDR2 and a CDR3;

the amino acid sequence of CDR1 is SEQ ID No:1, the amino acid sequence of the CDR2 is SEQ ID No:4, the amino acid sequence of the CDR3 is SEQ ID No:7, preparing a base material;

Or the amino acid sequence of the CDR1 is SEQ ID No:2, the amino acid sequence of the CDR2 is SEQ ID No:5, the amino acid sequence of the CDR3 is SEQ ID No:8, 8;

Or the amino acid sequence of the CDR1 is SEQ ID No:3, the amino acid sequence of the CDR2 is SEQ ID No:6, the amino acid sequence of the CDR3 is SEQ ID No:9.

Further, the anti-human PD-1 nanobody comprises FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 which are sequentially arranged.

Further, the amino acid sequence of the FR1 is SEQ ID No: 10. SEQ ID No:11 or SEQ ID No:12, any one of which is shown in the figure;

The amino acid sequence of the FR2 is SEQ ID No: 13. SEQ ID No:14 or SEQ ID No:15, or 15;

The amino acid sequence of the FR3 is SEQ ID No: 16. SEQ ID No:17 or SEQ ID No:18, respectively;

the amino acid sequence of FR4 is shown as SEQ ID No: 19.

In addition, the invention also provides an isolated nucleic acid molecule comprising the nucleotide sequence of the antigen binding polypeptide, wherein the nucleotide sequence is shown in SEQ ID No: 20. SEQ ID No:21 or SEQ ID No:22, as shown in any of the figures.

In addition, the invention also provides a recombinant expression vector comprising the isolated nucleic acid molecule.

In addition, the invention also provides a host cell comprising the isolated nucleic acid molecule or the recombinant expression vector; the host cell is a mammalian cell.

In addition, the invention also provides a pharmaceutical composition comprising the antigen binding polypeptide and a pharmaceutically acceptable carrier.

In addition, the invention also provides a fusion protein, which is a chimeric antigen receptor, and the extracellular domain of the chimeric antigen receptor comprises the antigen binding polypeptide; the chimeric antigen receptor also includes a transmembrane domain and an intracellular domain.

In addition, the invention also provides an application of the excited anti-human PD-1 antigen binding polypeptide in preparing medicaments, which comprises an application of the antigen binding polypeptide, the isolated nucleic acid molecule, the recombinant expression vector or the host cell in preparing medicaments; the medicament is useful for treating or preventing autoimmune diseases.

Compared with the prior art, the invention has the beneficial effects that:

The anti-human PD-1 antigen binding polypeptide has the advantages of excellent antigen binding specificity, capability of specifically activating a PD-1 signal path and no influence on the combination of PD-L1 and PD-1 in a human body while having the excellent performance of a nano antibody, such as small molecular weight and high humanization degree. Has the following advantages:

(1) The agonist anti-human PD-1 antigen binding polypeptide can specifically bind to human PD-1, and does not influence the binding of PD-1 and PD-L1 in vivo, so that the agonist activity of the anti-PD-1 antibody has a superposition effect.

(2) The active anti-human PD-1 antigen binding polypeptide provided by the invention specifically binds to human PD-1, and can activate a PD-1 signal path and inhibit T cell activation.

(3) Compared with monoclonal antibodies, particularly chimeric antibodies, the anti-human PD-1 antigen binding polypeptides of the invention have higher safety and lower immunogenicity.

(4) The anti-human PD-1 antigen binding polypeptide provided by the invention has small molecular weight, so that the anti-human PD-1 antigen binding polypeptide has extremely strong tissue penetration force, can directly penetrate through certain in-vivo barriers, and is beneficial to diagnosis and targeted treatment of autoimmune diseases.

It should be understood that the description in this summary is not intended to limit the critical or essential features of the embodiments of the invention, nor is it intended to limit the scope of the invention. Other features of the present invention will become apparent from the description that follows.

Drawings

Other features, objects and advantages of the present invention will become more apparent upon reading of the detailed description of non-limiting embodiments, made with reference to the accompanying drawings in which:

FIG. 1 is a graph of the effective concentration (EC 50 value) of binding of anti-human PD-1 nanobodies and control antibodies to PD-1 as determined by ELISA.

