CN114835810A - anti-PD-1 nano antibody and application thereof - Google Patents
anti-PD-1 nano antibody and application thereof Download PDFInfo
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- CN114835810A CN114835810A CN202210337061.1A CN202210337061A CN114835810A CN 114835810 A CN114835810 A CN 114835810A CN 202210337061 A CN202210337061 A CN 202210337061A CN 114835810 A CN114835810 A CN 114835810A
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Abstract
The invention provides a novel anti-PD-1 nanobody which is a fusion protein of a nanobody VHH chain and IgG4-Fc comprising a modified hinge region. The nano antibody has no floccule after expression and purification, good uniformity, high binding affinity to the 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
Technical Field
The invention belongs to the technical field of biological pharmacy, and particularly relates to a nano antibody aiming at a programmed death receptor 1(PD-1) and application thereof.
Background
Programmed death receptor 1(PD-1) belongs to immunoglobulin superfamily CD28/B7, is I type transmembrane glycoprotein composed of 288 amino acids, and is an immunosuppressive receptor. PD-1 is structurally composed of an extracellular domain, a hydrophobic transregion 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 simultaneously contains 4 sites capable of being glycosylated; the cytoplasmic tail contains amino-and carboxy-terminal tyrosine residues that form an Immunoreceptor Tyrosine Inhibition Motif (ITIM) and an Immunoreceptor Tyrosine Switch Motif (ITSM), respectively.
The main ligand of PD-1 is PD-L1, which is also called B7-H1, and 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 region, wherein the cytoplasmic tail region is involved in intracellular signal transduction; the IgV and IgC regions are involved in intercellular signal transduction.
When PD-1 binds to PD-L1, phosphorylation of phosphatidylinositol-3-kinase, further activation of protein kinase B, activation of stimulatory T cell signaling pathways, glucose metabolism, secretion of interferon, and the like occur, which results in blocking of downstream signals of T cell activation, thereby effectively inhibiting transcription of T cells, and finally inhibiting immune function of T cells, which plays an important role in negative regulation of immune response. Therefore, the blocking of the PD-1/PD-L1 signal channel can lead the activation of T cells to be up-regulated, and activate endogenous anti-tumor immune response, thereby playing the role of treating tumors.
(Pabolizumab) is a globally leading anti-PD-1 mab developed by the company mshondri, and is marketed in the united states, europe, and china in 9 months 2014, 7 months 2015, and 7 months 2018, respectively. The heavy-pound tumor immunotherapy shows the favorable curative effect in various cancers, can be used for treating melanoma, colorectal cancer, head and neck cancer, non-small cell lung cancer, classical Hodgkin lymphoma, bladder cancer, gastric cancer and the like, and has 13 approved indications at present. Keytruda sold worldwide in 2021 as $ 171.8 billion, an absolute TOP1 product among a large number of anti-tumor monoclonal antibody drugs. However, Keytruda, while having great commercial success, is traditionalThe monoclonal antibody has certain disadvantages in the aspects of stability, production cost and the like compared with the nano antibody.
Nanobodies (Nb), heavy chain single domain antibodies VHH (variable domain of heavy chain of heavy-chain antibodies), 2.5nm in diameter and 4nm in length, are the smallest naturally occurring fragments that can bind to an antigen. The nano antibody can be expressed in mammals and can also be expressed 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 a wider temperature and pH range, and has certain advantages compared with the traditional antibody.
CN107814845A, a prior patent of the applicant, discloses an anti-PD-1 nanobody, which describes: immunizing camel by using human PD-1 antigen protein to obtain an immune nano antibody gene library; coupling PD-1 protein molecules on an enzyme label plate, displaying the space structure of PD-1 protein, and then screening the obtained immune nano antibody gene library by using the antigen in the form through a phage display technology so as to obtain a PD-1 specific nano antibody gene; then the gene is transferred into escherichia coli to obtain a nano antibody strain which can be efficiently expressed in the escherichia coli and has high specificity. In addition, CN107814845A discloses humanized anti-PD-1 nanobody obtained by humanized modification of camelid-derived nanobody.
However, in CN107814845A, during vector construction, a nano antibody VHH sequence is synthesized on a pFUSE-hIgG1-Fc2 vector, pFUSE-Nb-hIgG1-Fc2 plasmid is extracted and then transfected into a eukaryotic cell HEK293 for expression and purification, and the VHH-IgG1-Fc antibody is obtained. However, after the antibody is purified, a product sample is detected by appearance to find obvious floccules; the CE-SDS detection shows obvious splitting peaks, which indicates that the antibody has relatively poor uniformity, and the applicant considers that the antibody can hardly reach the standard of candidate molecules for drug development, and the applicant needs to improve the antibody according to the thinking and the method of the drug development so as to meet the requirements of the drug development and the commercial production.
Disclosure of Invention
The invention aims to provide an improved anti-PD-1 nano antibody based on a nano antibody sequence disclosed in CN107814845A, the nano antibody retains high affinity to an antigen PD-1 but has obviously improved uniformity, and has good drug efficacy, the drug efficacy is obviously improved, the nano antibody meets the requirement of taking the nano antibody as a drug development candidate molecule, and the nano antibody 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 IgG4-Fc comprising a modified hinge region, wherein:
(i) the anti-PD-1 nano-antibody VHH chain comprises a CDR1 shown in SEQ ID NO.2, a CDR2 shown in SEQ ID NO.3, a CDR3 shown in SEQ ID NO.4 and a framework region FR2 between a CDR1 and a CDR2 shown in SEQ ID NO. 28; and is
(ii) The modified hinge region in the anti-PD-1 nano antibody comprises an amino acid sequence shown in 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 in 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-IgG1-Fc) of a VHH chain comprising the amino acid sequence shown in SEQ ID NO.1, which the skilled person can routinely determine the VHH chain CDR1, CDR2 and CDR3 segments it comprises. Combinations of VHH chain CDR1, CDR2, and CDR3 partitioned in a manner known in the art are encompassed by the present 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 VHH chain and the amino acid sequence of FR2 of 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 linker peptide are respectively as follows:
the amino acid sequence of the modified hinge region is shown as SEQ ID NO. 9:
ERKSSVECPPCP
the sequence of the hinge region in the IgG4-Fc before modification is shown in SEQ ID NO. 6:
ESKYGPPCPPCP
the amino acid sequence of the connecting peptide is shown in 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 nano antibody provided by the invention is an anti-human PD-1 nano antibody.
