CN114835810B - anti-PD-1 nano antibody and application thereof - Google Patents

anti-PD-1 nano antibody and application thereof Download PDF

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CN114835810B
CN114835810B CN202210337061.1A CN202210337061A CN114835810B CN 114835810 B CN114835810 B CN 114835810B CN 202210337061 A CN202210337061 A CN 202210337061A CN 114835810 B CN114835810 B CN 114835810B
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CN114835810A (en
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吴幼玲
钟山
谢岩生
黄凯
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Zhejiang Teruisi Pharmaceutical Inc
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Zhejiang Teruisi Pharmaceutical Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Electro-optical investigation, e.g. flow cytometers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/567—Framework region [FR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G01N2333/70521—CD28, CD152

Abstract

The present invention provides a novel anti-PD-1 nanobody, which is a fusion protein of a nanobody VHH chain and an IgG4-Fc comprising a modified hinge region. The nano antibody has no floccule after expression and purification, good uniformity, high binding affinity to antigen PD-1 and good in-vivo tumor inhibition effect, and is suitable for industrial production and commercial application. The invention also provides application of the nano antibody.

Description

anti-PD-1 nano antibody and application thereof
Technical Field
The invention belongs to the technical field of biopharmaceuticals, and particularly relates to a nano antibody aiming at programmed death receptor 1 (PD-1) and application thereof.
Background
Programmed death receptor 1 (PD-1) belongs to the immunoglobulin superfamily CD28/B7, is a type I transmembrane glycoprotein composed of 288 amino acids, and is an immunosuppressive receptor. PD-1 consists of an extracellular domain, a hydrophobic cross-region and a cytoplasmic region, wherein the extracellular domain comprises an IgV (immunoglobulin variable region) domain, has 22-33% homology with other costimulatory molecules such as cytotoxic T lymphocyte antigen 4 (CTLA-4) and the like, and simultaneously contains 4 sites capable of being glycosylated; the cytoplasmic tail contains two tyrosine residues, amino-and carboxy-terminal, which constitute an Immunoreceptor Tyrosine Inhibitory Motif (ITIM) and an Immunoreceptor Tyrosine Switching Motif (ITSM), respectively.
The primary ligand of PD-1 is PD-L1, which is also known as B7-H1, which is a type I transmembrane glycoprotein encoded by the human CD274 gene. PD-L1 contains an IgV-like region, an IgC (immunoglobulin constant region) -like region, a transmembrane region and a cytoplasmic tail, wherein the cytoplasmic tail is associated with intracellular signal transduction; the IgV and IgC regions are involved in signal transduction between cells.
When PD-1 and PD-L1 are combined, downstream signals of T cell activation are blocked by phosphorylation of phosphatidylinositol-3-kinase, further activation of protein kinase B, activation of a stimulatory T cell signal pathway, glucose metabolism, secretion of interferon and the like, so that transcription of the T cells is effectively inhibited, and finally immune functions of the T cells are inhibited, and the method plays an important role in negative regulation of immune response. Thus, blocking the PD-1/PD-L1 signaling pathway can up-regulate T cell activation, activate endogenous anti-tumor immune response, and thereby exert therapeutic effects on tumors.
(/>Palbociclib) is a globally leading anti-PD-1 mab developed by the company moesadong, which was marketed in the united states, europe and china in month 9, 2015, 7 and 2018, respectively. The heavy-weight tumor immunotherapy shows a happy curative effect in various cancers, can be used for treating melanoma, colorectal cancer, head and neck cancer, non-small cell lung cancer, classical Hodgkin's lymphoma, bladder cancer, gastric cancer and the like, and has 13 currently available indications. Key truda sold worldwide in 2021 for up to $171.8 hundred million, becoming an absolute TOP1 product in a large number of anti-tumor monoclonal antibodies. However, keytruda has been commercially successful, but it is a conventional monoclonal antibody, and has certain disadvantages in terms of stability, production cost, and the like, compared with nanobodies.
Nanobody (Nb), a heavy chain single domain antibody VHH (variable domain of heavy chain of heavy-chain antibody), is 2.5nm in diameter and 4nm long, and is the smallest fragment that naturally occurs and that binds to an antigen. The nano antibody can be expressed in mammals and in an escherichia coli expression system, the expressed antibody has lower immunogenicity, is easier to produce in batches at low cost, has higher stability in wider temperature and pH range, and has certain advantages compared with the traditional antibody.
CN107814845a, a prior patent to the applicant, discloses an anti-PD-1 nanobody, which describes: immunizing a camel by utilizing a humanized PD-1 antigen protein to obtain an immune nanobody gene bank; coupling PD-1 protein molecules on an ELISA plate, displaying the spatial structure of PD-1 protein, and screening the obtained immune nanobody gene library by using phage display technology with the antigen in the form, thereby obtaining PD-1 specific nanobody genes; then the gene is transferred into escherichia coli, and the nano antibody strain which can be efficiently expressed in the escherichia coli and has high specificity is obtained. In addition, CN107814845a also discloses humanized anti-PD-1 nanobodies obtained by humanizing camelid-derived nanobodies.
However, in CN107814845A, when constructing a vector, a nanobody VHH sequence is synthesized on a pFUSE-hIgG1-Fc2 vector, pFUSE-Nb-hIgG1-Fc2 plasmid is extracted, and then the plasmid is transfected into a eukaryotic cell HEK293 for expression and purification, so that the VHH-IgG1-Fc antibody is obtained. However, after the antibody is purified, the product sample is detected to have obvious floccules through appearance detection; the CE-SDS detection shows obvious splitting peaks, which indicates that the uniformity of the antibody is relatively poor, and the applicant considers that the antibody is difficult to reach the standard of candidate molecules for drug development, and the antibody needs to be improved according to the thinking and the method of drug development so as to meet the requirements of drug development and commercial production.
Disclosure of Invention
The invention aims to provide an improved anti-PD-1 nanobody based on a nanobody sequence disclosed in CN107814845A, wherein the nanobody has high affinity to an antigen PD-1, obviously improved uniformity, good drug curative effect and obviously improved drug curative effect, meets the requirement of taking the nanobody as a drug development candidate molecule, is suitable for industrial production and has clinical application potential.
The technical scheme of the invention is as follows:
in one aspect, the invention provides an anti-PD-1 nanobody consisting of a VHH chain and an IgG4-Fc comprising a modified hinge region, wherein:
(i) The anti-PD-1 nanobody VHH chain comprises a CDR1 shown as SEQ ID NO.2, a CDR2 shown as SEQ ID NO.3, a CDR3 shown as SEQ ID NO.4 and a framework region FR2 between the CDR1 and the CDR2 shown as SEQ ID NO. 28; and is also provided with
(ii) The modified hinge region in the anti-PD-1 nanobody comprises an amino acid sequence shown as SEQ ID NO.9 or consists of a connecting peptide and a hinge region sequence in IgG4-Fc, wherein the connecting peptide comprises an amino acid sequence shown as SEQ ID NO.12, SEQ ID NO.21 or SEQ ID NO. 24.
In the present invention, the terms "anti-PD-1 nanobody", "anti-PD-1 antibody" and "fusion protein" are used interchangeably depending on the context.
The anti-PD-1 nanobody VHH chain of the invention comprises CDR1, CDR2 and CDR3 derived from SEQ ID NO.1.
SEQ ID NO.1：
QVQLQESGGGLVQPGGSLRLSCAASGSTYLRFSMGWFRQVPGKGLEGVAAIGGDGRTSYADSVKGRFTISKDNSKNTLYLDMNSLRAEDTAVYYCAAAVLLDGSFSLLAPLVPYKYDYWGQGTLVTVSS
CN107814845A discloses a reference nanobody (VHH-IgG 1-Fc) comprising a VHH chain comprising the amino acid sequence shown in SEQ ID NO.1, and the VHH chain CDR1, CDR2 and CDR3 segments it comprises can be routinely determined by a person skilled in the art. VHH chain CDR1, CDR2 and CDR3 combinations, partitioned by methods known in the art, are all encompassed within the scope of the invention.