FIG. 2 is a schematic of affinity of anti-human PD-1 nanobodies and control antibodies to CHO cells over-expressing PD-1 on their surfaces at high concentrations (10 ug/ml) as measured by flow cytometry.

FIG. 3 is a schematic of affinity of anti-human PD-1 nanobodies and control antibodies to CHO cells over-expressing PD-1 on their surface at low concentrations (1.11 ug/ml) as measured by flow cytometry.

FIG. 4 is a graph showing the detection inhibition concentration (IC ₅₀ value) of the T cell activation inhibition function activity by the anti-human PD-1 nanobody and the control antibody measured by ELISA.

FIG. 5 is a graph showing the binding of candidate antibody 2-I-G8 epitope detection-PD 1/PDL 1.

FIG. 6 is a graph showing the binding of candidate antibody 2-G-F1 epitope detection-PD 1/PDL 1.

FIG. 7 is a graph showing the binding of candidate antibody 2-I-F6 epitope detection-PD 1/PDL 1.

FIG. 8 is a graph of candidate antibody 2-I-G8 affinity detection.

FIG. 9 is a graph of candidate antibody 2-I-F6 affinity detection.

FIG. 10 is a graph showing affinity detection of candidate antibody 2-G-F1.

Detailed Description

The invention is described in further detail below with reference to the drawings and examples. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting of the invention. It should be noted that, for convenience of description, only the portions related to the invention are shown in the drawings.

It should be noted that, without conflict, the embodiments of the present invention and features of the embodiments may be combined with each other. The invention will be described in detail below with reference to the drawings in connection with embodiments.

The embodiment of the invention provides an agonistic anti-human PD-1 antigen binding polypeptide, wherein the antigen binding polypeptide is an anti-human PD-1 nano antibody and comprises a CDR1, a CDR2 and a CDR3;

The amino acid sequence of CDR1 is SEQ ID No:1, amino acid sequence of CDR2 is SEQ ID No:4, amino acid sequence of CDR3 is SEQ ID No:7, preparing a base material;

Or CDR1 has the amino acid sequence of SEQ ID No:2, amino acid sequence of CDR2 is SEQ ID No:5, amino acid sequence of CDR3 is SEQ ID No:8, 8;

Or CDR1 has the amino acid sequence of SEQ ID No:3, amino acid sequence of CDR2 is SEQ ID No:6, amino acid sequence of CDR3 is SEQ ID No:9

SEQ ID No:1 is:

GRTFNNKS；

SEQ ID No:2 is:

GSFFEIYH；

SEQ ID No:3 is:

EFTLNYYA；

SEQ ID No:4 is:

ITWSGNT；

SEQ ID No:5 is:

ITSDGVT；

SEQ ID No:6 is:

ISNSGSRT；

SEQ ID No:7 is:

AADRNPWEIINTKTDYYES；

SEQ ID No:8 is:

NFRSGLNDY；

SEQ ID No:9 is:

AADFKCSWTSNWAESVQY。

In a preferred embodiment, the anti-human PD-1 nanobody comprises FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 arranged in sequence.

In a preferred embodiment, the amino acid sequence of FR1 is SEQ ID No: 10. SEQ ID No:11 or SEQ ID No:12, any one of which is shown in the figure;

SEQ ID No:10 is:

DVQLVESGGGLVQPGGSLRLSCAAS；

SEQ ID No:11 is:

QVQLVESGGGLVQPGGSLRLSCVVS；

SEQ ID No:12 is:

QVQLVESGGGLVQAGGSLRLSCAAS；

the amino acid sequence of FR2 is SEQ ID No: 13. SEQ ID No:14 or SEQ ID No:15, or 15;

SEQ ID No:13 is:

MAWFRQAPGKEREFVAG；

SEQ ID No:14 is:

MGWYRQNSGKEREFFAL；

SEQ ID No:15 is:

IVWFRQAPGKEREEISC；

The amino acid sequence of FR3 is SEQ ID No: 16. SEQ ID No:17 or SEQ ID No:18, respectively;

SEQ ID No:16 is:

GYSHSMKDRSTISRDNAKTTVYLQMNRLVPEDTAVYYC；

SEQ ID No:17 is:

NYVPYVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC；

SEQ ID No:18 is:

SYAHSVKDRFSISRDNAKSTVYLQMNSLKPEDTAVYYC；

The amino acid sequence of FR4 is shown as SEQ ID No: 19;

SEQ ID No:19 is:

WGQGTQVTVSS。

In addition, the embodiment of the invention also provides an isolated nucleic acid molecule, which comprises the nucleotide sequence of the antigen binding polypeptide, wherein the nucleotide sequence is shown in SEQ ID No: 20. SEQ ID No:21 or SEQ ID No:22, respectively;

SEQ ID No:20 is:

GATGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGCGCAGCCTCTGGACGCACCTTCAACAACAAATCCATGGCCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTCGCAGGTATTACTTGGAGTGGGAACACTGGCTATTCACACTCCATGAAAGACCGATCCACCATCTCCAGAGACAACGCCAAGACTACGGTGTATCTGCAAATGAACAGGCTGGTACCTGAGGACACGGCCGTCTATTACTGCGCTGCAGATCGGAACCCATGGGAAATTATCAACACAAAGACTGATTACTATGAGTCGTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA;

SEQ ID No:21 is:

CAGGTACAGCTGGTGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGTAGTGTCTGGAAGTTTCTTCGAAATCTATCACATGGGCTGGTACCGCCAGAATTCAGGGAAGGAGCGCGAGTTCTTCGCACTGATTACTAGTGATGGTGTGACAAACTATGTGCCCTACGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTCTATTACTGTAATTTCAGGAGTGGACTGAATGACTATTGGGGTCAGGGAACCCAGGTCACCGTCTCCTCA;

SEQ ID No:22 is:

CAGGTACAGCTGGTGGAGTCTGGGGGAGGCTTGGTGCAGGCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGAATTCACTTTAAATTATTATGCCATAGTCTGGTTCCGCCAGGCCCCAGGGAAGGAGCGTGAGGAGATCTCATGTATTAGTAATAGTGGTAGTAGGACAAGCTATGCACACTCCGTGAAGGACCGATTCAGTATCTCCAGAGACAACGCCAAGAGCACCGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACAGCCGTTTATTACTGTGCAGCAGATTTTAAGTGTTCGTGGACGTCGAATTGGGCCGAGTCTGTACAGTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA.

In addition, the embodiment of the invention also provides a recombinant expression vector, which comprises the isolated nucleic acid molecule; recombinant expression vectors may contain promoters, ribosome binding sites for translation initiation, transcription terminators and other regulatory elements; the promoter may be a promoter suitable for expression in a producer cell.

In addition, embodiments of the present invention also provide a host cell comprising the isolated nucleic acid molecule described above or the recombinant expression vector described above; the host cell is a mammalian cell; host cells can be used for the production of antigen binding polypeptides; host cells can be transiently or non-transiently transfected with the expression vectors of the invention; the host cell is selected from CHO or a derived cell line thereof, 293T or a derived cell line thereof.

In some embodiments, the antigen binding polypeptide is a single domain antibody (VHH) or a heavy chain antibody.

In some embodiments, the antigen binding polypeptide is a humanized antibody and/or a fully human antibody.

In some embodiments, the antigen binding polypeptide is an antibody fragment, e.g., fv, fab, fab ', scFv, diabody, or F (ab') ₂.

In some embodiments, the antigen binding polypeptide is a multispecific antibody, such as a bispecific antibody or a trispecific antibody.

In some embodiments, the antigen binding polypeptide is a VHH antibody.

In some embodiments, the antigen binding polypeptide is a VHH-Fc antibody in which a VHH is fused to an Fc region of a human immunoglobulin, wherein the human immunoglobulin is preferably IgG, more preferably IgG1.

In addition, the embodiment of the invention also provides a pharmaceutical composition, which comprises the antigen binding polypeptide and a pharmaceutically acceptable carrier.

In addition, the embodiment of the invention also provides a fusion protein, wherein the fusion protein is a Chimeric Antigen Receptor (CAR), and an extracellular domain of the chimeric antigen receptor comprises the antigen binding polypeptide; chimeric antigen receptors also include transmembrane and intracellular domains.