Further, in the anti-PD-1 nanobody provided by the present invention, the VHH chain comprises the above-described CDR1, CDR2 and CDR3 and 4 Framework Regions (FRs) therebetween, each of which is arranged in the manner of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR 4. Preferably, in the anti-PD-1 nanobody provided by the present 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 present invention, in the anti-PD-1 nanobody provided by the present 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 nano antibody provided by the invention comprises an amino acid sequence shown in 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 VHH chain and IgG4-Fc comprising a modified hinge region, and the full-length amino acid sequence of the nanobody is shown in 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 present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding an amino acid sequence comprised in an anti-PD-1 nanobody provided by the present invention. The amino acid sequence may be any of the sequences provided above. The nucleotide sequence of the invention can 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 by the invention. Preferably, the vector may be an expression vector. According to a particular embodiment of the invention, the vector is a eukaryotic cell expression vector or a prokaryotic cell expression vector.
In a further aspect, the invention provides a host cell comprising, e.g., transformed or transfected with, a vector as provided herein, or having integrated into its genome a nucleic acid molecule as provided herein. According to a particular 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 present invention provides a composition comprising an anti-PD-1 nanobody, a nucleic acid molecule, a vector and/or a host cell provided by the present invention.
In still another aspect, the invention provides use of the anti-PD-1 nanobody, the nucleic acid molecule, the vector, the host cell and/or the composition for the preparation 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 preferably, the disease is lung cancer, stomach cancer, liver cancer, melanoma, cervical cancer, colorectal cancer, bladder cancer, breast cancer, leukemia, lymphoma, renal cell carcinoma, cecum cancer, pancreatic cancer, bile duct cancer, head and neck cancer, merkel cell carcinoma, ovarian cancer, nasopharyngeal cancer, glioma, esophageal cancer, bone cancer or prostate cancer. Preferably, the disease is colorectal cancer.
Accordingly, the present invention also provides a method of treating a PD-1 or PD-L1-associated disease, the method 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 as provided by the present invention. Preferably, the disease is a tumour (including a malignant tumour or cancer) or an infectious disease. Further preferably, the disease is lung cancer, stomach cancer, liver cancer, melanoma, cervical cancer, colorectal cancer, bladder cancer, breast cancer, leukemia, lymphoma, renal cell carcinoma, cecum cancer, pancreatic cancer, bile duct cancer, head and neck cancer, merkel cell carcinoma, ovarian cancer, nasopharyngeal cancer, glioma, esophageal cancer, bone cancer or prostate cancer. The subject is a vertebrate, preferably a mammal, e.g. a human, domestic and agricultural animal and a zoo, sports or pet animal, such as sheep, dog, horse, cat, cow, rat, pig, macaque. According to a particular embodiment of the invention, the subject is a human.
In still another aspect, the invention provides use of the anti-PD-1 nanobody, the nucleic acid molecule, the vector, the host cell and/or the composition in preparation of a PD-1 detection reagent. Preferably, the detection reagent is a flow cytometry-based or enzyme-linked immunosorbent assay detection reagent.
Accordingly, the present invention also provides a PD-1 detection reagent comprising an anti-PD-1 nanobody, a nucleic acid molecule, a vector, a host cell and/or a composition provided by the present invention. Preferably, the detection reagent is a detection reagent based on flow cytometry or enzyme-linked immunosorbent assay.
The present invention also provides a method of detecting PD-1 in a sample from a subject, the method comprising contacting an anti-PD-1 nanobody, a nucleic acid molecule, a vector, a host cell and/or a composition provided by the present invention with the sample.
In yet another aspect, the present invention provides a kit comprising an anti-PD-1 nanobody, a nucleic acid molecule, a vector, a host cell and/or a composition provided by the present invention. Preferably, the kit is a detection kit, such as 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 IgG4-Fc comprising a modified hinge region, and is an improved nanobody based on the nanobody disclosed in CN 107814845A.
Considering that the eukaryotic cell HEK293 adopted when the antibody is expressed in CN107814845A is not a strain for industrial production of the antibody, IgG1 adopted is not a conventional antibody subtype aiming at PD-1 (anti-PD-1 monoclonal antibody therapeutic drugs are all IgG4 types, so that only blocking function is reserved, and ADCC effector function is weakened), therefore, the inventor synthesizes a VHH chain sequence of a nano antibody in CN107814845A on IgG4-Fc and adopts industrially used CHO cells for expression and purification, and finds that the purified VHH-IgG4-Fc (hereinafter referred to as PR antibody) has obvious floccule and also has obvious splitting peaks through CE-SDS detection, which indicates that the floccule and the uniformity of the antibody optimized by the IgG4-Fc are not obviously improved, and the research on the drug property of the antibody based on the VHH sequence, industrial production and clinical treatment application are difficult to realize.
In view of this, the inventors of the present invention further optimized the sequence from the research and development perspective of pharmaceutical properties, especially specific amino acid substitutions for multiple amino acid sites in VHH chain, hinge region and Fc region, screened and obtained novel anti-PD-1 nanobody with better activity and more application value by detecting physicochemical and biochemical properties (including homogeneity, binding affinity, antigen activity blocking effect and in vivo tumor suppression experiment) of the obtained protein, especially the nanobody has no floc after expression and purification, has good homogeneity, high binding affinity for the antigen PD-1, significantly improved in vivo tumor suppression effect, especially the curative effect can be combined with that of in vivo tumor suppression experiment(PaboLinzuzumab) which meets the requirements of using the linzuzumab as a drug development candidate molecule, is suitable for industrial production and has clinical application potential.
