CN106928368B - FAP nano antibody Nb57 - Google Patents
FAP nano antibody Nb57 Download PDFInfo
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- CN106928368B CN106928368B CN201511024916.1A CN201511024916A CN106928368B CN 106928368 B CN106928368 B CN 106928368B CN 201511024916 A CN201511024916 A CN 201511024916A CN 106928368 B CN106928368 B CN 106928368B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
- B01J20/24—Naturally occurring macromolecular compounds, e.g. humic acids or their derivatives
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
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- 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
- C07K2317/565—Complementarity determining region [CDR]
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- 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]
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- 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®
Abstract
The invention discloses an FAP nano antibody aiming at FAP polypeptide molecular epitope, also discloses a gene sequence for coding the FAP nano antibody, an expression vector and a host cell capable of expressing the FAP nano antibody, and also discloses application of the FAP nano antibody. The FAP nano antibody, the gene sequence, the host cell and the like disclosed by the invention can be efficiently expressed in escherichia coli, have good specificity and high affinity with FAP immunoreaction, and can be applied to preparation of FAP detection reagents or anti-tumor drugs and the like.
Description
Technical Field
The invention belongs to the technical field of biomedicine or biopharmaceutical, and particularly relates to a nano antibody (Nb57) aiming at FAP polypeptide epitope molecules, a coding sequence and application thereof.
Background
Fibroblast Activation Protein (FAP) is a surface antigen specifically expressed by tumor-associated fibroblasts, has the activities of dipeptidyl peptidase and collagenase, has stable genome, and has the effects of promoting the growth, invasion and immunosuppression of tumor cells4、9.5×104、9.7×104The relative molecular mass of the dimer is 1.7 × 105. The FAP is structurally divided into a cytoplasmic region, a transmembrane region and an extracellular region, wherein the cytoplasmic region is a short peptide chain consisting of the first 6 amino acids, the transmembrane region is a hydrophobic transmembrane segment consisting of 19 amino acids and playing a role in fixation, however, most of the amino acid sequences 20-761 in FAP molecules are exposed in a microenvironment outside cells, and the part is the extracellular region and is a key enzyme catalytic region. FAP has dipeptide-based peptidase activity and collagenase activity, and can degrade dipeptide and type I collagen in a matrix. FAP has 50% homology with DPPIV (dipeptidyl peptidase IV/CD26) belonging to the family of dipeptidyl peptidases, all have exopeptidase activity and often form a complex. Thus, it is speculated that FAP may promote tumor development by modulating certain peptide growth factors in the tumor stroma, modifying active peptides and altering their functions.
In malignant tumors, FAP-positive fibroblasts account for only 2% of the total tumor cells, but when removed, rapid necrosis of both cancer cells and stromal cells occurs. Therefore, the FAP has very important effect on the generation and development of malignant tumors, and has wide application prospect in the aspect of tumor treatment. Therapeutic strategies targeting the tumor microenvironment, and in particular targeting FAP, play a very important role in tumor therapy. In the process of generating and developing tumors, tumor cells not only passively escape the attack of an immune system, but also actively inhibit the normal functions of immune cells in the growth environment of the tumor cells, and interact with the microenvironment where the tumor cells are located to form an inhibitory tumor microenvironment and promote the malignant development of the tumors.
Several FAP-specific monoclonal antibodies have been developed and are beginning to find application in clinical diagnosis and therapy, with improved affinity for tumor tissue. However, the traditional method has the defects of poor antibody stability, low sensitivity, high production cost and the like.
The nano antibody technology is an antibody engineering revolution carried out by biomedical scientists on the basis of traditional antibodies by applying molecular biology technology and combining the concept of nano particle science, so as to develop the latest and smallest antibody molecules, which are originally discovered in camel blood by leiman. While the common antibody proteins consist of two heavy chains and two light chains, the novel antibodies found in camelid blood have only two heavy chains and no light chains, these "heavy chain antibodies" bind tightly to targets such as antigens as normal antibodies, but do not aggregate together into clumps as single chain antibodies do. The nano antibody based on the 'heavy chain antibody' has the advantages of molecular weight of only 1/10 of common antibodies, more flexible chemical property, good stability, high solubility, easy expression and easy acquisition, and is easy to couple with other molecules. However, no suitable nanobody has been developed for FAP in the prior art.