In some embodiments, the transmembrane domain of the CAR may be from a transmembrane domain commonly used in the art, e.g., from a transmembrane domain of a T cell costimulatory molecule.

In some embodiments, the intracellular signaling domain of the CAR may comprise an immunoreceptor tyrosine-based activation motif (ITAM).

In some embodiments, the intracellular signaling domain may comprise an intracellular domain selected from the group consisting of: fcγ receptor (fcγr), fcε receptor (fcεr), fcα receptor (fcαr), neonatal Fc receptor (FcRn)、CD3、CD3ζ、CD3γ、CD3δ、CD3ε、CD4、CD5、CD8、CD21、CD22、CD28、CD32、CD40L(CD154)、CD45、CD66d、CD79a、CD79b、CD80、CD86、CD278( also known as ICOS), CD247 ζ, CD247 η, DAP10, DAP12, FYN, LAT, lck, MAPK, MHC complex, NFAT, NF- κ B, PLC- γ, iC3b, C3dg, C3d and Zap70.

In some embodiments, the CAR may further comprise a co-stimulatory domain.

In some embodiments, the co-stimulatory domain may comprise the following signaling domains: MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activating molecules (SLAM proteins), activating NK cell receptors or Toll ligand receptors.

In addition, the embodiment of the invention also provides an application of the agonist anti-human PD-1 antigen binding polypeptide in preparing a medicament, which is characterized by comprising the application of the antigen binding polypeptide, the isolated nucleic acid molecule, the recombinant expression vector or the host cell in preparing the medicament; the medicine is used for treating or preventing autoimmune diseases.

The antigen binding polypeptides or anti-human PD-1 nanobodies of the invention are capable of specifically binding to PD-1 with high affinity, thereby activating the signaling pathway of PD-1/PD-L1, and are therefore particularly suitable for use in the treatment of autoimmune diseases and other related diseases.

In some embodiments, the antigen binding polypeptides or anti-human PD-1 nanobodies of the invention are used to treat a subject suffering from an autoimmune disease. For example, a subject with an autoimmune disease has an elevated level of PD-1 expression as compared to T cells in a healthy subject.

The subject is a vertebrate, preferably a mammal, such as a human. Mammals include, but are not limited to, mice, apes, humans, farm animals, sports animals, and pets. The subject is to be understood in a broad sense to also encompass tissues, cells, and their progeny of a biological entity obtained in vivo or cultured in vitro.

The antigen binding polypeptides, anti-human PD-1 nanobodies or fusion proteins of the invention may be included in a pharmaceutical composition. The pharmaceutical compositions may be used for prophylactic and/or therapeutic treatment.

In therapeutic applications, the pharmaceutical compositions of the invention may be administered to a subject already suffering from a disease in an amount sufficient to cure or at least partially arrest the progression of symptoms of the disease. The amount effective for treatment may vary depending on the severity of the disease, the course of the disease, past therapy, the health status of the subject, body weight, response to the drug, and judgment of the treating physician. The antigen binding polypeptides, anti-human PD-1 nanobodies or fusion proteins of the invention can be used in combination with other therapeutic agents, e.g., sequentially or simultaneously.

In prophylactic applications, the pharmaceutical compositions of the present invention may be administered before, during or after the onset of the disease. For example, the pharmaceutical composition of the present invention may be used as a prophylactic agent in order to prevent occurrence of diseases.

The antigen binding polypeptides, anti-human PD-1 nanobodies, fusion proteins or pharmaceutical compositions of the invention may be administered by injection, e.g., direct injection, stereotactic injection, injection by micropump infusion system. Injection may be performed by intravenous, parenteral, intraperitoneal and/or subcutaneous routes to deliver the antigen binding polypeptide, anti-human PD-1 nanobody, fusion protein or pharmaceutical composition to cells, tissues or organs of a subject.

Example 1

Obtaining anti-human PD-1 nanobodies by phage display

In this example, a variety of antibody-encoding fragments were obtained by immunizing alpaca with human PD-1 as antigen, and candidate antibody clones were subsequently screened by phage library and mammalian expression system. Specifically, specific steps of the screening process for anti-human PD-1 nanobodies are shown below.