According to a specific embodiment of the present invention, in the anti-PD-1 nanobody provided by the present invention, the amino acid sequences of CDR1, CDR2 and CDR3 of the VHH chain and the amino acid sequence of FR2 of the framework region are respectively as follows:
SEQ ID NO.2：
GSTYLRFSMG
SEQ ID NO.3：
IGGDGRT
SEQ ID NO.4：
AAAVLLDGSFSLLAPLVPYKYDY
SEQ ID NO.28：
WFRQVPGKEREGVAA
according to a specific embodiment of the present invention, in the anti-PD-1 nanobody provided by the present invention, the amino acid sequence of the modified hinge region or the amino acid sequence of the connecting peptide is as follows:
the amino acid sequence of the modified hinge region is shown in SEQ ID NO. 9:
ERKSSVECPPCP
the hinge region sequence in the IgG4-Fc before modification is shown in SEQ ID NO. 6:
ESKYGPPCPPCP
the amino acid sequence of the connecting peptide is shown as SEQ ID NO.12, SEQ ID NO.21 or SEQ ID NO. 24:
SEQ ID NO.12：
EPKIPQPQPKPQPQPQPQPKPQPKPEPE
SEQ ID NO.21：
EPKIPQPQPK
SEQ ID NO.24：
EPKIPQPQPKPQPKPEPE
the linker peptide was attached to the N-terminus of the pre-modified IgG4-Fc hinge region shown in SEQ ID NO. 6.
Preferably, the anti-PD-1 nanobody provided by the invention is an anti-human PD-1 nanobody.
Further, in the anti-PD-1 nanobody provided by the invention, the VHH chain comprises the CDR1, CDR2 and CDR3 and 4 Framework Regions (FRs) therebetween, and the arrangement mode of each region is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Preferably, in the anti-PD-1 nanobody provided by the invention, the VHH chain comprises FR1 shown in SEQ ID NO.27, FR3 shown in SEQ ID NO.29 and FR4 shown in SEQ ID NO. 30.
SEQ ID NO.27：
QVQLQESGGGLVQPGGSLRLSCAAS
SEQ ID NO.29：
SYADSVKGRFTISKDNSKNTLYLDMNSLRAEDTAVYYC
SEQ ID NO.30：
WGQGTLVTVSS
According to a specific embodiment of the invention, in the anti-PD-1 nanobody provided by the invention, the VHH chain comprises the amino acid sequence shown in SEQ ID NO. 7.
SEQ ID NO.7：
QVQLQESGGGLVQPGGSLRLSCAASGSTYLRFSMGWFRQVPGKEREGVAAIGGDGRTSYADSVKGRFTISKDNSKNTLYLDMNSLRAEDTAVYYCAAAVLLDGSFSLLAPLVPYKYDYWGQGTLVTVSS
Preferably, the anti-PD-1 nanobody provided by the invention comprises an amino acid sequence shown as SEQ ID NO.10, SEQ ID NO.13, SEQ ID NO.22 or SEQ ID NO. 25.
SEQ ID NO.10：
QVQLQESGGGLVQPGGSLRLSCAASGSTYLRFSMGWFRQVPGKEREGVAAIGGDGRTSYADSVKGRFTISKDNSKNTLYLDMNSLRAEDTAVYYCAAAVLLDGSFSLLAPLVPYKYDYWGQGTLVTVSSERKSSVECPPCP
SEQ ID NO.13：
QVQLQESGGGLVQPGGSLRLSCAASGSTYLRFSMGWFRQVPGKEREGVAAIGGDGRTSYADSVKGRFTISKDNSKNTLYLDMNSLRAEDTAVYYCAAAVLLDGSFSLLAPLVPYKYDYWGQGTLVTVSSEPKIPQPQPKPQPQPQPQPKPQPKPEPEESKYGPPCPPCP
SEQ ID NO.22：
QVQLQESGGGLVQPGGSLRLSCAASGSTYLRFSMGWFRQVPGKEREGVAAIGGDGRTSYADSVKGRFTISKDNSKNTLYLDMNSLRAEDTAVYYCAAAVLLDGSFSLLAPLVPYKYDYWGQGTLVTVSSEPKIPQPQPKESKYGPPCPPCP
SEQ ID NO.25：
QVQLQESGGGLVQPGGSLRLSCAASGSTYLRFSMGWFRQVPGKEREGVAAIGGDGRTSYADSVKGRFTISKDNSKNTLYLDMNSLRAEDTAVYYCAAAVLLDGSFSLLAPLVPYKYDYWGQGTLVTVSSEPKIPQPQPKPQPKPEPEESKYGPPCPPCP
Further, according to a specific embodiment of the present invention, the anti-PD-1 nanobody provided by the present invention consists of a VHH chain and IgG4-Fc comprising a modified hinge region, and the full-length sequence of the amino acid of the nanobody is shown as SEQ ID NO.11, SEQ ID NO.14, SEQ ID NO.23 or SEQ ID NO. 26.
SEQ ID NO.11：
QVQLQESGGGLVQPGGSLRLSCAASGSTYLRFSMGWFRQVPGKEREGVAAIGGDGRTSYADSVKGRFTISKDNSKNTLYLDMNSLRAEDTAVYYCAAAVLLDGSFSLLAPLVPYKYDYWGQGTLVTVSSERKSSVECPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
SEQ ID NO.14：
QVQLQESGGGLVQPGGSLRLSCAASGSTYLRFSMGWFRQVPGKEREGVAAIGGDGRTSYADSVKGRFTISKDNSKNTLYLDMNSLRAEDTAVYYCAAAVLLDGSFSLLAPLVPYKYDYWGQGTLVTVSSEPKIPQPQPKPQPQPQPQPKPQPKPEPEESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
SEQ ID NO.23：
QVQLQESGGGLVQPGGSLRLSCAASGSTYLRFSMGWFRQVPGKEREGVAAIGGDGRTSYADSVKGRFTISKDNSKNTLYLDMNSLRAEDTAVYYCAAAVLLDGSFSLLAPLVPYKYDYWGQGTLVTVSSEPKIPQPQPKESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
SEQ ID NO.26：
QVQLQESGGGLVQPGGSLRLSCAASGSTYLRFSMGWFRQVPGKEREGVAAIGGDGRTSYADSVKGRFTISKDNSKNTLYLDMNSLRAEDTAVYYCAAAVLLDGSFSLLAPLVPYKYDYWGQGTLVTVSSEPKIPQPQPKPQPKPEPEESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
In another aspect, the invention provides a nucleic acid molecule comprising a nucleotide sequence encoding an amino acid sequence comprised in an anti-PD-1 nanobody provided herein. The amino acid sequence may be any of the sequences provided above. The nucleotide sequence of the present invention may be a DNA sequence or an RNA sequence. The nucleic acid molecule may be a DNA molecule or an RNA molecule, and may be single-stranded or double-stranded.
In yet another aspect, the invention provides a vector comprising a nucleic acid molecule provided herein. Preferably, the vector may be an expression vector. According to a specific embodiment of the invention, the vector is a eukaryotic or prokaryotic expression vector.
In a further aspect, the invention provides a host cell comprising a vector provided herein, e.g. transformed or transfected with the vector, or having integrated in its genome a nucleic acid molecule provided herein. According to a specific embodiment of the invention, the host cell is a eukaryotic cell, such as a fungal, insect, plant or animal cell, or a prokaryotic cell, such as a bacterial cell.
In another aspect, the invention provides a composition comprising an anti-PD-1 nanobody, a nucleic acid molecule, a vector, and/or a host cell provided herein.
In yet another aspect, the invention provides the use of the anti-PD-1 nanobody, nucleic acid molecule, vector, host cell and/or composition in the manufacture of a medicament for the treatment of PD-1 or PD-L1-related diseases. Preferably, the disease is a tumor or cancer, or an infectious disease. Further preferred, the disease is lung cancer, stomach cancer, liver cancer, melanoma, cervical cancer, colorectal cancer, bladder cancer, breast cancer, leukemia, lymphoma, renal cell carcinoma, blind bowel cancer, pancreatic cancer, cholangiocarcinoma, head and neck cancer, merkel cell carcinoma, ovarian cancer, nasopharyngeal carcinoma, glioma, esophageal cancer, bone cancer or prostate cancer. Preferably, the disease is colorectal cancer.
Accordingly, the present invention also provides a method of treating PD-1 or a PD-L1-related disease, comprising administering to a subject in need thereof an anti-PD-1 nanobody, a nucleic acid molecule, a vector, a host cell, and/or a composition provided by the invention. Preferably, the disease is a tumor (including malignancy or cancer) or an infectious disease. Further preferred, the disease is lung cancer, stomach cancer, liver cancer, melanoma, cervical cancer, colorectal cancer, bladder cancer, breast cancer, leukemia, lymphoma, renal cell carcinoma, blind bowel cancer, pancreatic cancer, cholangiocarcinoma, head and neck cancer, merkel cell carcinoma, ovarian cancer, nasopharyngeal carcinoma, glioma, esophageal cancer, bone cancer or prostate cancer. The subject is a vertebrate, preferably a mammal, e.g., a human, livestock and farm animal, and zoo, sports or pet animal, such as sheep, dog, horse, cat, cow, rat, pig, macaque. According to a specific embodiment of the invention, the subject is a human.