Disclosure of Invention
The invention aims to solve the technical problem of providing an FAP nano antibody, a DNA molecule for coding the FAP nano antibody, application of the nano antibody and the like aiming at the defect that the nano antibody aiming at FAP epitope is lacked in the prior art.
The technical scheme adopted by the invention for solving the technical problems is as follows: in a first aspect of the present invention, a FAP nanobody is provided, which is a nanobody against FAP epitopes and includes two sequences as set forth in SEQ ID NO: 9, or a VHH chain of the amino acid sequence set forth in 9. The FAP nano antibody can be abbreviated as Nb 57.
In a second aspect of the invention, there is provided a VHH chain directed against FAP nanobody, comprising framework region FRs and complementarity determining region CDRs, said framework region FRs comprising the amino acid sequences of FRs of the following group: SEQ ID NO: FR1 as shown in 1, SEQ ID NO: FR2 as shown in 2, SEQ ID NO: FR3 as shown in SEQ ID NO: FR4 shown in FIG. 4; the complementarity determining region CDR includes the amino acid sequences of CDRs of the group: SEQ ID NO: 5, CDR1 shown in SEQ ID NO: 6, CDR2 shown in SEQ ID NO: CDR3 shown in FIG. 7. The structure of the framework region is relatively conserved, and the framework region mainly plays a role in maintaining the structure of the protein; the CDR structure is relatively diverse and is primarily responsible for antibody recognition.
Preferably, the VHH chain of the FAP nanobody has the amino acid sequence of SEQ ID NO: 9, or a pharmaceutically acceptable salt thereof.
In a third aspect of the invention, there is provided a DNA molecule for encoding a protein selected from the group consisting of: the FAP nanobody Nb57 disclosed by the invention or the VHH chain aiming at the FAP nanobody disclosed by the invention.
Preferably, said DNA molecule has the sequence of SEQ ID NO: 8.
The FAP nano antibody provided by the invention can be prepared in a large scale by a phage amplification or genetic engineering recombinant expression mode. Phage amplification refers to the mass propagation and production of phage particles displaying FAP nano antibody Nb57 by using phage displaying FAP nano antibody Nb57 through a biological amplification mode. The gene engineering recombination expression mode refers to that the gene coding the FAP nano antibody Nb57 is cloned to an expression vector to carry out mass preparation of the nano antibody in a protein expression mode.
In a fourth aspect of the invention, there is provided an expression vector comprising SEQ ID NO: 8.
In a fifth aspect of the invention, a host cell is provided, which can express the FAP nanobody Nb57 of the invention.
In a sixth aspect of the invention, the invention provides application of the FAP nanobody Nb57 in preparation of FAP detection reagents. The FAP nano antibody Nb57 can replace the traditional FAP antibody to prepare FAP detection reagent for detecting FAP polypeptide molecules. The invention also provides application of the FAP nano antibody Nb57 in preparation of antitumor drugs. The invention also correspondingly provides an FAP detection reagent or an anti-tumor drug, which comprises the FAP nano antibody Nb 57.
In a seventh aspect of the invention, the FAP nanobody Nb57 provided by the invention is used in immunological detection for non-treatment purposes. The immunological detection types comprise immunological analysis detection types based on antigen-antibody specific reaction, such as enzyme-linked immunosorbent assay, colloidal gold immunochromatography, immunodot hybridization and the like. When the FAP nano antibody Nb57 is applied, phage particles which are obtained by phage amplification and display FAP nano antibody Nb57 are directly used for analysis and detection, and FAP nano antibody Nb57 is subjected to prokaryotic organism or eukaryotic organism expression and then is subjected to immunological detection and analysis in the form of protein.