A. Preparation of antigens

According to the amino acid sequence and nucleotide sequence of human PD-1, analyzing and designing an antigen capable of effectively inducing alpaca to generate a specific antibody aiming at human PD-1, connecting His at the C end of the antigen to obtain a modified antigen, and marking the modified antigen as a human PD-1-His antigen, wherein the amino acid sequence is shown as SEQ ID No:23, the nucleic acid sequence is shown as SEQ ID No: shown at 24.

B. Immune alpaca

And d, completely and uniformly mixing the human PD-1-His antigen obtained in the step a with an equal volume of Freund's adjuvant, and performing subcutaneous injection on the alpaca.

Specifically, alpaca was primed with 250 μg of an emulsified mixture of Human PD-1 hFC antigen and an equal volume of freund's complete adjuvant on day 1 and 3 boosts with 250 μg of an emulsified mixture of Human PD-1 hFC antigen and an equal volume of freund's complete adjuvant on days 14, 28, 42, respectively. After 7 days of booster immunization, 5ml alpaca peripheral blood was taken separately to detect anti-PD-1 serum titers in the blood by ELISA.

The specific procedure for ELISA detection is as follows. The Human PD-1 his antigen was diluted to 1 μg/mL with 0.05M carbonate buffer (ph=9.6) and added to the culture plate in an amount of 100 μl/well. The plates were coated overnight at 4 ℃. The coating solution was discarded and washed 5 times with PBST. 3% skimmed milk 200 [ mu ] L is added into each hole, and the mixture is sealed for 2 hours at 37 ℃. Wash 5 times with PBST buffer. 100. Mu.L/well of serum dilution (dilution of 1:1000 ratio) was added and incubated for 1h at 37 ℃. Wash 5 times with PBST, add 100. Mu.L of anti-alpaca IgG (HRP anti-Llama IgG) (H+L) antibody (Novus Co., ltd., cat#: NB7242, diluted 1:50000 with PBS) to each well and incubate at room temperature for 1H. Plates were washed 5 times with PBST. TMB chromogenic solution was added for development (100. Mu.L/well) and incubated at room temperature for 10-15min. The reaction was stopped by addition of stop solution (50. Mu.L/well) and the optical density was measured at 450 nm.

After 2 weeks of immunization 4, alpaca peripheral blood was collected 50mL and mononuclear cells were isolated.

C. Library construction

Extracting RNA of the PBMC obtained in the step b, and obtaining target gene fragments through nested PCR after reverse transcription. Cloning the target gene fragment into eukaryotic expression vectors, and transforming the obtained expression vectors into competent cells to construct a PD-1-VHH phage display library. The specific steps are as follows.

Using PBMC cells obtained by the screening in step B as a template, total cellular RNA was extracted using Trizol and reverse transcribed into cDNA using PRIMESCRIPT ™ II 1st Strand cDNA Synthesis Kit kit (TaKaRa, cat#: 6210B). The fragment of interest was obtained by nested PCR. The amplification system of the target fragment is shown in Table 1 below, and the amplification procedure is shown in Table 2 below.

TABLE 1 nest type PCR one-round reaction system

TABLE 2 nest type PCR two-round reaction system

The amplified two rounds of PCR products were added with recombinant arms and cloned into yeast display vector PYDISPLAY (akaco biotechnology (su) limited) and transformed into yeast competent cells by electric shock to obtain a yeast display library. The yeast display library is subjected to one round of magnetic bead sorting and two rounds of sorting, yeast liquid after sorting is coated on an SDCAA plate, monoclonal culture is selected, after induction and expression are carried out for 48 hours, the yeast liquid is incubated with Biotin-antigen and positive antibodies, and secondary antibodies are subjected to flow detection by using APC-strepitavidin (eBioscience, CAT#: 17-4317-82) and PE anti-human IgG (Invitrogen, cat#: 12-4998-82). Yeast clones bound to the target antigen were resuspended using 0.2% SDS, incubated at 95℃for 10min for lysis, the supernatant was centrifuged to obtain 0.5. Mu.L of the bacterial supernatant as a template for PCR amplification (remaining bacterial solution was stored at-20 ℃).