Drawings
Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:
FIG. 1: CE-SDS detection of VHH-IgG1-Fc and VHH-IgG4-Fc (PR antibody).
FIG. 2: and (3) comparing the sequences of the PR antibody and the optimized nano antibody.
FIG. 3: and (3) detecting results of the PR antibody and the optimized nano antibody by using the CE-SDS.
FIG. 4: SEC detection results of nanobodies N7 and N15 were optimized.
FIG. 5: tumor volume growth trend after administration.
FIG. 6: after the experiment, the mice had heavy tumors.
Detailed Description
The invention is illustrated below with reference to specific examples. It will be understood by those skilled in the art that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention in any way.
The experimental procedures in the following examples are conventional unless otherwise specified. The raw materials and reagent materials used in the following examples are all commercially available products unless otherwise specified.
In the following examples, the following procedure was used:
preparing and purifying a nano antibody sample:
a. synthesizing a gene sequence encoding a nano antibody sequence;
b. constructing the synthesized gene sequence on pcDNA3.1 vector, transforming into Escherichia coli DH5 alpha strain, shaking greatly, extracting pcDNA3.1 plasmid with Omega plasmid large extraction kit, filtering and sterilizing;
c. CHO-S cells were cultured to 5X 10 6 Per ml;
d. c, mixing the pcDNA3.1 plasmid obtained in the step b and a transfection reagent PEI in a ratio of 1:3 in a transfection medium ExpicHO TM Mixing with Expression Medium, standing for 20min, and addingIn CHO-S cells of step c, 6% CO at 37 ℃% 2 Culturing at 115rpm for 5 days;
e. centrifuging the culture product, and filtering with 0.2 μm filter membrane to obtain centrifugal supernatant;
f. purifying the centrifugal supernatant by adopting a ProA affinity chromatography, and washing away the foreign proteins and other impurities by using a Tris system buffer solution with the pH value of 7.0;
g. elution with 24mM acetic acid;
h. and (3) adjusting the pH of the obtained eluent back to 5.0 by using Tris Base, and filtering by using a 0.2-micron filter membrane to obtain a sample to be detected.
(II) appearance and visible foreign body detection of nano antibody sample
And (4) collecting the purified sample in a penicillin bottle, and observing the appearance of the nano antibody sample and the condition of visible foreign matters.
(III) CE-SDS detection of Nanobody samples:
a. solution preparation: respectively preparing a dilution buffer solution, DTT, NEM, a staining gel, a destaining solution, a molecular weight standard 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; adding 120mM N-ethyl maleimide (NEM) solution and non-reducing reagent (mixing the sample solution with ultrapure water) into the sample under non-reducing condition; heating ladder (step marker) and sample under reducing and non-reducing conditions, and adding to the chip; carrying out electrophoretic separation on a sample in a micro-channel filled with a polymer solution;
c. and (3) detection: based on laser-induced fluorescence of a fluorescent dye, a sample is added to a polymer solution and non-covalently bound to protein-SDS micelles;
d. and (4) analyzing results: the bioanalyzer software automatically sized according to ladder and calculated the percentage of each separation peak in the electropherogram.
SEC detection of (tetra) NanoAb samples
a. Preparation of a reagent: preparing solution a (100mM phosphate buffer, 100mM sodium sulfate, pH 2.8), solution B (100mM phosphate buffer, 100mM sodium sulfate, pH 7.0);
b. setting a detection system: operating a 'U3000 SEC' project and a '3000' chromatographic system, cleaning a pipeline A by using a solution A, cleaning a pipeline B by using a solution B, setting the column temperature to be 25 ℃, setting the temperature of a sample chamber to be 8 ℃, closing a cleaning valve, and balancing the system pressure after cleaning the system by using ultrapure water;
c. analyzing a sample: setting a sample sequence on Empower3, analyzing 1 needle of 2mg/ml for each sample after analyzing 6 needles of standard products, analyzing 1 needle of standard product after all the samples are analyzed, and sampling volume is 5 microliter; 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 nano antibody and application thereof, wherein the VHH sequence of the antibody is as follows:
SEQ ID NO.1(CDR1/CDR2/CDR 3: SEQ ID NO.2/SEQ ID NO.3/SEQ ID NO.4, underlined and shown in bold):
VHH-IgG4-Fc (PR antibody) was prepared according to the procedure described above "(preparation of Nanobody sample") wherein the amino acid sequence of IgG4-Fc is shown in SEQ ID NO. 5.
After purification of VHH-IgG1-Fc (see example 8 in CN 107814845A) and VHH-IgG4-Fc expressed in HEK293 disclosed in CN107814845A, and following the procedure described above for the appearance and detection of visible foreign bodies of the "(two) Nanobody samples, it was found that the purified antibody samples all exhibited significant flocs. Detection was carried out according to the procedure of "CE-SDS detection of three-nm antibody sample" above, and it was found that cleavage peaks were each detected, and the results are shown in FIG. 1A (VHH-IgG1-Fc) and 1B (VHH-IgG4-Fc), respectively.
Without being bound by any theory, it is believed that floc generation after purification, possibly due to specific amino acids of VHH, hinge region and Fc region, and poor homogeneity, further optimization of existing sequences from a pharmaceutical development perspective is required.
Example 2Sequence optimization design and preliminary screening of known anti-PD-1 nano antibody
In order to overcome the defects of floccule and poor uniformity after PR antibody purification, the PR antibody is optimized, and the optimization design scheme is shown in Table 1.