In an eighth aspect of the invention, the invention provides an application of the FAP nanobody Nb57 in preparation of a binding and adsorbing FAP reagent. For example, the FAP nanobody of the present invention may be prepared into immunoaffinity column, adsorbed to capture FAP, enriched for further research, etc.
The implementation of the invention has the following beneficial effects: the invention firstly synthesizes FAP polypeptide and makes it have immunogenicity, then couples FAP molecule on enzyme label plate, displays correct space structure of the protein, screens immune nano antibody gene library (camel heavy chain antibody phage display gene library) by using phage display technology with the antigen in this form, thereby obtains FAP specific nano antibody gene, transfers the gene into colon bacillus, and establishes nano antibody strain capable of high-efficiency expression in colon bacillus; the FAP nano antibody obtained by screening by the method has the immunoreaction characteristic with FAP, has good specificity and high affinity, and can be applied to preparation of FAP detection reagents or anti-tumor drugs and the like.
Drawings
The invention will be further described with reference to the accompanying drawings and examples, in which:
FIG. 1 is a schematic diagram of amino acid numbering and structure of FAP nanobody;
FIG. 2 is a DNA electrophoresis diagram of FAP nanobody;
FIG. 3 is an electrophoresis diagram of SDS-PAGE of FAP nano antibody after gel affinity chromatography on nickel column resin.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments.
The invention utilizes phage display technology to screen single-domain heavy-chain antibody (VHH) phage clones which can be specifically combined with target molecule FAP antigen from a camel source immunized single-domain heavy-chain antibody library, thereby obtaining a nano antibody strain which can be efficiently expressed in escherichia coli, namely FAP nano antibody Nb 57.
The invention will be further illustrated with reference to the following specific examples.
Example 1: construction of nanobody library for FAP:
(1) firstly, synthesizing FAP polypeptide, mixing 1mg FAP with Freund's adjuvant in equal volume, immunizing a Xinjiang dromedary camel once a week for 7 times in total, and stimulating B cells to express antigen-specific nano antibodies;
(2) after 7 times of immunization, extracting 100mL camel peripheral blood lymphocytes and extracting total RNA;
(3) synthesizing cDNA and amplifying VHH by using nested PCR;
(4) utilizing restriction enzymes PstI and NotI to cut 20ug pComb3 phage display vector (supplied by Biovector Chinese plasmid vector strain cell gene collection center) and 10ug VHH and connect the two fragments, (5) transforming the connection product into electrotransformation competent cell TG1 to construct FAP nano antibody library and determine the library capacity, wherein the size of the library capacity is 1.85 × 108。
Example 2: nano antibody screening process for FAP:
(1) the solution was dissolved in 100mM NaHCO3Coupling 20ug FAP in pH 8.2 on NUNC enzyme label plate, standing at 4 deg.C overnight;
(2) adding 100uL of 0.1% casein in the next day, and sealing for 1-3h at room temperature;
(3) after 1-3h, 100uL phage (5 × 10) was added11tfu immune camel nanometer antibody phage display gene bank), acting for 1-2h at room temperature;
(4) washing 4-6 times with 0.05% PBS + Tween-20 to wash away unbound phage;
(5) phages specifically binding to FAP were dissociated with 100mM TEA (triethylamine) and infected with escherichia coli TG1 in log phase growth, cultured for 1h at 37 ℃, phages were generated and purified for the next round of screening, and the same screening process was repeated for 3-5 rounds, gradually enriched.
Example 3: screening of specific single positive clones by phage enzyme-linked immunosorbent assay (ELISA):
(1) from the phage-containing cell culture dishes after the 3-5 rounds of selection described above, 96 individual colonies were picked and inoculated into TB medium containing 100. mu.g per ml of ampicillin (2.3 g of potassium dihydrogen phosphate, 12.52 g of dipotassium hydrogen phosphate, 12 g of peptone, 24 g of yeast extract, 4 ml of glycerol in 1L of TB medium), grown to a logarithmic phase, followed by addition of Isopropylthiogalactoside (IPTG) at a final concentration of 1 mmol and overnight incubation at 30-35 ℃.
