CN110804099B - Human CD70 antigen binding polypeptides - Google Patents
Human CD70 antigen binding polypeptides Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70575—NGF/TNF-superfamily, e.g. CD70, CD95L, CD153, CD154
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
- C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
- G01N2333/70575—NGF/TNF-superfamily, e.g. CD70, CD95L, CD153 or CD154
Abstract
The invention discloses a human CD70 antigen-binding polypeptide, which takes human CD70 as a target spot and develops the antigen-binding polypeptide with good specificity and higher affinity. Compared with the prior art, the invention successfully prepares the human CD70 antigen binding polypeptide (fusion protein), the polypeptide has good specificity and high affinity, and can be combined with human CD70 expressed on the surface of cells, and the anti-human CD70 monoclonal antigen binding polypeptide is a potential drug for tumor immunotherapy.
Description
Technical Field
The invention relates to a human CD70 antigen binding polypeptide, belonging to the technical field of antigen binding polypeptides.
Background
The CD70 molecule, tumor necrosis factor receptor superfamily 7 factor (TNFSF 7). The polypeptide chain length is 193 a.a., the molecular weight is 21.1KD, the polypeptide is a II type transmembrane glycoprotein (a soluble form is not seen yet), and 155 amino acids at the C end are positioned outside a membrane. Disulfide bonds are formed among the first cysteine, the third cysteine, the second cysteine and the fourth cysteine outside the cells, and the spatial structure of the extracellular part is maintained. Usually it is present on the membrane as a homotrimer.
In normal tissues, the cellular expression profile of CD70 is narrow, and is mainly expressed in B cells in germinal centers (inducing secretion of antibodies) and T cells in local lymphoid tissues, and is expressed at low levels in thymic medullary epithelial cells. However, its expression can be greatly increased by antigen induction and decreased with the attenuation of immune response, such as high expression of CD70 in T, B cells activated by antigen induction. Meanwhile, the expression of the polypeptide is regulated by cytokines, such as IL1a, IL12, TNFa and GM-CSF can up-regulate the expression of CD70, and IL4 and IL10 can down-regulate the expression of CD 70. In addition, in mature DCs, CD70 is expressed under the mediation of the TLR signaling pathway. These phenomena just suggest that CD70 is a costimulatory molecule required for complete activation of naive T cells and plays an important role in the regulation of immune responses. It interacts with the ligand CD27, not only acting as a secondary signal for T cell activation, but also the interaction between the two can regulate the proliferation and differentiation of immune cells such as B, NK.
CD70, transiently expressed in activated T and B lymphocytes and mature DC cells, is highly expressed in a variety of tumor tissues in pathological conditions. A large number of researches show that the CD70 is expressed at high level in a plurality of tumor tissues such as lymphoma, renal carcinoma, glioma, breast cancer, blood-borne malignant tumors (such as Hodgkin lymphoma, multiple myeloma, leukemia and the like). Expression of CD27 was also observed to increase or decrease in these tissues in synchrony with CD 70. Probably, the two can be used as a ligand/receptor pair to form a feedback loop to promote the proliferation and differentiation of lymphocytes and play a role in mediating diseases. After the interaction between CD70 and CD27, CD27 is activated, TRAF2/5 is combined with the intracellular segment to further activate NF-KB and c-Jun kinase signal channels, and the cell proliferation, survival, differentiation and the like are caused. In addition, it is also believed that binding of CD70 to CD27 may also cause the intracellular segment of CD27 to bind to Siva and thereby cause Caspase-mediated apoptosis.
In view of the above, the development of tumor immunotherapy by targeting CD70 to produce specific antibodies is of great interest. At present, aiming at a CD70 target, three application modes of CART treatment, antibody drug and antibody coupling drug are all developed, the most rapidly developed application mode is Cusatuzumab (Cusatuzumab) developed by combining the Qiangsheng and argEN-X, the phase I clinic is completed at present, and the result shows that the combination of Cusatuzumab and Vidaza can obviously reduce leukemia stem cells in the bone marrow of AML patients, and the reliability of the target is proved.