TABLE 1 optimization protocol for PR antibodies
Optimized domains | Optimized number (kind) |
CDR | 2 |
FR | 8 |
FR+CDR | 12 |
FR + Hinge (Hinge region) | 1 |
FR + polypeptide (linker peptide) | 3 |
Conformational Change (Fc + VHH, VHH attached to the C-terminus of Fc) | 1 |
Note: in table 1, CDR, optimization number 2 means that 2 kinds of sequence optimization schemes are designed for CDR regions. Similarly, 8 sequence optimization schemes are designed for the FR region; simultaneously, 8 sequence optimization schemes are designed for both FR and CDR regions; simultaneously, 1 sequence optimization scheme is designed aiming at both FR and Hinge regions; 3 schemes for increasing and connecting polypeptide (connecting peptide) while optimizing FR sequences are designed; the configuration change, namely the scheme that VHH is changed from the original N terminal connected to Fc to C terminal.
The optimized nanobody sample was prepared according to the procedure described in "(preparation of nanobody sample) above," and the sample detection was performed according to the procedure described in "(appearance and visible foreign substance detection of nanobody sample) above," and "(CE-SDS detection of nanobody sample) above. The results are shown in Table 2.
TABLE 2 detection results of optimized Nanobody samples
As can be seen from the CE-SDS detection results and the appearance of the purified samples, the samples were N7, N10, N12, N13, N14 and N15, which have good uniformity and no obvious floc formation.
Example 3Optimizing physicochemical and biochemical analysis of nano antibody
And performing physicochemical and biochemical analysis and further screening on the primarily screened N7, N10, N12, N13, N14 and N15. The sequences of the PR antibody and the optimized nanobody are shown below (the optimized sites are shown bold, italics and underlined; CDR regions are defined according to IMGT), and the sequence alignment is shown in FIG. 2. PR:
VHH (SEQ ID NO.1) (CDR1/CDR2/CDR 3: SEQ ID NO.2/SEQ ID NO.3/SEQ ID NO.4, underlined and bold):
IgG4-Fc (SEQ ID NO.5) (hinge region: SEQ ID NO.6, shown in bold):
N7:
VHH (SEQ ID NO.7) (FR1/CDR1/FR2/CDR2/FR3/CDR3/FR 4: 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 bold and FR2 mutated amino acids are underlined, italicized and bold):
IgG4-Fc (SEQ ID NO.8) (hinge region: SEQ ID NO.9, shown in bold, hinge region mutated amino acids shown underlined):
VHH + hinge region (SEQ ID NO. 10):
QVQLQESGGGLVQPGGSLRLSCAASGSTYLRFSMGWFRQVPGKEREGVAAIGGDGRTSYADSVKGRFTISKDNSKNTLYLDMNSLRAEDTAVYYCAAAVLLDGSFSLLAPLVPYKYDYWGQGTLVTVSSERKSSVECPPCP
full length antibody (SEQ ID NO. 11):
QVQLQESGGGLVQPGGSLRLSCAASGSTYLRFSMGWFRQVPGKEREGVAAIGGDGRTSYADSVKGRFTISKDNSKNTLYLDMNSLRAEDTAVYYCAAAVLLDGSFSLLAPLVPYKYDYWGQGTLVTVSSERKSSVECPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
N10:
VHH (SEQ ID NO.7) (FR1/CDR1/FR2/CDR2/FR3/CDR3/FR 4: 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 bold and FR2 mutated amino acids are underlined, italicized and bold):
linker peptide (SEQ ID NO. 12):
IgG4-Fc (SEQ ID NO.5) (hinge region: SEQ ID NO.6, shown in italics and bold):
VHH chain + linker peptide + hinge region (SEQ ID No. 13):
QVQLQESGGGLVQPGGSLRLSCAASGSTYLRFSMGWFRQVPGKEREGVAAIGGDGRTSYADSVKGRFTISKDNSKNTLYLDMNSLRAEDTAVYYCAAAVLLDGSFSLLAPLVPYKYDYWGQGTLVTVSSEPKIPQPQPKPQPQPQPQPKPQPKPEPEESKYGPPCPPCP
full length antibody (SEQ ID NO. 14):
QVQLQESGGGLVQPGGSLRLSCAASGSTYLRFSMGWFRQVPGKEREGVAAIGGDGRTSYADSVKGRFTISKDNSKNTLYLDMNSLRAEDTAVYYCAAAVLLDGSFSLLAPLVPYKYDYWGQGTLVTVSSEPKIPQPQPKPQPQPQPQPKPQPKPEPEESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
N12:
VHH (SEQ ID NO.15) (FR1/CDR1/FR2/CDR2/FR3/CDR3/FR 4: SEQ ID NO.31/SEQ ID NO.2/SEQ ID NO.32/SEQ ID NO.3/SEQ ID NO.33/SEQ ID NO.4/SEQ ID NO.30 wherein the CDR1, CDR2, CDR3 are underlined and bold and the FR1 and FR3 mutant amino acids are underlined, italicized and bold):
IgG4-Fc (SEQ ID NO.5) (hinge region: SEQ ID NO.6, shown in bold):
VHH chain + hinge region (SEQ ID No. 16):
QVQLQESGGGLVQPGGSLRLSSAASGSTYLRFSMGWFRQVPGKGLEGVAAIGGDGRTSYADSVKGRFTISKDNSKNTLYLDMNSLRAEDTAVYYSAAAVLLDGSFSLLAPLVPYKYDYWGQGTLVTVSSESKYGPPCPPCP
full length antibody (SEQ ID NO. 17):
QVQLQESGGGLVQPGGSLRLSSAASGSTYLRFSMGWFRQVPGKGLEGVAAIGGDGRTSYADSVKGRFTISKDNSKNTLYLDMNSLRAEDTAVYYSAAAVLLDGSFSLLAPLVPYKYDYWGQGTLVTVSSESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
N13:
VHH (SEQ ID NO.18) (FR1/CDR1/FR2/CDR2/FR3/CDR3/FR 4: 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 the CDR1, CDR2, CDR3 are underlined and bold and the FR1 and FR3 mutant amino acids are underlined, italicized and bold):
IgG4-Fc (SEQ ID NO.5) (hinge region: SEQ ID NO.6, shown in bold):
VHH chain + hinge region (SEQ ID NO. 19):
QVQLQESGGGLVQPGGSLRLSAAASGSTYLRFSMGWFRQVPGKGLEGVAAIGGDGRTSYADSVKGRFTISKDNSKNTLYLDMNSLRAEDTAVYYAAAAVLLDGSFSLLAPLVPYKYDYWGQGTLVTVSSESKYGPPCPPCP
full length antibody (SEQ ID NO. 20):
QVQLQESGGGLVQPGGSLRLSAAASGSTYLRFSMGWFRQVPGKGLEGVAAIGGDGRTSYADSVKGRFTISKDNSKNTLYLDMNSLRAEDTAVYYAAAAVLLDGSFSLLAPLVPYKYDYWGQGTLVTVSSSKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
N14:
VHH (SEQ ID NO.7) (FR1/CDR1/FR2/CDR2/FR3/CDR3/FR 4: 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 bold, FR2 mutant amino acids are underlined, italicized and bold)