D. Nanobody acquisition

Panning using the yeast display library from step c and selecting positive clones. The coding sequences of the selected VHH antibodies are fused to the Human IgG1 Fc coding sequences and constructed into antibody expression vectors to transfect eukaryotic cells for expression and purification. Finally, the anti-human PD-1 nanobody is obtained. The specific method is as follows.

Based on the FACS detection result of the candidate antibody, positive clones were selected and antibody expression was performed.

Taking out LVTransm transfection reagent and pcDNA3.4-human IgG1Fc antibody expression vector from the refrigerator, thawing at room temperature, and blowing up and down with a pipette to mix completely. The PBS buffer was removed and warmed to room temperature. Taking 2mL of PBS to one hole of a 6-hole plate, respectively adding 20 mug antibody expression vector, blowing up and down by a pipette, fully and uniformly mixing, adding 60 mu L LVTRANSM, immediately blowing up and down by the pipette, uniformly mixing, and standing for 10 minutes at room temperature.

The DNA/LVTransm complex was added to 20mL of 293F cells and mixed thoroughly with gentle shaking. The cells were placed in an incubator at 37℃with 5% CO ₂ and 130rpm for continued culture.

After continuous cultivation for 5-7 days, the culture supernatant was collected by centrifugation, filtered with a 0.45 μm filter membrane, and the filtrate was transferred to a sterile centrifuge tube and the antibody was purified using a Protein A column. After purification, 3 nano antibodies are obtained, which are respectively named as 2-G-F1, 2-I-F6 and 2-I-G8.

The VHH amino acid sequences and nucleotide sequences of nanobodies 2-G-F1, 2-I-F6 and 2-I-G8 are shown below.

TABLE 3 sequence information of anti-human PD-1 nanobodies

EXAMPLE 2 ELISA detection of binding of recombinant anti-human PD-1 antibody to target protein

1) The recombinant protein was diluted with sterile CBS to a final concentration of 1. Mu.g/mL, a new 96-well ELISA plate was taken, 100. Mu.L was added to each well and coated overnight at 4 ℃.

2) The antigen coating was removed and washed 5 times with PBST (0.05% tween 20).

3) 3% MPBS added at 200. Mu.L/well was blocked at 37℃for 2h;

4) After removal of the blocking buffer, the well plate was washed 5 times with PBST;

5) Adding 3 anti-PD-1 nanobodies 2-G-F1, 2-I-F6 and 2-I-G8 obtained in example 1, and incubating at room temperature for 1h with 50 ul/well of transfection supernatant or purified antibody (initial concentration 10 [ mu ] G/mL, 3-fold gradient dilution of 7 spots, 100 [ mu ] L/well), negative control well of PBS; meanwhile, anti-human PD-1 antibodies PD1B849 and PD1B878 (akacon biotechnology (su state) limited) were used as positive control antibodies;

6) Remove the liquid in the wells and wash 5 times with PBST;

7) Add 100. Mu.L/well HRP-Protein A antibody (1:50000 dilution), incubate at room temperature for 1h;

8) After removing the liquid from the wells, the well plate was washed 5 times with PBST;

9) Adding 100 mu L/hole TMB color development liquid;

10 Incubation for 10-15min at room temperature and in dark place;

11 Adding 50. Mu.L/Kong Zhongzhi of the solution;

12 OD450 values in wells were read using a microplate reader. Analysis of the data using GRAPHPAD PRISM software gave half the effective concentration (EC ₅₀ values), with specific results shown in table 4 and fig. 1.

TABLE 4 binding of nanobodies to PD-1 (EC 50)

The results in table 4 show that the EC ₅₀ values of the nanobodies of the invention are lower compared to the positive antibodies PD1B849 and PD1B878, indicating that the binding capacity of the nanobodies of the invention to PD-1 is stronger than the control antibody. FIG. 1 is an absorbance at 450 nm, indicating that the antibodies of the invention have better PD-1 binding activity than the reference antibody.