In a further aspect, the invention provides the use of said anti-PD-1 nanobody, nucleic acid molecule, vector, host cell and/or composition in the preparation of a PD-1 detection reagent. Preferably, the detection reagent is a detection reagent based on flow cytometry or enzyme-linked immunosorbent assay.
Accordingly, the invention also provides a PD-1 detection reagent comprising the anti-PD-1 nanobody, nucleic acid molecule, vector, host cell and/or composition provided by the invention. Preferably, the detection reagent is a detection reagent based on flow cytometry or enzyme-linked immunosorbent assay.
The invention also provides a method of detecting PD-1 in a sample from a subject, the method comprising contacting an anti-PD-1 nanobody, nucleic acid molecule, vector, host cell, and/or composition provided by the invention with the sample.
In yet another aspect, the invention provides a kit comprising an anti-PD-1 nanobody, a nucleic acid molecule, a vector, a host cell, and/or a composition provided herein. Preferably, the kit is a detection kit, for example a detection kit based on flow cytometry or enzyme-linked immunosorbent assay.
In another aspect, the present invention provides a method of producing the anti-PD-1 nanobody, the method comprising the steps of:
(a) Culturing the host cell provided by the invention, thereby obtaining a culture containing the anti-PD-1 nanobody; and, (b) recovering the anti-PD-1 nanobody from the culture.
Compared with the prior art, the invention provides an anti-PD-1 nanobody, which is a fusion protein of a nanobody VHH chain and an IgG4-Fc containing a modified hinge region, and is an improved nanobody based on the nanobody disclosed in CN 107814845A.
Considering that eukaryotic cell HEK293 adopted when expressing antibody in CN107814845A is not a strain for antibody industrialized production, igG1 adopted is not a conventional antibody subtype aiming at PD-1 (anti-PD-1 monoclonal antibody therapeutic drugs are of an IgG4 type, so that only blocking function is reserved and ADCC class effector function is weakened), the inventor synthesizes nanobody VHH chain sequence in CN107814845A onto IgG4-Fc and adopts industrially used CHO cells for expression and purification, and found that obvious floccules exist in the purified VHH-IgG4-Fc (hereinafter abbreviated as PR antibody), obvious splitting peaks appear through CE-SDS detection, and the floccules and uniformity of the antibody optimized by the IgG4-Fc are not obviously improved, so that research on antibody medicine formation, industrialized production and clinical treatment application based on the VHH sequence are difficult to realize.
In view of this, the inventors of the present invention further optimized the sequence from the viewpoint of pharmaceutical development, in particular, specific amino acid substitutions were made to a plurality of amino acid sites in VHH chain, hinge region and Fc region, and by examining physicochemical and biochemical properties (including uniformity, binding affinity, antigen activity blocking effect and in vivo tumor suppression experiment) of the obtained protein, novel anti-PD-1 nanobody having better activity and more application value was screened out, in particular, the nanobody was free of floc after expression and purification, had good uniformity, binding affinity to antigen PD-1 was high, in vivo tumor suppression effect was remarkably improved, in particular, efficacy was comparable to that of(/>Palbociclib) is matched with the requirement of taking the palbociclib as a candidate molecule for drug development, is suitable for industrial production, and has clinical application potential.
Drawings
Embodiments of the present invention are described in detail below with reference to the attached drawing figures, wherein:
fig. 1: CE-SDS detection results of VHH-IgG1-Fc and VHH-IgG4-Fc (PR antibody).
Fig. 2: sequence alignment of PR antibodies and optimized nanobodies.
Fig. 3: PR antibody and CE-SDS detection result of optimized nanobody.
Fig. 4: SEC detection results for nanobodies N7 and N15 were optimized.
Fig. 5: tumor volume trend after dosing.
Fig. 6: mice tumor weight after the end of the experiment.
Detailed Description
The invention is described below with reference to specific examples. It will be appreciated by those skilled in the art that these examples are for illustration of the invention only and are not intended to limit the scope of the invention in any way.
The experimental methods in the following examples are conventional methods unless otherwise specified. The raw materials, reagent materials and the like used in the examples described below are commercially available products unless otherwise specified.
In the following examples, the following procedure was used:
preparation and purification of nanobody samples:
a. synthesizing a gene sequence encoding a nanobody sequence;
b. constructing the synthesized gene sequence on a pcDNA3.1 vector, transforming into an escherichia coli DH5 alpha strain, shaking greatly, extracting pcDNA3.1 plasmid by using an Omega plasmid big extraction kit, and filtering and sterilizing;
c. CHO-S cells were cultured to 5X 10 6 Individual/ml;
d. the pcDNA3.1 plasmid obtained in step b and the transfection reagent PEI are placed in the transfection medium ExpiCHO in a ratio of 1:3 TM Expression Medium and standing for 20min, then adding into CHO-S cells of step c, and adding 6% CO at 37deg.C 2 Culturing at 115rpm for 5 days;
e. centrifuging the culture product, and filtering with a 0.2 μm filter membrane to obtain an supernatant;
f. purifying the supernatant by ProA affinity chromatography, and washing off the impurity proteins and other impurities by using a Tris system buffer with pH of 7.0;
g. elution with 24mM acetic acid;
h. and (3) the pH value of the obtained eluent is adjusted back to 5.0 by using Tris Base, and the eluent is filtered by using a 0.2 mu m filter membrane to obtain a sample to be detected.
Appearance of (II) nanobody sample and visible foreign matter detection
And after the sample is purified, collecting the sample in a penicillin bottle, and observing the appearance and the visible foreign matter condition of the nano antibody sample.
(III) CE-SDS detection of nanobody samples:
a. preparing a solution: respectively preparing a dilution buffer solution, DTT, NEM, a dyeing gel and a decoloring solution, a molecular weight standard substance and a denaturant;
b. sample treatment: adding ultrapure water and a reducing reagent (a sample buffer solution provided by a protein 230 kit and mixed with 1M DTT) into a sample, diluting and then denaturing; under non-reducing conditions, the sample was added with 120mM N-ethylmaleimide (NEM) solution and non-reducing reagent (sample solution was mixed with ultrapure water); heating the ladder (step marker) and the sample under the reducing and non-reducing conditions, and adding the heated ladder and the sample to the chip; carrying out electrophoresis separation on the sample in a microchannel filled with polymer solution;
c. and (3) detection: laser-induced fluorescence based on a fluorescent dye, the sample being added to the polymer solution and bound non-covalently to the protein-SDS micelles;
d. analysis of results: the bioanalyzer software automatically determines the size from the ladder (step marker) and calculates the percentage of each peak separated in the electropherogram.
SEC detection of (IV) nanobody samples
a. Preparation of the reagent: preparing a solution a (100 mM phosphate buffer, 100mM sodium sulfate, ph=2.8), a solution B (100 mM phosphate buffer, 100mM sodium sulfate, ph=7.0);
b. the detection system is provided with: operating a U3000SEC project and a 3000 chromatographic system, cleaning an A pipeline by using an A solution, cleaning a B pipeline by using a B solution, setting the column temperature to 25 ℃, setting the temperature of a sample chamber to 8 ℃, closing a cleaning valve, and balancing the system pressure after cleaning the system by using ultrapure water;
c. analyzing the sample: setting a sample sequence on an Empower3, analyzing 1 needle for 2mg/ml of each sample after analyzing 6 needles of standard substances, and analyzing 1 needle of standard substance after all samples are analyzed, wherein the sample injection volume is 5 microliters; then loading and running the sequence;
d. after the run was completed, the data was analyzed.
Example 1Detection of known anti-PD-1 nanobodies
CN107814845a discloses a new anti-PD-1 nanobody and its application, the VHH sequence of the antibody is as follows:
SEQ ID NO.1 (CDR 1/CDR2/CDR3: SEQ ID NO.2/SEQ ID NO.3/SEQ ID NO.4, underlined and bolded):
preparation of nanobody samples according to the procedure described above VHH-IgG4-Fc (PR antibody) was prepared, wherein the amino acid sequence of IgG4-Fc is shown in SEQ ID No.5.
After purification of VHH-IgG1-Fc (see CN107814845A example 8) and VHH-IgG4-Fc expressed in HEK293 as disclosed in CN107814845A, the appearance of the (second) nanobody samples and the detection of visible foreign matter as described above were observed and it was found that obvious floc appeared in both purified antibody samples. The detection was performed according to the CE-SDS detection procedure of the above "(III) nanobody sample, and it was found that split peaks were detected, and the results were shown in FIG. 1 at 1A respectively
VHH-IgG 1-Fc) and 1B (VHH-IgG 4-Fc).