Linker peptide (SEQ ID NO. 21):
IgG4-Fc (SEQ ID NO.5) (hinge region: SEQ ID NO.6, shown in bold):
VHH chain + linker peptide + hinge region (SEQ ID No. 22):
QVQLQESGGGLVQPGGSLRLSCAASGSTYLRFSMGWFRQVPGKEREGVAAIGGDGRTSYADSVKGRFTISKDNSKNTLYLDMNSLRAEDTAVYYCAAAVLLDGSFSLLAPLVPYKYDYWGQGTLVTVSSEPKIPQPQPKESKYGPPCPPCP
full length antibody (SEQ ID NO. 23):
QVQLQESGGGLVQPGGSLRLSCAASGSTYLRFSMGWFRQVPGKEREGVAAIGGDGRTSYADSVKGRFTISKDNSKNTLYLDMNSLRAEDTAVYYCAAAVLLDGSFSLLAPLVPYKYDYWGQGTLVTVSSEPKIPQPQPKESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
N15:
VHH (SEQ ID NO.7) (FR1/CDR1/FR2/CDR2/FR3/CDR3/FR 4: 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 bold, FR2 mutant amino acids are underlined, italicized and bold)
Linker peptide (SEQ ID NO. 24):
IgG4-Fc (SEQ ID NO.5) (hinge region: SEQ ID NO.6, shown in bold):
VHH chain + linker peptide + hinge region (SEQ ID No. 25):
QVQLQESGGGLVQPGGSLRLSCAASGSTYLRFSMGWFRQVPGKEREGVAAIGGDGRTSYADSVKGRFTISKDNSKNTLYLDMNSLRAEDTAVYYCAAAVLLDGSFSLLAPLVPYKYDYWGQGTLVTVSSEPKIPQPQPKPQPKPEPEESKYGPPCPPCP
full length antibody (SEQ ID NO. 26):
QVQLQESGGGLVQPGGSLRLSCAASGSTYLRFSMGWFRQVPGKEREGVAAIGGDGRTSYADSVKGRFTISKDNSKNTLYLDMNSLRAEDTAVYYCAAAVLLDGSFSLLAPLVPYKYDYWGQGTLVTVSSEPKIPQPQPKPQPKPEPEESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
3.1 CE-SDS detection
Sample detection was performed according to the "CE-SDS detection of Nanobody samples" procedure above. The results are shown in table 3 and 3a (pr), 3B (N7), 3C (N10), 3D (N12), 3E (N13), 3F (N14) and 3G (N15) in fig. 3.
TABLE 3 CE-SDS detection results of optimized Nanobody samples
3.2 SEC detection
The sample detection was performed according to the "SEC detection for four-nanometer antibody samples" procedure above. The results are shown in Table 4 and 4A (N7) and 4B (N15) in FIG. 4.
TABLE 4 SEC detection results for optimized 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)
And (3) detecting and optimizing the binding capacity of the nano antibody to the antigen PD-1 protein by ELISA. The method comprises the following steps:
a. antigen coating: taking 100 mu L of human PD-1 protein (Acro PD1-HB2F2) with the concentration of 20ng/mL per well, incubating for 2 hours at room temperature, and washing the plate for 5 times;
b. preparing reference products and test products: after the reference substance and the test substance are diluted to 1000ng/mL, carrying out gradient dilution;
c. reaction: adding the sample after gradient dilution into the 96-well plate pre-coated in the step a according to 100 mu L/well, covering a plate cover, incubating at room temperature for 2 hours, 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 a TMB chromogenic substrate into the sample plate, covering the cover plate, and incubating for 10-15 minutes at room temperature;
f. terminating the color development reaction: adding 2M sulfuric acid into the sample plate, and stopping the color reaction;
g. reading a plate: and (4) placing the sample plate in a microplate reader, and reading the plate.
The results are shown in Table 5.
TABLE 5 antigen binding assay results for optimized 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 assay of antigenic Activity
Detecting the blocking effect of the optimized nano antibody on the activity of the antigen PD-1 protein. The method comprises the following steps:
a. 100mL of FBS, 10mL of NEAA, 10mL of streptomycin solution, 4mL of Hygromycin B (50mg/L) and 800 μ L of Puromycin (10mg/L) are added into 1L of DMEM/F12 basal medium to prepare CHO-PD-L1-CD3L cell complete medium; CHO-PD-L1-CD3L cells were inoculated into the medium and subcultured every 2 days to logarithmic growth phase;
100mL of FBS, 10mL of NEAA, 10mL of streptomycin solution, 8mL of Hygromycin B (50mg/L) and 400 mu L of Puromycin (10mg/L) are added into 1L of 1640 basic culture medium to prepare a Jurkat-PD-1-NFAT cell complete culture medium; Jurkat-PD-1-NFAT cells are inoculated into a culture medium, and subculture is carried out to logarithmic phase every 2 days;
b. CHO-PD-L1-CD3L cells were digested and centrifuged, and the cells were resuspended to 5X 10 with DMEM/F12 complete medium 5 Adding 100 mu L of the cells/mL into a 96-well white bottom plate per well, and incubating for 16 +/-2 h in an incubator at 37 +/-2 ℃ and (5 +/-1)% CO 2;
c. pre-diluting the test sample, the reference sample and the quality control sample to 100 mu g/mL, and performing gradient dilution by using an analysis culture medium (RPMI1640 basic culture medium + 2% FBS);
d. taking out the cell culture plate in the step b, sucking the supernatant and discarding, and adding the diluted test sample, reference sample and quality control product into the cell culture plate, wherein each well is 50 mu L;
e. Jurkat-PD-1-NFAT cells were removed and the cells were resuspended to 2X 10 with assay medium 6 Per mL; adding the Jurkat cell suspension to the culture plate of step d at 50. mu.L per well, and incubating for 6h in an incubator;
f. taking out a Bio-Glo Luciferase Assay System reagent 1h in advance, standing at room temperature, and adding 100 mu L of reagent into each hole of the cell plate; and incubating for 2-3 min in a dark place at room temperature, and reading by using an enzyme-linked immunosorbent assay.