In view of the important immunoregulation function of the CD70/CD27 molecules, the regulation and control of CD70 inevitably causes a series of signal transmission and physiological reactions of T cells, even B cells, NK cells and other lymphocytes, so that the CD-modified polypeptide is an ideal immunotherapy target, and reasonable application is possible to mobilize multiple cellular immunity. Meanwhile, the target is taken, and the development direction of the medicine is more (CART treatment, antibody medicine and antibody coupling medicine, even multispecific antibody and the like).
The antibodies in clinical experiments at present include Cusatuzumab, Vorsetuzumab and humanized antibodies in antibody conjugate drugs, and the antibodies have antibody structures such as a variable region and a constant region, wherein the variable region can be still subdivided into a framework region and a hypervariable region, so that the variable region is still a traditional antibody and is not a polypeptide. In the traditional antibody medicine, an antibody humanization link is required in the development process so as to reduce the immunogenicity in human bodies through antibody engineering. With the current state of the art, antibody humanization remains a challenging task with few technical hurdles, high engineering risks, time, labor, and expense.
Disclosure of Invention
The purpose of the invention is as follows: aiming at the problems in the prior art, the invention discloses a human CD70 antigen-binding polypeptide.
The technical scheme is as follows: in order to achieve the purpose, the invention adopts the following technical scheme:
a human CD70 antigen binding peptide, wherein the polypeptide is single chain structure and comprises an antigen binding part with the amino acid sequence shown as SEQ ID NO. 1.
As one embodiment of the invention, the human CD70 antigen binding peptide comprises a signal peptide and an antigen binding site, and the amino acid sequence of the antigen binding peptide is shown as SEQ ID NO. 2.
Alternatively, the human CD70 antigen binding peptide comprises a signal peptide, an antigen binding site and a human Fc region, and the amino acid sequence of the human CD70 antigen binding peptide is shown as SEQ ID NO. 3.
Or, the human CD70 antigen-binding peptide comprises a signal peptide, an antigen-binding site, a flexible connecting peptide and a human Fc region, and the amino acid sequence of the human CD70 antigen-binding peptide is shown in SEQ ID NO. 4.
The preparation method of the human CD70 antigen-binding peptide is a method for screening by adopting a phage display peptide library. The specific method comprises the following steps:
commercial recombinant human CD70 His-tag protein and phage display 12 peptide library were used as starting materials. The CD70 His-tag protein was coated on an immune tube, incubated with a 12-peptide library, and unbound/weakly bound phage were washed off, and phage bound to the CD70 His-tag protein were eluted. This was repeated several times, each time the coating amount of the CD70 His-tagged protein and the washing conditions were changed, and the phages with weak binding were eliminated successively and more phages with strong binding were retained as much as possible. The obtained phage with strong binding ability were monocloned using plaque formation assay, ELISA screening was performed using monoclonal culture supernatants, and nucleic acid sequence determination was performed on the monoclonals. Establishing a coding sequence (in one embodiment of the invention, connecting a signal peptide of the secreted luciferase, a dodecapeptide, namely an antigen binding site, human IgG2 Fc and a His tag coding sequence), reading in frame, and constructing a mammalian expression vector. CHO-K1 cells were transfected and the supernatant was removed for flow cytometry. And (3) a large number of positive expression vectors are transfected and subjected to flow assay, the fusion protein is obtained by purifying the supernatant by using a Ni-NTA affinity chromatography column, and the binding property of the fusion protein and CD70 is identified.
A nucleic acid molecule comprising a sequence encoding said human CD70 antigen binding peptide.
A vector comprising said nucleic acid molecule.
A host cell comprising said nucleic acid molecule or said vector.
A kit comprising said human CD70 antigen binding peptide.