Without being bound by any theory, it is believed that floc generation after purification may be due to specific amino acids of VHH, hinge and Fc regions, and that uniformity is poor, and existing sequences need to be further optimized from a pharmaceutical development perspective.
Example 2Sequence optimization design and primary screening of known anti-PD-1 nano antibody
In order to solve the defects of floccules and poor uniformity after PR antibody purification, PR antibodies are optimized, and the optimal design scheme is shown in Table 1.
TABLE 1 optimization protocol for PR antibodies
Optimized domains Optimizing number (seed)
CDR 2
FR 8
FR+CDR 12
FR+finger (Hinge region) 1
FR+polypeptide (connecting peptide) 3
Configuration change (Fc+VHH, VHH attached to C-terminal of Fc) 1
Note that: in table 1, CDR, optimization number 2, indicates that 2 sequence optimization schemes were designed for CDR regions. Similarly, 8 sequence optimization schemes are designed for the FR region; meanwhile, 8 sequence optimization schemes are designed for both FR and CDR regions; meanwhile, 1 sequence optimization scheme is designed for both FR and Hinge regions; 3 schemes for adding a polypeptide (connecting peptide) while optimizing FR sequences are designed; configuration change, i.e. the scheme in which VHH is changed from the N-terminal to C-terminal of original Fc.
Preparation of the (one) nanobody samples the optimized nanobody samples were prepared according to the procedure described above, and sample detection was performed according to the appearance of the (two) nanobody samples and the visible foreign matter detection and CE-SDS detection of the (three) nanobody samples described above. The results are shown in Table 2.
TABLE 2 optimization of detection results for nanobody samples
From the CE-SDS detection result and the appearance of the purified samples, the uniformity of N7, N10, N12, N13, N14 and N15 is good, and no obvious floccules are formed.
Example 3Optimizing physicochemical and biochemical analysis of nanobodies
And carrying out physicochemical and biochemical analysis and further screening on the preliminarily screened N7, N10, N12, N13, N14 and N15. The sequences of PR antibodies and optimized nanobodies are shown below (bold, italic and underlined as optimized sites; CDR regions are defined in terms of IMGT) and the sequence alignment is shown in FIG. 2.PR:
VHH (SEQ ID NO. 1) (CDR 1/CDR2/CDR3: SEQ ID NO.2/SEQ ID NO.3/SEQ ID NO.4, underlined and bolded):
IgG4-Fc (SEQ ID NO. 5) (hinge region: SEQ ID NO.6, shown in bold):
N7：
VHH (SEQ ID NO. 7) (FR 1/CDR1/FR2/CDR2/FR3/CDR3/FR4: SEQ ID NO.27/SEQ ID NO.2/SEQ ID NO.28/SEQ ID NO.3/SEQ ID NO.29/SEQ ID NO.4/SEQ ID NO.30, wherein CDR1, CDR2, CDR3 are underlined and bolded, and the FR2 mutant amino acids are underlined, italics and bolded):
IgG4-Fc (SEQ ID NO. 8) (hinge region: SEQ ID NO.9, shown in bold, hinge region mutant amino acids underlined):
vhh+ hinge region (SEQ ID No. 10):
QVQLQESGGGLVQPGGSLRLSCAASGSTYLRFSMGWFRQVPGKEREGVAAIGGDGRTSYADSVKGRFTISKDNSKNTLYLDMNSLRAEDTAVYYCAAAVLLDGSFSLLAPLVPYKYDYWGQGTLVTVSSERKSSVECPPCP
antibody full length (SEQ ID NO. 11):
QVQLQESGGGLVQPGGSLRLSCAASGSTYLRFSMGWFRQVPGKEREGVAAIGGDGRTSYADSVKGRFTISKDNSKNTLYLDMNSLRAEDTAVYYCAAAVLLDGSFSLLAPLVPYKYDYWGQGTLVTVSSERKSSVECPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
N10：
VHH (SEQ ID NO. 7) (FR 1/CDR1/FR2/CDR2/FR3/CDR3/FR4: SEQ ID NO.27/SEQ ID NO.2/SEQ ID NO.28/SEQ ID NO.3/SEQ ID NO.29/SEQ ID NO.4/SEQ ID NO.30, wherein CDR1, CDR2, CDR3 are underlined and bolded, and the FR2 mutant amino acids are underlined, italics and bolded):
connecting peptide (SEQ ID NO. 12):
IgG4-Fc (SEQ ID NO. 5) (hinge region: SEQ ID NO.6, shown in italics and bolded):
VHH chain + connecting peptide + hinge region (SEQ ID No. 13):
QVQLQESGGGLVQPGGSLRLSCAASGSTYLRFSMGWFRQVPGKEREGVAAIGGDGRTSYADSVKGRFTISKDNSKNTLYLDMNSLRAEDTAVYYCAAAVLLDGSFSLLAPLVPYKYDYWGQGTLVTVSSEPKIPQPQPKPQPQPQPQPKPQPKPEPEESKYGPPCPPCP
antibody full length (SEQ ID No. 14):
QVQLQESGGGLVQPGGSLRLSCAASGSTYLRFSMGWFRQVPGKEREGVAAIGGDGRTSYADSVKGRFTISKDNSKNTLYLDMNSLRAEDTAVYYCAAAVLLDGSFSLLAPLVPYKYDYWGQGTLVTVSSEPKIPQPQPKPQPQPQPQPKPQPKPEPEESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
N12：
VHH (SEQ ID NO. 15) (FR 1/CDR1/FR2/CDR2/FR3/CDR3/FR4: SEQ ID NO.31/SEQ ID NO.2/SEQ ID NO.32/SEQ ID NO.3/SEQ ID NO.4/SEQ ID NO.30, wherein CDR1, CDR2, CDR3 are underlined and bolded, and FR1 and FR3 mutant amino acids are underlined, italics and bolded):
IgG4-Fc (SEQ ID NO. 5) (hinge region: SEQ ID NO.6, shown in bold):
VHH chain+hinge region (SEQ ID No. 16):
QVQLQESGGGLVQPGGSLRLSSAASGSTYLRFSMGWFRQVPGKGLEGVAAIGGDGRTSYADSVKGRFTISKDNSKNTLYLDMNSLRAEDTAVYYSAAAVLLDGSFSLLAPLVPYKYDYWGQGTLVTVSSESKYGPPCPPCP
antibody full length (SEQ ID NO. 17):
QVQLQESGGGLVQPGGSLRLSSAASGSTYLRFSMGWFRQVPGKGLEGVAAIGGDGRTSYADSVKGRFTISKDNSKNTLYLDMNSLRAEDTAVYYSAAAVLLDGSFSLLAPLVPYKYDYWGQGTLVTVSSESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
N13：
VHH (SEQ ID NO. 18) (FR 1/CDR1/FR2/CDR2/FR3/CDR3/FR4: SEQ ID NO.34/SEQ ID NO.2/SEQ ID NO.32/SEQ ID NO.3/SEQ ID NO.35/SEQ ID NO.4/SEQ ID NO.30, wherein CDR1, CDR2, CDR3 are underlined and bolded, and the FR1 and FR3 mutant amino acids are underlined, italics and bolded):
IgG4-Fc (SEQ ID NO. 5) (hinge region: SEQ ID NO.6, shown in bold):
VHH chain+hinge region (SEQ ID No. 19):
QVQLQESGGGLVQPGGSLRLSAAASGSTYLRFSMGWFRQVPGKGLEGVAAIGGDGRTSYADSVKGRFTISKDNSKNTLYLDMNSLRAEDTAVYYAAAAVLLDGSFSLLAPLVPYKYDYWGQGTLVTVSSESKYGPPCPPCP
antibody full length (SEQ ID NO. 20):
QVQLQESGGGLVQPGGSLRLSAAASGSTYLRFSMGWFRQVPGKGLEGVAAIGGDGRTSYADSVKGRFTISKDNSKNTLYLDMNSLRAEDTAVYYAAAAVLLDGSFSLLAPLVPYKYDYWGQGTLVTVSSSKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
N14：
VHH (SEQ ID NO. 7) (FR 1/CDR1/FR2/CDR2/FR3/CDR3/FR4: SEQ ID NO.27/SEQ ID NO.2/SEQ ID NO.28/SEQ ID NO.3/SEQ ID NO.29/SEQ ID NO.4/SEQ ID NO.30, wherein CDR1, CDR2, CDR3 are underlined and bolded, and the FR2 mutant amino acids are underlined, italics and bolded
Connecting peptide (SEQ ID NO. 21):
IgG4-Fc (SEQ ID NO. 5) (hinge region: SEQ ID NO.6, shown in bold):
VHH chain + connecting peptide + hinge region (SEQ ID No. 22):
QVQLQESGGGLVQPGGSLRLSCAASGSTYLRFSMGWFRQVPGKEREGVAAIGGDGRTSYADSVKGRFTISKDNSKNTLYLDMNSLRAEDTAVYYCAAAVLLDGSFSLLAPLVPYKYDYWGQGTLVTVSSEPKIPQPQPKESKYGPPCPPCP
antibody full length (SEQ ID NO. 23):
QVQLQESGGGLVQPGGSLRLSCAASGSTYLRFSMGWFRQVPGKEREGVAAIGGDGRTSYADSVKGRFTISKDNSKNTLYLDMNSLRAEDTAVYYCAAAVLLDGSFSLLAPLVPYKYDYWGQGTLVTVSSEPKIPQPQPKESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
N15：
VHH (SEQ ID NO. 7) (FR 1/CDR1/FR2/CDR2/FR3/CDR3/FR4: SEQ ID NO.27/SEQ ID NO.2/SEQ ID NO.28/SEQ ID NO.3/SEQ ID NO.29/SEQ ID NO.4/SEQ ID NO.30, wherein CDR1, CDR2, CDR3 are underlined and bolded, and the FR2 mutant amino acids are underlined, italics and bolded