EXAMPLE 3 FACS detection of candidate antibody binding to over-expressed PD-1 cells

Recovering CHO-S, CHO-S-PD1 cell strain from liquid nitrogen, and regulating cell state to logarithmic growth phase;

dividing the cells into a plurality of parts, wherein the number of each part of cells is 3×105 cells;

incubating candidate antibodies (10 ug/ml and 1.11 ug/ml) or 100 μl of transfection supernatant with target cells, mixing well, and incubating at room temperature for 1h;

Centrifugation at 800 Xg for 3min at room temperature, removal of antibody-containing supernatant, washing of cells 3 times with PBS;

Adding a secondary antibody PE anti human IgG (1:5000 dilution), fully mixing, and incubating for 30min at room temperature in a dark place;

Centrifuging at 800 Xg at room temperature for 3min, removing the supernatant containing the secondary antibody, and washing the cells 3 times by using PBS; flow assays were performed using 500 μl PBS to resuspend cells. The specific results are shown in fig. 2 and 3. Figures 2 and 3 show the Mean Fluorescence Intensity (MFI) values of the individual antibodies, demonstrating the greater binding capacity of the anti-human PD-1 nanobodies of the invention to CHO cells overexpressing human PD-1 compared to PD1B 878.

The results in FIGS. 2 and 3 show that the candidate antibodies 2-G-F1, 2-I-F6, 2-I-G8 are able to bind efficiently to CHO-S-PD1 cells compared to the positive antibodies PD1B849 and PD1B 878.

Example 4 candidate antibody work detection assay

1) Antibody gradient dilution: starting at the highest concentration of 200. Mu.g/mL, the antibody was diluted 5-fold (60. Mu.L of antibody stock was added to 240. Mu.L of RPMI1640 serum-free medium) and 9 gradients were serially diluted, with concentration gradients of 2X 100, 2X 20, 2X 4, 2X 0.8, 2X 0.16, 2X 0.032, 2X 0.0064, 2X 0.00128, 2X 0.000256 and 0. Mu.g/mL in this order. mu.L of anti-PD1 antibody to be detected (corresponding to final concentrations of 100, 20, 4, 0.8, 0.16, 0.032, 0.0064, 0.00128, 0.000256 and 0. Mu.g/mL in the wells) was added to each well.

2) CHO-S-muromomab-CD32B cells were used, the cell density was adjusted to 1X 10-6/mL with 1640+10% FBS medium, and added to 96-well plates, and each well was inoculated with 5X 10-4/50. Mu.L target cells. Incubate for 1h at 37 ℃.

3) Effector cells Jurkat-PD1 (Full) -NFAT-Luciferase were used to inoculate 2X 10. Sup..sup.4/50. Mu.L effector cells per well at a cell density of 4X 10. Sup..sup.5/mL in 1640+10% FBS medium. Only Jurkat-PD1 (Full) -NFAT-Luciferase was set as background wells.

4) After 18h of co-cultivation, 20. Mu.L of Bright-Glo (Promega, CAT#: e2610 Reagents, read Luciferase fluorescence values. Analysis of the data using GRAPHPAD PRISM software gave half-maximal inhibitory concentrations (IC ₅₀ values), with specific results shown in table 5 and fig. 4.

TABLE 5 Activity assay of nanobodies to inhibit T cell activation

FIG. 4 shows that candidate antibodies 2-G-F1, 2-I-F6 and 2-I-G8 are effective in inhibiting the blocking of CHO-S-muromomab-CD32B activation of Jurkat-PD1-NFAT-Luciferase cells compared to positive antibodies PD1B849 and PD1B 878. The results in Table 5 show that IC ₅₀ of the nanobody of the invention is superior or nearly so to the control antibody, where 2-I-G8 is stronger than 2-G-F1 and 2-I-F6.

Example 5 candidate antibody epitope detection-PD 1/PDL1 binding

1) PD1-His was cured using an HIS1K sensor for 250s.

2) The buffer is PBST (PBS+0.02% tween 20), the candidate antibody (100 nM 2-G-F1,2-I-G8, 2-I-F6) is combined, and after balancing, PDL1-Fc is combined at a concentration of 200nM; control group: PBST was bound followed by PDL1-Fc at a concentration of 200 nM.