The results are shown in Table 6.
TABLE 6 test results for optimizing the blocking effect of the Nanobody on the antigen Activity
3.5 binding affinity (fortebio)
Detecting the binding affinity (fortebio) of the optimized nano-antibody to the antigen PD-1 protein. The method comprises the following steps:
a detection instrument: octet K2, pall-fortebio
Chip: protein A (manufacturer: Pall Fortebio, cat number: 18-5010)
Buffer solution: pH 7.4 PBST (pH 7.4 PBS, Twen200.05% v/v)
Software version: fortebio data analysis 10.0
The antibody to be detected (PR, N7, N10, N12, N13, N14 and N15) is captured through the specificity of a Protein A chip, the signal reaches 3nm, and the Protein is combined with PD-1 Protein with different concentrations diluted in a gradient manner.
Wherein, the protein of the sample to be detected is diluted by PBST buffer solution to the final concentration of 5 ug/mL. Lyophilized PD-1 protein with ddH 2 O was diluted to 250ug/mL and then diluted with PBST with a maximum concentration of 50nM, 2-fold concentration gradient, for a total of 7 concentrations.
The results are shown in Table 7.
TABLE 7 optimization of Nanobody binding affinity to antigen
The results of the data from 3.1 to 3.5 are summarized in Table 8.
TABLE 8 optimization of the results of physicochemical and biochemical analyses of Nanobodies
Example 4Detection of tumor inhibition effect of optimized nano antibody
The therapeutic effect of the optimized nanobody of the present invention on subcutaneous tumors was examined in a humanized mouse of C57BL/6hPD 1.
MC38-hPD-L1 tumor cells (colon cancer cells) are transplanted subcutaneously in a C57BL/6J-hPD1 humanized mouse, and an MC38-hPD-L1 transplantation tumor model is established. The method comprises the following steps:
1. the mice are isolated and adaptively raised for one week before test treatment;
2. MC38-hPD-L1 cells were cultured at 37 ℃ in 5% CO 2 Culturing and amplifying in the incubator, collecting cells in logarithmic growth phase and suspending inAdding matrigel into PBS, adjusting cell concentration to 1 × 10 7 Individual cells/mL;
3. injecting the cell suspension into the right side of the C57BL/6J-hPD1 humanized mouse subcutaneously by a 1mL syringe, and injecting 100 μ L of each mouse;
4. the mean tumor volume to be determined was about 112mm 3 In time, mice with too large, too small or irregular tumor shapes were eliminated.
Mice were divided into 5 groups by the random block method, i.e., a control group (group 1), Keytruda (group 2), PR antibody group (group 3), optimized nanobody N7 group (group 4), and optimized nanobody N15 group (group 5), with 8 mice per group. The administration was performed according to the protocol in Table 9, with each group administered intraperitoneally at a volume of 10ml/kg for 3 weeks, 2 times per week, and 6 times total. The antibody of each group is prepared into a use concentration of 1mg/ml, and the control group adopts normal saline, and the administration is started on the grouping day.
TABLE 9 dosing regimen
Tumor size was measured 2 times per week, mouse body weight was weighed, and mouse life status was 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 major axis; b represents the tumor minor axis.
2. Relative Tumor Volume (RTV)
RTV=V t /V initial ×100(%)
Wherein, V initial When administered in groups (i.e. d) initial ) Measurement of the resulting tumor volume, V t Tumor volume at each measurement.
3. Relative tumor proliferation rate T/C (%)
T/C(%)=(T RTV /C RTV )×100%
Wherein, T RTV Relative tumor volume, C, in the treatment groups RTV Relative tumor volumes for the solvent set are indicated.
4. Tumor volume tumor inhibition rate (TGI)
TGI=[1-(TV t -TV initial )/(CV t -CV initial )]×100%
Wherein, TV t Represents the tumor volume at each measurement in the treatment group; TV (television) initial Represents the tumor volume of the treatment group when administered in groups; CV of t Represents the tumor volume at each measurement of the control group; CV of initial The tumor volume of the control group at the time of group administration is shown.
Results are expressed as Mean and standard error (Mean ± SEM), and differences in tumor volume between control and administered groups were analyzed using T-test, with p <0.05 indicating statistical differences.
First, the change in tumor volume in mice is shown in table 10 and fig. 5.
TABLE 10 mouse tumor volume (mm) 3 )
Note: data are expressed as Mean + -SEM
"-": animal euthanasia
On day 14 after the start of the administration, the mean tumor volume of the control group was 4057.43. + -. 288.73mm 3 . Tumor volumes of groups 2, 3, 4 and 5 were 884.86 + -194.57 mm, respectively 3 、1584.55±186.82mm 3 、672.45±101.01mm 3 815.51 + -131.88 mm 3 The 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 (P) in tumor volume compared to the control group<0.01)。
Tumor growth inhibition of the antibodies was calculated for female humanized mice engrafted with hPD-L1 tumor based on tumor volume measured at day 14. The results are shown in tables 11 and 12.