Connecting peptide (SEQ ID NO. 24):
IgG4-Fc (SEQ ID NO. 5) (hinge region: SEQ ID NO.6, shown in bold):
VHH chain + connecting peptide + hinge region (SEQ ID No. 25):
QVQLQESGGGLVQPGGSLRLSCAASGSTYLRFSMGWFRQVPGKEREGVAAIGGDGRTSYADSVKGRFTISKDNSKNTLYLDMNSLRAEDTAVYYCAAAVLLDGSFSLLAPLVPYKYDYWGQGTLVTVSSEPKIPQPQPKPQPKPEPEESKYGPPCPPCP
antibody full length (SEQ ID NO. 26):
QVQLQESGGGLVQPGGSLRLSCAASGSTYLRFSMGWFRQVPGKEREGVAAIGGDGRTSYADSVKGRFTISKDNSKNTLYLDMNSLRAEDTAVYYCAAAVLLDGSFSLLAPLVPYKYDYWGQGTLVTVSSEPKIPQPQPKPQPKPEPEESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
3.1 CE-SDS assay
Sample detection was performed following the CE-SDS detection procedure for the "(second) nanobody samples above. The results are shown in tables 3 and 3A (PR), 3B (N7), 3C (N10), 3D (N12), 3E (N13), 3F (N14) and 3G (N15) in fig. 3.
TABLE 3 optimization of CE-SDS detection results of nanobody samples
3.2 SEC detection
Sample detection was performed following the SEC detection "procedure for the" (fourth) nanobody sample, above. The results are shown in Table 4 and 4A (N7) and 4B (N15) in FIG. 4.
TABLE 4 optimization of SEC detection results for nanobody samples
Sample ID Monomer%
PR 90.7
N7 94.5
N10 96.4
N12 93.8
N13 96.2
N14 97.5
N15 97.9
3.3 antigen binding (ELISA)
The binding capacity of the nanobody to the antigen PD-1 protein was optimized by ELISA detection. The method comprises the following steps:
a. antigen coating: taking 100 mu L/well of human PD-1 protein (Acro PD1-HB2F 2) with the concentration of 20ng/mL, incubating the mixture in a 96-well plate at room temperature for 2 hours, and washing the plate for 5 times;
b. preparing a reference product and a test product: after the reference substance and the test substance are diluted to 1000ng/mL, carrying out gradient dilution;
c. the reaction: c, adding the sample subjected to gradient dilution into the 96-well plate pre-coated in the step a according to 100 mu L/well, covering a plate cover, incubating for 2 hours at room temperature, and washing the plate for 5 times;
d. adding a detection antibody: transferring the diluted detection antibody to the sample plate in the step c according to 100 mu L/hole, covering a cover plate, incubating for 1 hour at room temperature, and washing the plate for 5 times;
e. adding a chromogenic substrate: adding TMB chromogenic substrate into the sample plate, covering the sample plate, and incubating for 10-15 minutes at room temperature;
f. the color reaction was terminated: adding 2M sulfuric acid into the sample plate to terminate the color reaction;
g. reading a plate: and placing the sample plate in an enzyme labeling instrument, and reading the plate.
The results are shown in Table 5.
TABLE 5 optimization of antigen binding assay results for nanobodies
Sample ID EC50(ng/ml)
PR 15.89
N7 9.98
N10 14.89
N12 15.30
N13 19.92
N14 15.12
N15 11.07
3.4 blocking detection of antigen Activity
Detecting and optimizing the blocking effect of the nano antibody on the activity of the antigen PD-1 protein. The method comprises the following steps:
a. a complete medium of CHO-PD-L1-CD3L cells was prepared by adding 100mL of FBS, 10mL of NEAA, 10mL of a solution of penicillin, 4mL of Hygromycin B (50 mg/L) and 800. Mu.L of Puromycin (10 mg/L) to 1L of DMEM/F12 basal medium; inoculating CHO-PD-L1-CD3L cells into a culture medium, and carrying out cell subculture every 2 days until logarithmic growth phase;
adding 100mL of FBS, 10mL of NEAA, 10mL of a green streptomycin solution, 8mL of Hygromycin B (50 mg/L) and 400 mu L of Puromycin (10 mg/L) into 1L of a 1640 basal medium to prepare a Jurkat-PD-1-NFAT cell complete medium; jurkat-PD-1-NFAT cells were inoculated into the medium, and the cells were subcultured every 2 days to logarithmic growth phase;
b. CHO-PD-L1-CD3L cells were digested and centrifuged and resuspended to 5X 10 with DMEM/F12 complete medium 5 mu.L of each well is added into a white-bottomed 96-well plate, and incubated for 16+/-2 hours in a (5+/-1)% CO2 incubator at 37+/-2 ℃;
c. pre-diluting the test sample, the reference sample and the quality control sample to 100 mug/mL, and performing gradient dilution by using an analysis culture medium (RPMI 1640 basic culture medium+2% FBS);
d. taking out the cell culture plate in the step b, sucking the supernatant, discarding, and adding the diluted test sample, the diluted reference sample and the diluted quality control sample into the supernatant, wherein the amount of the diluted test sample, the diluted reference sample and the diluted quality control sample is 50 mu L per hole;
e. Jurkat-PD-1-NFAT cells were removed and the cells resuspended to 2X 10 with assay medium 6 individual/mL; adding 50 mu L of the Jurkat cell suspension to the culture plate in the step d, and incubating for 6 hours in an incubator;
f. taking out the Bio-Glo Luciferase Assay System reagent 1h in advance, standing at room temperature, and adding 100 mu L of the reagent into a cell plate per well; and (3) incubating for 2-3 min at room temperature in a dark place, and then reading by using an enzyme-labeled instrument.
The results are shown in Table 6.
TABLE 6 detection results for optimizing blocking effect of nanobodies on antigen Activity
3.5 binding affinity (fortebio)
The binding affinity (fortebio) of the optimized nanobody to the antigen PD-1 protein was detected. The method comprises the following steps:
detection instrument: octet K2, pall-fortebio
And (3) a chip: protein A (manufacturer: pall Fortebio, cat# 18-5010)
Buffer solution: pH 7.4 PBST (pH 7.4 PBS,Tween20 0.05%v/v)
Software version: fortebio data analysis 10.0.0
The antibodies to be detected (PR, N7, N10, N12, N13, N14, N15) are specifically captured through a Protein A chip, the signal reaches 3nm, and the antibodies are combined with PD-1 proteins with different concentrations in gradient dilution.
Wherein the protein of the sample to be tested is diluted with PBST buffer to a final concentration of 5ug/mL. Lyophilized PD-1 protein was subjected to ddH 2 O was diluted to 250ug/mL, then with PBST, at a maximum concentration of 50nM,2-fold concentration gradient dilution, 7 total concentrations.
The results are shown in Table 7.
TABLE 7 optimization of nanobody binding affinity to antigen
The results of 3.1 to 3.5 pieces of test data are summarized in Table 8.
TABLE 8 optimization of physicochemical and Biochemical analysis results of nanobodies
Example 4Optimizing tumor inhibition detection of nanobodies
The therapeutic effect of the optimized nanobodies of the invention on subcutaneous tumors was tested in C57BL/6hPD1 humanized mice.