3) And (3) detection: the temperature was measured at 25℃for 60s equilibrium combined with 180 s.

The results of FIGS. 5-7 show that the binding epitopes of the nanobodies 2-G-F1, 2-I-F6 and 2-I-G8 of the invention to PD-1 are different from the binding epitopes of PD-L1 and PD-1, and that the nanobodies of the invention do not affect the binding of PD1 to PD-L1.

Example 6 candidate antibody affinity detection

1) PD1-His was cured using an HIS1K sensor at a concentration of 5ug/ml for a curing time of 250s.

2) The buffer was PBST (PBS+0.02% tween 20) and the detection antibodies were diluted to 100, 50, 25, 12.5,6.25, 0nM.

3) Affinity detection: equilibrium is carried out for 60s, 180s is combined, 180s is dissociated, and the detection temperature is 25 oC.

TABLE 6 nanobody affinity assay

The results in Table 6 and FIGS. 8-10 show that nanobodies 2-I-G8, 2-G-F1 and 2-I-F6 of the invention all have affinities above nM.

According to the invention, the agonist-type anti-human PD-1 antigen binding polypeptide targeting PD-1 is screened out by combining a phage display technology with a high-throughput expression platform. The anti-human PD-1 antigen binding polypeptide has the advantages of simplicity in humanization, high affinity, high stability, capability of being expressed by microorganisms, low immunogenicity, good solubility, strong penetrability, capability of recognizing hidden epitopes and the like, and is beneficial to diagnosis and targeted treatment of autoimmune diseases.

In the description of the present specification, the terms "connected," "mounted," "secured," and the like are to be construed broadly, and for example, "connected" may be a fixed connection, a removable connection, or an integral connection; can be directly connected or indirectly connected through an intermediate medium. The specific meaning of the above terms in the present application can be understood by those of ordinary skill in the art according to the specific circumstances.

In the description of the present specification, the terms "one embodiment," "some embodiments," and the like, mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present application. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

The above is only a preferred embodiment of the present application, and is not intended to limit the present application, but various modifications and variations can be made to the present application by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the protection scope of the present application.

Claims

1. An agonist anti-human PD-1 antigen-binding polypeptide, wherein the antigen-binding polypeptide is an anti-human PD-1 nanobody, comprising CDR1, CDR2, and CDR3;

2. The agonist anti-human PD-1 antigen-binding polypeptide of claim 1, wherein the anti-human PD-1 nanobody comprises FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4, which are arranged in sequence.

3. The agonist anti-human PD-1 antigen-binding polypeptide of claim 2, wherein the amino acid sequence of FR1 is SEQ ID No: 10. SEQ ID No:11 or SEQ ID No:12, any one of which is shown in the figure;

the amino acid sequence of FR4 is shown as SEQ ID No: 19.

4. An isolated nucleic acid molecule comprising the nucleotide sequence of the antigen-binding polypeptide of any one of claims 1-3, wherein the nucleotide sequence is set forth in SEQ ID No: 20. SEQ ID No:21 or SEQ ID No:22, as shown in any of the figures.

5. A recombinant expression vector comprising the isolated nucleic acid molecule of claim 4.

6. A host cell comprising the isolated nucleic acid molecule of claim 4 or the recombinant expression vector of claim 5; the host cell is a mammalian cell.

7. A pharmaceutical composition comprising the antigen-binding polypeptide of any one of claims 1-3 and a pharmaceutically acceptable carrier.

8. A fusion protein, wherein the fusion protein is a chimeric antigen receptor, the extracellular domain of which comprises the antigen-binding polypeptide of any one of claims 1-3; the chimeric antigen receptor also includes a transmembrane domain and an intracellular domain.

9. Use of an agonistic anti-human PD-1 antigen-binding polypeptide in the manufacture of a medicament, comprising the antigen-binding polypeptide of any one of claims 1-3, the isolated nucleic acid molecule of claim 4, the recombinant expression vector of claim 5, or the host cell of claim 6 in the manufacture of a medicament; the medicament is useful for treating or preventing autoimmune diseases.