TABLE 11 analysis of tumor growth inhibition
TABLE 12 comparison of tumor volumes between groups
Note: data are expressed as Mean + -SEM
Tumor volumes between the vehicle and treatment groups were compared using T-test analysis, indicating p <0.0001 and p < 0.05.
Second, the tumor weight of the mice
At the end of the experiment, the animals were euthanized, the tumor mass was removed and weighed, and the tumor weights of the mice were as shown in FIG. 6, specifically, the mean tumor weight of the control group was 7.0907 + -0.8349 g, and the mean tumor weights of the test samples Keytruda, PR, N7 and N15 were 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 of tables 10 to 12 and FIGS. 5 and 6 show that, in addition to the test substance PR, the treatment with the test substances N7, N15 and Keytruda antibody alone achieved excellent antitumor effects on the control group in the hPD-L1-MC38 tumor model. N7, N15 and Keytruda were significantly superior to PR antibody in antitumor effect, and N7 and N15 showed comparable antitumor effect in hPD-L1-MC38 tumor model compared to Keytruda. As Keytruda serving as a PD-1/PD-L1 monoclonal antibody medicament is approved by domestic and foreign drug administration for treating up to 13 tumors or cancers, great clinical benefits are brought to patients, and great commercial success is achieved, the anti-PD-1 nano antibody also has wide clinical treatment prospects as a replaceable nano antibody medicament with the drug effect not inferior to that of the anti-PD-1 nano antibody. In addition, the anti-PD-1 nano antibody provides a drug candidate molecule which better meets the requirement of drug development compared with PR.
The above description of the specific embodiments of the present invention is not intended to limit the present invention, and those skilled in the art may make various changes and modifications according to the present invention without departing from the spirit of the present invention, which is defined by the scope of the appended claims.
Sequence listing
<110> Zhejiang Terui Si pharmaceutical Co., Ltd
<120> anti-PD-1 nano antibody and application 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
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<211> 10
<212> PRT
<213> Artificial sequence (artificial sequence)
<220>
<223> VHH, CDR1
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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 (12)
1. An anti-PD-1 nanobody, characterized in that it consists of a VHH chain and an IgG4-Fc comprising a modified hinge region, wherein:
(i) the anti-PD-1 nano antibody VHH chain comprises a CDR1 shown in SEQ ID NO.2, a CDR2 shown in SEQ ID NO.3, a CDR3 shown in SEQ ID NO.4 and a framework region FR2 between a CDR1 and a CDR2 shown in SEQ ID NO. 28;
and is
(ii) The modified hinge region in the anti-PD-1 nano antibody comprises an amino acid sequence shown in SEQ ID NO.9, or consists of connecting peptide and an IgG4-Fc hinge region sequence shown in SEQ ID NO.6, wherein the connecting peptide comprises an amino acid sequence shown in SEQ ID NO.12, SEQ ID NO.21 or SEQ ID NO. 24.
2. The anti-PD-1 nanobody according to claim 1, wherein the nanobody VHH chain comprises the CDRs 1, 2 and 3 and 4 Framework Regions (FRs) therebetween, each region being arranged in the form of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and the FRs 1, FR3 and FR4 comprise the amino acid sequences shown in SEQ ID No.27, SEQ ID No.29 and SEQ ID No.30, respectively.
3. The anti-PD-1 nanobody according to claim 2, characterized in that the nanobody VHH chain comprises the amino acid sequence shown in SEQ ID No. 7.
4. The anti-PD-1 nanobody according to claim 3, characterized in that it comprises the amino acid sequence shown in SEQ ID No.10, SEQ ID No.13, SEQ ID No.22 or SEQ ID No. 25.
5. The anti-PD-1 nanobody according to claim 4, characterized in that the nanobody has the full-length amino acid sequence shown in SEQ ID No.11, SEQ ID No.14, SEQ ID No.23 or SEQ ID No. 26.
6. Use of the nanobody of any one of claims 1 to 5 in the preparation of a medicament for the treatment of a PD-1 or PD-L1-related disease.
7. Use according to claim 6, wherein the disease is a tumor or cancer.
8. The use according to claim 7, wherein the tumor or cancer is lung cancer, stomach cancer, liver cancer, melanoma, cervical cancer, colorectal cancer, bladder cancer, breast cancer, leukemia, lymphoma, renal cell carcinoma, cecum cancer, pancreatic cancer, cholangiocarcinoma, head and neck cancer, merkel cell carcinoma, ovarian cancer, nasopharyngeal cancer, glioma, esophageal cancer, bone cancer, or prostate cancer.
9. Use according to claim 8, wherein the disease is colorectal cancer.
10. A PD-1 detection reagent, wherein the PD-1 detection reagent comprises the anti-PD-1 nanobody of any one of claims 1 to 5.
11. The PD-1 detection reagent according to claim 10, wherein the PD-1 detection reagent is a flow cytometry-based or enzyme-linked immunosorbent assay detection reagent.