MC38-hPD-L1 tumor cells (colon cancer cells) were transplanted subcutaneously into C57BL/6J-hPD1 humanized mice, and MC38-hPD-L1 engraftment tumor models were established. The method comprises the following steps:
1. mice are isolated and adaptively raised for one week before test treatment;
2. MC38-hPD-L1 cells were incubated at 37℃with 5% CO 2 Culturing and amplifying in incubator, collecting logarithmic phase cell, re-suspending in PBS, adding matrigel, and adjusting cell concentration to 1×10 7 Individual cells/mL;
3. the cell suspension was injected subcutaneously into the right side of C57BL/6J-hPD1 humanized mice with a 1mL syringe, each injected 100. Mu.L;
4. the tumor volume to be averaged was about 112mm 3 In this case, mice with excessively large volumes, excessively small volumes or irregularly shaped tumors were eliminated.
The mice were divided into 5 groups by random block method, which are control group (group 1), keytruda (group 2), PR antibody group (group 3), optimized nanobody N7 group (group 4), optimized nanobody N15 group (group 5), 8 each. The doses were according to the protocol in Table 9, each group was given intraperitoneally, each group at a volume of 10ml/kg, for 3 weeks, 2 times per week, and 6 times total. Wherein each group of antibodies is prepared into a use concentration of 1mg/ml, the control group adopts physiological saline, and the administration is started on the grouping day.
TABLE 9 dosing regimen
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The tumor diameters were measured 2 times a week, the weights of the mice were weighed, the living states of the mice were observed, and abnormal conditions were recorded. The following calculation formula is adopted:
1. tumor volume (Tumor volume, TV)
TV＝1/2×a×b 2
Wherein: a represents the tumor long diameter; b represents the tumor minor diameter.
2. Relative tumor volume (Relative tumor volume, RTV)
RTV＝V t /V initial ×100(％)
Wherein V is initial For grouped administration (i.e. d initial ) Measuring the volume of the obtained tumor, V t Tumor volume at each measurement.
3. Relative tumor proliferation Rate T/C (%)
T/C(％)＝(T RTV /C RTV )×100％
Wherein T is RTV Representing relative tumor volume of treatment group, C RTV Represents the relative tumor volume of the solvent group.
4. Tumor volume tumor inhibition (TGI)
TGI＝[1-(TV t -TV initial )/(CV t -CV initial )]×100％
Wherein, TV t Tumor volume at each measurement of treatment group; TV set initial Tumor volume for treatment group at the time of group dosing;CV t tumor volume at each measurement of control group is indicated; CV (CV) initial Tumor volumes of the control group at the time of group administration are shown.
Results are expressed as Mean and standard error (mean±sem) and the difference in tumor volume between control and dosing groups was analyzed using T-test, p <0.05 indicating that there was a statistical difference.
1. The tumor volume changes in mice are shown in Table 10 and FIG. 5.
TABLE 10 mouse tumor volume (mm 3 )
Note that: data are expressed as mean±sem
"-": euthanasia of animals
On day 14 after the start of administration, the average tumor volume of the control group was 4057.43.+ -. 288.73mm 3 . Tumor volumes of group 2, group 3, group 4 and group 5 were 884.86.+ -. 194.57mm, respectively 3 、1584.55±186.82mm 3 、672.45±101.01mm 3 And 815.51 + -131.88 mm 3 Tumor inhibition rates were 80.42%, 62.69%, 85.79% and 82.16%, respectively. Group 2, group 3, group 4 and group 5 showed significant differences in tumor volume compared to the control group (P<0.01)。
The tumor growth inhibition capacity of the antibodies on female humanized mice transplanted with hPD-L1 tumors was calculated based on the tumor volumes measured on day 14. The results are shown in tables 11 and 12.
TABLE 11 analysis of tumor growth inhibition rate
TABLE 12 comparison of tumor volumes between groups
Note that: data are expressed as mean±sem
Tumor volumes between the vehicle and treatment groups were compared using T-test analysis, with p <0.0001 and p <0.05.
2. Tumor weight of mice
At the end of the test, animals were euthanized, tumor mass removed and weighed, the tumor weights of the mice are shown in FIG. 6, and the average tumor weights of the control group are 7.0907 + -0.8349 g, and the average tumor weights of the tested samples Keytruda, PR, N and N15 are 3.0556 + -0.9384 g, 5.1787 + -0.3078 g, 2.4394 + -0.7359 g and 3.0210 + -0.8778 g, respectively.
The experimental results in tables 10 to 12 and fig. 5 and 6 show that the test subjects N7, N15 and Keytruda antibodies alone, in addition to the test subject PR, each obtained excellent antitumor effect on the hPD-L1-MC38 tumor model in the control group. N7, N15 and Keystuda are obviously superior to PR antibodies in anti-tumor effect, and N7 and N15 show equivalent anti-tumor effect on hPD-L1-MC38 tumor model compared with Keystuda. In view of the fact that Keystuda is used as a PD-1/PD-L1 monoclonal antibody and is approved by domestic and foreign drug administration institutions for treatment of up to 13 tumors or cancers, great clinical benefit is brought to patients, great commercial success is achieved, and the anti-PD-1 nanobody disclosed by the invention has broad clinical treatment prospect as an alternative nanobody drug with the drug effect not being inferior to that of the anti-PD-1 nanobody. In addition, the anti-PD-1 nanobody provides a drug candidate molecule which meets the drug development requirements better than PR.
The above description of the embodiments of the present invention is not intended to limit the present invention, and those skilled in the art can make various changes or modifications according to the present invention without departing from the spirit of the present invention, and shall fall within the scope of the appended claims.
Sequence listing
<110> Zhejiang terylesi pharmaceutical Co., ltd
<120> an anti-PD-1 nanobody and use thereof
<130> LC22110008
<160> 35
<170> PatentIn version 3.5
<210> 1
<211> 129
<212> PRT
<213> Artificial sequence (artificial sequence)
<220>
<223> VHH
<400> 1
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Tyr Leu Arg Phe
20 25 30
Ser Met Gly Trp Phe Arg Gln Val Pro Gly Lys Gly Leu Glu Gly Val
35 40 45
Ala Ala Ile Gly Gly Asp Gly Arg Thr Ser Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Lys Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Asp Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Ala Ala Val Leu Leu Asp Gly Ser Phe Ser Leu Leu Ala Pro Leu Val
100 105 110
Pro Tyr Lys Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
115 120 125
Ser
<210> 2
<211> 10
<212> PRT
<213> Artificial sequence (artificial sequence)
<220>
<223> VHH, CDR1
<400> 2
Gly Ser Thr Tyr Leu Arg Phe Ser Met Gly
1 5 10
<210> 3
<211> 7
<212> PRT
<213> Artificial sequence (artificial sequence)
<220>
<223> VHH, CDR2
<400> 3
Ile Gly Gly Asp Gly Arg Thr
1 5
<210> 4
<211> 23
<212> PRT
<213> Artificial sequence (artificial sequence)
<220>
<223> VHH, CDR3
<400> 4
Ala Ala Ala Val Leu Leu Asp Gly Ser Phe Ser Leu Leu Ala Pro Leu
1 5 10 15
Val Pro Tyr Lys Tyr Asp Tyr
20
<210> 5
<211> 229
<212> PRT
<213> Artificial sequence (artificial sequence)
<220>
<223> IgG4-Fc
<400> 5
Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe
1 5 10 15
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
20 25 30
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
35 40 45
Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val
50 55 60
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser
65 70 75 80
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
85 90 95
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser
100 105 110
Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
115 120 125
Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln
130 135 140
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
145 150 155 160
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
165 170 175
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu
180 185 190
Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser
195 200 205
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
210 215 220
Leu Ser Leu Gly Lys
225
<210> 6
<211> 12
<212> PRT
<213> Artificial sequence (artificial sequence)
<220>
<223> hinge
<400> 6
Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro
1 5 10
<210> 7
<211> 129
<212> PRT
<213> Artificial sequence (artificial sequence)
<220>
<223> VHH
<400> 7
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Tyr Leu Arg Phe
20 25 30
Ser Met Gly Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Ala Ile Gly Gly Asp Gly Arg Thr Ser Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Lys Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Asp Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Ala Ala Val Leu Leu Asp Gly Ser Phe Ser Leu Leu Ala Pro Leu Val
100 105 110
Pro Tyr Lys Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
115 120 125
Ser
<210> 8
<211> 229
<212> PRT
<213> Artificial sequence (artificial sequence)
<220>
<223> IgG4-Fc
<400> 8
Glu Arg Lys Ser Ser Val Glu Cys Pro Pro Cys Pro Ala Pro Glu Phe
1 5 10 15
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
20 25 30
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
35 40 45
Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val
50 55 60
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser
65 70 75 80
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
85 90 95
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser
100 105 110
Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
115 120 125
Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln
130 135 140
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
145 150 155 160
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
165 170 175
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu
180 185 190
Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser
195 200 205
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