(2) Crude antibody is obtained by using a permeation method, and the antibody is transferred to an ELISA plate coated by the antigen and is placed for 1-1.5 hours at room temperature.
(3) Unbound antibody was washed away with PBST, and a primary anti-mouse anti-HA antibody (mouse anti-HA tagganty, available from Beijing kang, century Biotechnology Co., Ltd.) was added and allowed to stand at room temperature for 1-1.5 hours.
(4) Unbound antibody was washed away with PBST, and anti-mouse alkali line phosphataseconjugate (goat anti-mouse alkaline phosphatase-labeled antibody, available from Eimei technologies, Ltd.) was added and allowed to stand at room temperature for 1 to 1.5 hours.
(5) Unbound antibody was washed away with PBST, and absorbance was read on an ELISA instrument at 405nm by adding an alkaline phosphatase developing solution.
(6) And when the OD value of the sample well is more than 3 times larger than that of the control well, judging the sample well to be a positive clone well.
(7) The bacteria of the positive cloning wells were shaken in LB liquid containing 100. mu.g per ml to extract the plasmid and sequenced.
Through the experiment, the invention obtains 6 positive cloning holes which show more than 3 times of binding force with antigen, and the positive cloning holes are regarded as primary screening positive cloning strains. Analyzing the gene sequences of the primary screening positive clones according to the Vector NTI of the sequence alignment software, regarding the strains with the same CDR1, CDR2 and CDR3 sequences as the same clones, and regarding the strains with different sequences as different clones, so as to obtain the amino acid sequences of VHH chains of a group of antibodies, such as SEQ ID NO: shown at 9. The FAP nanobody is numbered as Nb57, and the amino acid numbering and structural diagram are shown in FIG. 1.
Please refer to fig. 2, which is a DNA electrophoresis diagram of the FAP nanobody. The DNA bands of the first gel well labeled M in the figure are: DNA molecular markers of 1000bp, and the DNA bands of gel holes marked from left to right by 1-24 are FAP nano antibody DNA electrophoresis bands. The DNA of the 1-24 gel holes is the PCR product of FAP nano antibody DNA in the positive clone strain, and the PCR product band is about 500 bp.
Example 4: the nano antibody is expressed and purified in host bacterium escherichia coli:
(1) the amino acid sequence obtained by the previous sequencing analysis is shown as SEQ ID NO: the plasmid of the clone shown in 9 is electrically transformed into escherichia coli WK6, and is coated on a culture plate containing LA + glucose, namely ampicillin and glucose, and is cultured overnight at 37 ℃;
(2) selecting a single colony, inoculating the single colony in 5mL LB culture solution containing ampicillin, and carrying out shake culture at 37 ℃ overnight;
(3) inoculating 1mL of overnight strain into 330mL of TB culture solution, carrying out shake culture at 37 ℃, adding IPTG (isopropyl-beta-thiogalactoside) when the OD value reaches 0.6-1, and carrying out shake culture at 30-35 ℃ overnight;
(4) centrifuging and collecting bacteria;
(5) obtaining antibody crude extract by using an osmosis method;
(6) the protein with purity of more than 90 percent can be prepared by nickel column ion affinity chromatography.
Please refer to fig. 3, which is an electrophoresis diagram of SDS-PAGE of FAP nanobody after purification by nickel column resin gel affinity chromatography. The reference mark M represents a protein molecular marker, and the reference mark Nb57 represents an SDS-PAGE band of the FAP nano antibody Nb57 obtained in the embodiment 4 of the invention, and the unit in the figure is KDa. The result shows that the size of the FAP nano antibody Nb57 is about 15KDa, and the purity of the FAP nano antibody Nb57 can reach more than 95% after the purification process.
The invention also carries out kinetic affinity analysis on the FAP nano antibody Nb57 purified in the example 4, and the experiment shows that the binding rate constant is 3.01 × 104(Unit M)-1S-1) Dissociation rate constant of 1.23 × 10-3(unit S)-1) Thereby calculatingAn equilibrium dissociation constant of 4.09 × 10 was obtained-8(unit M). Compared with affinity data of other clones selected from a phage library, the FAP nano antibody Nb57 has a low equilibrium dissociation constant, represents that the antibodies and antigens are difficult to dissociate, and has strong affinity.