12. A kit comprising the PD-1 detection reagent according to claim 10 or 11.
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CN202210337061.1A CN114835810B (en) | 2022-03-31 | 2022-03-31 | anti-PD-1 nano antibody and application thereof |
PCT/CN2023/085322 WO2023186061A1 (en) | 2022-03-31 | 2023-03-31 | Anti-pd-1 nanobody, use thereof and method thereof for treating disease |
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN114984207A (en) * | 2022-05-09 | 2022-09-02 | 浙江特瑞思药业股份有限公司 | anti-PD-1 nano antibody preparation |
WO2023186061A1 (en) * | 2022-03-31 | 2023-10-05 | 浙江特瑞思药业股份有限公司 | Anti-pd-1 nanobody, use thereof and method thereof for treating disease |
Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1509293A (en) * | 2001-03-07 | 2004-06-30 | Ĭ��ר������˾ | Expression technology for proteins containing hybrid isotype antibody moiety |
CN103755804A (en) * | 2014-01-27 | 2014-04-30 | 南通市伊士生物技术有限责任公司 | Nano antibody for type A H3N2 influenza virus and application thereof |
US20170137517A1 (en) * | 2015-11-18 | 2017-05-18 | Edward Bowman | PD1 and/or LAG3 BINDERS |
CN108452318A (en) * | 2017-02-17 | 2018-08-28 | 浙江特瑞思药业股份有限公司 | Target the antibody coupling drug and its preparation method and application of CD20 |
US20190023793A1 (en) * | 2016-08-04 | 2019-01-24 | Innovent Biologics (Suzhou) Co., Ltd. | Anti-pd-l1 nanobody and use thereof |
CN112409483A (en) * | 2019-08-22 | 2021-02-26 | 浙江道尔生物科技有限公司 | anti-PD-L1 nano antibody |
CN113527512A (en) * | 2013-07-31 | 2021-10-22 | 美国安进公司 | Growth differentiation factor 15(GDF-15) constructs |
Family Cites Families (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
NZ563193A (en) * | 2005-05-09 | 2010-05-28 | Ono Pharmaceutical Co | Human monoclonal antibodies to programmed death 1(PD-1) and methods for treating cancer using anti-PD-1 antibodies alone or in combination with other immunotherapeutics |
CN107216389B (en) * | 2016-03-18 | 2022-03-29 | 和迈生物科技有限公司 | anti-PD-L1 nano antibody and coding sequence and application thereof |
CN107814845B (en) * | 2016-09-14 | 2021-02-09 | 浙江特瑞思药业股份有限公司 | Novel anti-PD-1 nano antibody and application thereof |
CN108205063B (en) * | 2016-12-20 | 2021-05-28 | 浙江特瑞思药业股份有限公司 | Detection method (filter membrane plate ELISA) for anti-CD 20 monoclonal antibody and antigen binding activity |
TWI788340B (en) * | 2017-04-07 | 2023-01-01 | 美商必治妥美雅史谷比公司 | Anti-icos agonist antibodies and uses thereof |
CN108299561B (en) * | 2018-01-02 | 2020-11-13 | 暨南大学 | PD-1 nano antibody and clone expression method and application thereof |
CN116987191A (en) * | 2019-06-27 | 2023-11-03 | 启愈生物技术(上海)有限公司 | anti-PD-L1 nano antibody, fc fusion protein thereof and application |
JP2022550243A (en) * | 2019-09-30 | 2022-12-01 | シチュアン ケルン-バイオテック バイオファーマシューティカル カンパニー リミテッド | Anti-PD-1 antibody and use thereof |
CN112574309B (en) * | 2019-12-05 | 2023-06-16 | 启愈生物技术(上海)有限公司 | anti-PD-L1 nano antibody and application thereof |
US20230235059A1 (en) * | 2020-06-26 | 2023-07-27 | Sorrento Therapeutics, Inc. | Anti-pd1 antibodies and uses thereof |
CN115028726B (en) * | 2022-03-31 | 2024-01-09 | 浙江特瑞思药业股份有限公司 | anti-PD-1 nano antibody and application thereof |
-
2022
- 2022-03-31 CN CN202210679078.5A patent/CN115028726B/en active Active
- 2022-03-31 CN CN202210337061.1A patent/CN114835810B/en active Active
-
2023
- 2023-03-31 WO PCT/CN2023/085322 patent/WO2023186061A1/en unknown
Patent Citations (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1509293A (en) * | 2001-03-07 | 2004-06-30 | Ĭ��ר������˾ | Expression technology for proteins containing hybrid isotype antibody moiety |
CN113527512A (en) * | 2013-07-31 | 2021-10-22 | 美国安进公司 | Growth differentiation factor 15(GDF-15) constructs |
CN103755804A (en) * | 2014-01-27 | 2014-04-30 | 南通市伊士生物技术有限责任公司 | Nano antibody for type A H3N2 influenza virus and application thereof |
US20170137517A1 (en) * | 2015-11-18 | 2017-05-18 | Edward Bowman | PD1 and/or LAG3 BINDERS |
US20190330340A1 (en) * | 2015-11-18 | 2019-10-31 | Merck Sharp & Dohme Corp. | Pd1 and/or lag3 binders |
US20190023793A1 (en) * | 2016-08-04 | 2019-01-24 | Innovent Biologics (Suzhou) Co., Ltd. | Anti-pd-l1 nanobody and use thereof |
CN108452318A (en) * | 2017-02-17 | 2018-08-28 | 浙江特瑞思药业股份有限公司 | Target the antibody coupling drug and its preparation method and application of CD20 |
CN112409483A (en) * | 2019-08-22 | 2021-02-26 | 浙江道尔生物科技有限公司 | anti-PD-L1 nano antibody |
Non-Patent Citations (2)
Title |
---|
DORA MUGOLI CHIGOHO等: "Site-Specific Radiolabeling of a Human PD-L1 Nanobody via Maleimide–Cysteine Chemistry", PHARMACEUTICALS, vol. 14, no. 6, pages 550 * |
王淑静等: "肿瘤免疫治疗PD-1/PD-L1靶向核素分子探针的研究进展", 肿瘤防治研究, vol. 47, no. 1, pages 70 - 76 * |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2023186061A1 (en) * | 2022-03-31 | 2023-10-05 | 浙江特瑞思药业股份有限公司 | Anti-pd-1 nanobody, use thereof and method thereof for treating disease |
CN114984207A (en) * | 2022-05-09 | 2022-09-02 | 浙江特瑞思药业股份有限公司 | anti-PD-1 nano antibody preparation |
CN114984207B (en) * | 2022-05-09 | 2024-01-26 | 浙江特瑞思药业股份有限公司 | anti-PD-1 nano antibody preparation |
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CN115028726A (en) | 2022-09-09 |
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