210 215 220
Leu Ser Leu Gly Lys
225
<210> 9
<211> 12
<212> PRT
<213> Artificial sequence (artificial sequence)
<220>
<223> hinge
<400> 9
Glu Arg Lys Ser Ser Val Glu Cys Pro Pro Cys Pro
1 5 10
<210> 10
<211> 141
<212> PRT
<213> Artificial sequence (artificial sequence)
<220>
<223> VHH+hinge
<400> 10
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Tyr Leu Arg Phe
20 25 30
Ser Met Gly Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Ala Ile Gly Gly Asp Gly Arg Thr Ser Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Lys Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Asp Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Ala Ala Val Leu Leu Asp Gly Ser Phe Ser Leu Leu Ala Pro Leu Val
100 105 110
Pro Tyr Lys Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
115 120 125
Ser Glu Arg Lys Ser Ser Val Glu Cys Pro Pro Cys Pro
130 135 140
<210> 11
<211> 358
<212> PRT
<213> Artificial sequence (artificial sequence)
<220>
<223> N7
<400> 11
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Tyr Leu Arg Phe
20 25 30
Ser Met Gly Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Ala Ile Gly Gly Asp Gly Arg Thr Ser Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Lys Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Asp Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Ala Ala Val Leu Leu Asp Gly Ser Phe Ser Leu Leu Ala Pro Leu Val
100 105 110
Pro Tyr Lys Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
115 120 125
Ser Glu Arg Lys Ser Ser Val Glu Cys Pro Pro Cys Pro Ala Pro Glu
130 135 140
Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
145 150 155 160
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
165 170 175
Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly
180 185 190
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn
195 200 205
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
210 215 220
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro
225 230 235 240
Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
245 250 255
Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn
260 265 270
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
275 280 285
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
290 295 300
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg
305 310 315 320
Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys
325 330 335
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
340 345 350
Ser Leu Ser Leu Gly Lys
355
<210> 12
<211> 28
<212> PRT
<213> Artificial sequence (artificial sequence)
<220>
<223> linker
<400> 12
Glu Pro Lys Ile Pro Gln Pro Gln Pro Lys Pro Gln Pro Gln Pro Gln
1 5 10 15
Pro Gln Pro Lys Pro Gln Pro Lys Pro Glu Pro Glu
20 25
<210> 13
<211> 169
<212> PRT
<213> Artificial sequence (artificial sequence)
<220>
<223> VHH+linker+hinge
<400> 13
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Tyr Leu Arg Phe
20 25 30
Ser Met Gly Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Ala Ile Gly Gly Asp Gly Arg Thr Ser Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Lys Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Asp Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Ala Ala Val Leu Leu Asp Gly Ser Phe Ser Leu Leu Ala Pro Leu Val
100 105 110
Pro Tyr Lys Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
115 120 125
Ser Glu Pro Lys Ile Pro Gln Pro Gln Pro Lys Pro Gln Pro Gln Pro
130 135 140
Gln Pro Gln Pro Lys Pro Gln Pro Lys Pro Glu Pro Glu Glu Ser Lys
145 150 155 160
Tyr Gly Pro Pro Cys Pro Pro Cys Pro
165
<210> 14
<211> 386
<212> PRT
<213> Artificial sequence (artificial sequence)
<220>
<223> N10
<400> 14
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Tyr Leu Arg Phe
20 25 30
Ser Met Gly Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Ala Ile Gly Gly Asp Gly Arg Thr Ser Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Lys Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Asp Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Ala Ala Val Leu Leu Asp Gly Ser Phe Ser Leu Leu Ala Pro Leu Val
100 105 110
Pro Tyr Lys Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
115 120 125
Ser Glu Pro Lys Ile Pro Gln Pro Gln Pro Lys Pro Gln Pro Gln Pro
130 135 140
Gln Pro Gln Pro Lys Pro Gln Pro Lys Pro Glu Pro Glu Glu Ser Lys
145 150 155 160
Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly
165 170 175
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
180 185 190
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu
195 200 205
Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
210 215 220
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg
225 230 235 240
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
245 250 255
Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu
260 265 270
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
275 280 285
Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu
290 295 300
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
305 310 315 320
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
325 330 335
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp
340 345 350
Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His
355 360 365
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu
370 375 380
Gly Lys
385
<210> 15
<211> 129
<212> PRT
<213> Artificial sequence (artificial sequence)
<220>
<223> VHH
<400> 15
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Ser Ala Ala Ser Gly Ser Thr Tyr Leu Arg Phe
20 25 30
Ser Met Gly Trp Phe Arg Gln Val Pro Gly Lys Gly Leu Glu Gly Val
35 40 45
Ala Ala Ile Gly Gly Asp Gly Arg Thr Ser Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Lys Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Asp Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Ser Ala
85 90 95
Ala Ala Val Leu Leu Asp Gly Ser Phe Ser Leu Leu Ala Pro Leu Val
100 105 110
Pro Tyr Lys Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
115 120 125
Ser
<210> 16
<211> 141
<212> PRT
<213> Artificial sequence (artificial sequence)
<220>
<223> VHH+hinge
<400> 16
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Ser Ala Ala Ser Gly Ser Thr Tyr Leu Arg Phe
20 25 30
Ser Met Gly Trp Phe Arg Gln Val Pro Gly Lys Gly Leu Glu Gly Val
35 40 45
Ala Ala Ile Gly Gly Asp Gly Arg Thr Ser Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Lys Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Asp Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Ser Ala
85 90 95
Ala Ala Val Leu Leu Asp Gly Ser Phe Ser Leu Leu Ala Pro Leu Val
100 105 110
Pro Tyr Lys Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
115 120 125
Ser Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro
130 135 140
<210> 17
<211> 358
<212> PRT
<213> Artificial sequence (artificial sequence)
<220>
<223> N12
<400> 17
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Ser Ala Ala Ser Gly Ser Thr Tyr Leu Arg Phe
20 25 30
Ser Met Gly Trp Phe Arg Gln Val Pro Gly Lys Gly Leu Glu Gly Val
35 40 45
Ala Ala Ile Gly Gly Asp Gly Arg Thr Ser Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Lys Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Asp Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Ser Ala
85 90 95
Ala Ala Val Leu Leu Asp Gly Ser Phe Ser Leu Leu Ala Pro Leu Val
100 105 110
Pro Tyr Lys Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
115 120 125
Ser Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu
130 135 140
Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
145 150 155 160
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
165 170 175
Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly
180 185 190
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn
195 200 205
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
210 215 220
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro
225 230 235 240
Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
245 250 255
Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn
260 265 270
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
275 280 285
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
290 295 300
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg
305 310 315 320
Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys
325 330 335
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
340 345 350
Ser Leu Ser Leu Gly Lys
355
<210> 18
<211> 129
<212> PRT
<213> Artificial sequence (artificial sequence)
<220>
<223> VHH
<400> 18
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Ala Ala Ala Ser Gly Ser Thr Tyr Leu Arg Phe
20 25 30
Ser Met Gly Trp Phe Arg Gln Val Pro Gly Lys Gly Leu Glu Gly Val
35 40 45
Ala Ala Ile Gly Gly Asp Gly Arg Thr Ser Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Lys Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Asp Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Ala Ala
85 90 95
Ala Ala Val Leu Leu Asp Gly Ser Phe Ser Leu Leu Ala Pro Leu Val
100 105 110
Pro Tyr Lys Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
115 120 125
Ser
<210> 19
<211> 141
<212> PRT
<213> Artificial sequence (artificial sequence)
<220>
<223> VHH+hinge
<400> 19
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Ala Ala Ala Ser Gly Ser Thr Tyr Leu Arg Phe
20 25 30
Ser Met Gly Trp Phe Arg Gln Val Pro Gly Lys Gly Leu Glu Gly Val
35 40 45
Ala Ala Ile Gly Gly Asp Gly Arg Thr Ser Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Lys Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Asp Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Ala Ala
85 90 95
Ala Ala Val Leu Leu Asp Gly Ser Phe Ser Leu Leu Ala Pro Leu Val
100 105 110
Pro Tyr Lys Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
115 120 125
Ser Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro
130 135 140
<210> 20
<211> 357
<212> PRT
<213> Artificial sequence (artificial sequence)
<220>
<223> N13
<400> 20
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Ala Ala Ala Ser Gly Ser Thr Tyr Leu Arg Phe
20 25 30
Ser Met Gly Trp Phe Arg Gln Val Pro Gly Lys Gly Leu Glu Gly Val
35 40 45