While the invention has been described with reference to specific embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the scope of the invention. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the invention without departing from the essential scope thereof. Therefore, it is intended that the invention not be limited to the particular embodiments disclosed, but that the invention will include all embodiments falling within the scope of the appended claims.
Claims (9)
1. The FAP nanobody is a nanobody aiming at FAP epitope, and the amino acid sequence of VHH chain of the FAP nanobody is shown in SEQ ID NO: shown at 9.
2. A VHH chain directed against a FAP nanobody comprising framework region FRs and complementarity determining region CDRs, wherein the framework region FRs comprise the amino acid sequences of FRs of the group: SEQ ID NO: FR1 as shown in 1, SEQ ID NO: FR2 as shown in 2, SEQ ID NO: FR3 as shown in SEQ ID NO: FR4 shown in FIG. 4; the complementarity determining region CDR includes the amino acid sequences of CDRs of the group: SEQ ID NO: 5, CDR1 shown in SEQ ID NO: 6, CDR2 shown in SEQ ID NO: CDR3 shown in FIG. 7.
3. A DNA molecule for encoding a protein selected from the group consisting of: the FAP nanobody of claim 1, or the VHH chain directed against the FAP nanobody of claim 2.
4. The DNA molecule of claim 3, having a nucleotide sequence as set forth in SEQ ID NO: shown in fig. 8.
5. An expression vector comprising the nucleotide sequence of SEQ ID NO: 8.
6. A host cell capable of expressing the FAP nanobody of claim 1.
7. Use of the FAP nanobody of claim 1 in the preparation of FAP detection reagents or antitumor drugs.
8. An FAP detection reagent or an antitumor drug, comprising the FAP nanobody of claim 1.
9. Use of the FAP nanobody of claim 1 for the preparation of a binding-adsorbing FAP reagent.
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Citations (4)
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|---|---|---|---|---|
| EP1806365A1 (en) * | 2006-01-05 | 2007-07-11 | Boehringer Ingelheim International GmbH | Antibody molecules specific for fibroblast activation protein and immunoconjugates containing them |
| WO2011040972A1 (en) * | 2009-10-02 | 2011-04-07 | Ludwig Institute For Cancer Research Ltd. | Anti-fibroblast activation protein antibodies and methods and uses thereof |
| CN103154038A (en) * | 2010-08-13 | 2013-06-12 | 罗切格利卡特公司 | Anti-fap antibodies and methods of use |
| CN104447988A (en) * | 2014-12-11 | 2015-03-25 | 东南大学 | Bactrian camel-derived ApoE nano antibody as well as coding sequence and application thereof |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1806365A1 (en) * | 2006-01-05 | 2007-07-11 | Boehringer Ingelheim International GmbH | Antibody molecules specific for fibroblast activation protein and immunoconjugates containing them |
| WO2011040972A1 (en) * | 2009-10-02 | 2011-04-07 | Ludwig Institute For Cancer Research Ltd. | Anti-fibroblast activation protein antibodies and methods and uses thereof |
| CN103154038A (en) * | 2010-08-13 | 2013-06-12 | 罗切格利卡特公司 | Anti-fap antibodies and methods of use |
| CN104447988A (en) * | 2014-12-11 | 2015-03-25 | 东南大学 | Bactrian camel-derived ApoE nano antibody as well as coding sequence and application thereof |
Non-Patent Citations (2)
| Title |
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| Introduction to Heavy Chain Antibodies and Derived Nanobodies;Cecile Vincke et al.;《Methods in Molecular Biology》;20121231;第911卷;第15-26页 * |
| 小鼠成纤维细胞激活蛋白多克隆抗体的制备及应用;江冠民等;《现代肿瘤医学》;20140131;第22卷(第1期);第29-33页 * |
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