Ala Ala Ile Gly Gly Asp Gly Arg Thr Ser Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Lys Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Asp Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Ala Ala
85 90 95
Ala Ala Val Leu Leu Asp Gly Ser Phe Ser Leu Leu Ala Pro Leu Val
100 105 110
Pro Tyr Lys Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
115 120 125
Ser Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe
130 135 140
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
145 150 155 160
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
165 170 175
Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val
180 185 190
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser
195 200 205
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
210 215 220
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser
225 230 235 240
Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
245 250 255
Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln
260 265 270
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
275 280 285
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
290 295 300
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu
305 310 315 320
Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser
325 330 335
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
340 345 350
Leu Ser Leu Gly Lys
355
<210> 21
<211> 10
<212> PRT
<213> Artificial sequence (artificial sequence)
<220>
<223> linker
<400> 21
Glu Pro Lys Ile Pro Gln Pro Gln Pro Lys
1 5 10
<210> 22
<211> 151
<212> PRT
<213> Artificial sequence (artificial sequence)
<220>
<223> VHH+linker+hinge
<400> 22
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Tyr Leu Arg Phe
20 25 30
Ser Met Gly Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Ala Ile Gly Gly Asp Gly Arg Thr Ser Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Lys Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Asp Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Ala Ala Val Leu Leu Asp Gly Ser Phe Ser Leu Leu Ala Pro Leu Val
100 105 110
Pro Tyr Lys Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
115 120 125
Ser Glu Pro Lys Ile Pro Gln Pro Gln Pro Lys Glu Ser Lys Tyr Gly
130 135 140
Pro Pro Cys Pro Pro Cys Pro
145 150
<210> 23
<211> 368
<212> PRT
<213> Artificial sequence (artificial sequence)
<220>
<223> N14
<400> 23
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Tyr Leu Arg Phe
20 25 30
Ser Met Gly Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Ala Ile Gly Gly Asp Gly Arg Thr Ser Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Lys Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Asp Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Ala Ala Val Leu Leu Asp Gly Ser Phe Ser Leu Leu Ala Pro Leu Val
100 105 110
Pro Tyr Lys Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
115 120 125
Ser Glu Pro Lys Ile Pro Gln Pro Gln Pro Lys Glu Ser Lys Tyr Gly
130 135 140
Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser
145 150 155 160
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
165 170 175
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro
180 185 190
Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
195 200 205
Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val
210 215 220
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
225 230 235 240
Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr
245 250 255
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
260 265 270
Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
275 280 285
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
290 295 300
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
305 310 315 320
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser
325 330 335
Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
340 345 350
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys
355 360 365
<210> 24
<211> 18
<212> PRT
<213> Artificial sequence (artificial sequence)
<220>
<223> linker
<400> 24
Glu Pro Lys Ile Pro Gln Pro Gln Pro Lys Pro Gln Pro Lys Pro Glu
1 5 10 15
Pro Glu
<210> 25
<211> 159
<212> PRT
<213> Artificial sequence (artificial sequence)
<220>
<223> VHH+linker+hinge
<400> 25
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Tyr Leu Arg Phe
20 25 30
Ser Met Gly Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Ala Ile Gly Gly Asp Gly Arg Thr Ser Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Lys Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Asp Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Ala Ala Val Leu Leu Asp Gly Ser Phe Ser Leu Leu Ala Pro Leu Val
100 105 110
Pro Tyr Lys Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
115 120 125
Ser Glu Pro Lys Ile Pro Gln Pro Gln Pro Lys Pro Gln Pro Lys Pro
130 135 140
Glu Pro Glu Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro
145 150 155
<210> 26
<211> 376
<212> PRT
<213> Artificial sequence (artificial sequence)
<220>
<223> N15
<400> 26
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Tyr Leu Arg Phe
20 25 30
Ser Met Gly Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Ala Ile Gly Gly Asp Gly Arg Thr Ser Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Lys Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Asp Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Ala Ala Val Leu Leu Asp Gly Ser Phe Ser Leu Leu Ala Pro Leu Val
100 105 110
Pro Tyr Lys Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
115 120 125
Ser Glu Pro Lys Ile Pro Gln Pro Gln Pro Lys Pro Gln Pro Lys Pro
130 135 140
Glu Pro Glu Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala
145 150 155 160
Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
165 170 175
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
180 185 190
Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val
195 200 205
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
210 215 220
Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
225 230 235 240
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly
245 250 255
Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
260 265 270
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr
275 280 285
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
290 295 300
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
305 310 315 320
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
325 330 335
Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe
340 345 350
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
355 360 365
Ser Leu Ser Leu Ser Leu Gly Lys
370 375
<210> 27
<211> 25
<212> PRT
<213> Artificial sequence (artificial sequence)
<220>
<223> VHH, FR1
<400> 27
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser
20 25
<210> 28
<211> 15
<212> PRT
<213> Artificial sequence (artificial sequence)
<220>
<223> VHH, FR2
<400> 28
Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu Gly Val Ala Ala
1 5 10 15
<210> 29
<211> 38
<212> PRT
<213> Artificial sequence (artificial sequence)
<220>
<223> VHH, FR3
<400> 29
Ser Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Lys Asp Asn
1 5 10 15
Ser Lys Asn Thr Leu Tyr Leu Asp Met Asn Ser Leu Arg Ala Glu Asp
20 25 30
Thr Ala Val Tyr Tyr Cys
35
<210> 30
<211> 11
<212> PRT
<213> Artificial sequence (artificial sequence)
<220>
<223> VHH, FR4
<400> 30
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
1 5 10
<210> 31
<211> 25
<212> PRT
<213> Artificial sequence (artificial sequence)
<220>
<223> VHH, FR1
<400> 31
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Ser Ala Ala Ser
20 25
<210> 32
<211> 15
<212> PRT
<213> Artificial sequence (artificial sequence)
<220>
<223> VHH, FR2
<400> 32
Trp Phe Arg Gln Val Pro Gly Lys Gly Leu Glu Gly Val Ala Ala
1 5 10 15
<210> 33
<211> 38
<212> PRT
<213> Artificial sequence (artificial sequence)
<220>
<223> VHH, FR3
<400> 33
Ser Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Lys Asp Asn
1 5 10 15
Ser Lys Asn Thr Leu Tyr Leu Asp Met Asn Ser Leu Arg Ala Glu Asp
20 25 30
Thr Ala Val Tyr Tyr Ser
35
<210> 34
<211> 25
<212> PRT
<213> Artificial sequence (artificial sequence)
<220>
<223> VHH, FR1
<400> 34
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Ala Ala Ala Ser
20 25
<210> 35
<211> 38
<212> PRT
<213> Artificial sequence (artificial sequence)
<220>
<223> VHH, FR3
<400> 35
Ser Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Lys Asp Asn
1 5 10 15
Ser Lys Asn Thr Leu Tyr Leu Asp Met Asn Ser Leu Arg Ala Glu Asp
20 25 30
Thr Ala Val Tyr Tyr Ala
35

Claims (8)

1. An anti-PD-1 nanobody, wherein the anti-PD-1 nanobody consists of a VHH chain and an IgG4-Fc comprising a modified hinge region, wherein:
(i) The anti-PD-1 nanobody VHH chain comprises a CDR1 shown as SEQ ID NO.2, a CDR2 shown as SEQ ID NO.3, a CDR3 shown as SEQ ID NO.4 and a framework region FR2 between the CDR1 and the CDR2 shown as SEQ ID NO. 28;
and is also provided with
(ii) The amino acid sequence of the modified hinge region in the anti-PD-1 nanometer antibody is shown as SEQ ID NO. 9.
2. The anti-PD-1 nanobody of claim 1, wherein the anti-PD-1 nanobody is an anti-human PD-1 nanobody.
3. Use of an anti-PD-1 nanobody according to claim 1 or 2 in the manufacture of a medicament for the treatment of a tumor, wherein the tumor is lung cancer, gastric cancer, liver cancer, melanoma, cervical cancer, colorectal cancer, bladder cancer, breast cancer, lymphoma, renal cell carcinoma, blind bowel cancer, pancreatic cancer, cholangiocarcinoma, head and neck cancer, merkel cell carcinoma, ovarian cancer, nasopharyngeal carcinoma, glioma, esophageal cancer, bone cancer or prostate cancer.
4. The use according to claim 3, wherein the tumour is colorectal cancer.
5. Use of the anti-PD-1 nanobody of claim 1 or 2 in the preparation of a PD-1 detection reagent.
6. A PD-1 detection reagent, wherein the PD-1 detection reagent comprises the anti-PD-1 nanobody of claim 1 or 2.
7. The PD-1 detection reagent of claim 6, wherein the PD-1 detection reagent is a detection reagent based on flow cytometry or enzyme-linked immunosorbent assay.
8. A kit comprising the PD-1 detection reagent of claim 6 or 7.
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