Use of said human CD70 antigen binding polypeptide in the preparation of a kit for detecting the presence or level of CD70 in a sample.
The human CD70 antigen-binding peptide is applied to the preparation of anti-cancer or cancer detection or medicines.
The invention takes CD70 as a target spot to develop antigen-binding polypeptide. The antigen-binding polypeptides are all protein substances like antibodies, and all have an antigen-binding function. Thus, antigen-binding polypeptides have the same function and drug development direction as antibodies. However, unlike antibodies, the polypeptide is a novel target conjugate, and there is no report on the development of antigen-binding polypeptides targeting CD 70. Compared with an antibody, the polypeptide of the invention is 12 peptide, has the advantages of extremely low immunogenicity, no immunity, avoidance of the antibody humanization link, easy blood vessel penetration, various possible administration modes (oral administration, intramuscular injection and blood injection), easy storage and transportation and the like. Meanwhile, in the implementation process of the invention, a method for screening the phage display peptide library is adopted, and the phage display peptide library is applied to the development of CD70 targets, which is also an innovation of the invention.
The technical effects are as follows: compared with the prior art, the invention successfully prepares the human CD70 antigen binding polypeptide (fusion protein), the polypeptide has good specificity and high affinity, and can be combined with human CD70 expressed on the surface of cells, and the anti-human CD70 monoclonal antigen binding polypeptide is a potential drug for tumor immunotherapy.
Description of the drawings:
FIG. 1: the phase crude culture solution and the human CD70 are combined to obtain the ELISA detection result. In the phage panning process, duplicate validation was performed with 50ul and 5ul aliquots of LB medium containing the phage virus particles.
FIG. 2: the structure of a vector used for transfection of a CHO-K1 cell line for overexpression of the human CD70 antigen-binding polypeptide fusion protein is shown, MCS is a multiple cloning site, and a target gene is inserted into the position.
FIG. 3: and (3) detecting the CHO-K1 overexpression human-derived CD70 antigen-binding polypeptide fusion protein by SDS-PAGE. The left and right lanes in the figure are untransfected and post-transfected CHO-K1 cells, respectively, with expressed fusion protein boxed.
FIG. 4: the human CD70 and the antigen-binding polypeptide fusion protein are combined with the flow detection result. The black and red bars in the figure represent the binding signals to CHO-K1 cell line and human CD70 cell line overexpressing CHO-K1 cell line, respectively.
FIG. 5: and (3) carrying out titer ELISA detection on the purified human-derived CD70 antigen-binding polypeptide fusion protein. The purified fusion protein configured by concentration gradient was conjugated to human CD70 and reacted with HRP-labeled anti-human Fc antibody.
Detailed Description
The present invention will be further illustrated with reference to the following specific examples.
Examples
(1) Phage display peptide library panning
1) Activation of host bacteria ER2738 (purchased from NEB): sterile LB plates (1g/100ml peptone, 0.5g/100ml yeast powder, 0.5g/100ml NaCl) containing 20ug/ml tetracycline were prepared and cultured overnight by streaking ER2738 in a 37 ℃ incubator.
2) Coating: human CD70 protein (purchased from Acro Biosystem) was diluted at a concentration of 50ug/ml in 0.1M NaHCO3(pH8.6), 1.5ml of the solution was added to an immunization tube and the tube was coated overnight at 4 ℃. After the coating liquid is removed, the solution is sealed with a sealing liquid [0.1M NaHCO ]3(pH8.6),5mg/ml BSA]Incubation for 1 hour, after completion with TBST [50mM Tris-Cl (pH7.5),150mM NaCl, 0.1% Tween-20]Washing for 4 times.
3) Combining: get 1011pfu phage virions (purchased from NEB) were added to an immune tube containing 1.5ml TBST and incubated for 1 hour. The coating solution was removed and washed with TBST 4 times.
4) And (3) elution: repeatedly washing the immune tube with 1ml of eluent [0.2M glycine-hydrochloric acid (pH2.2) ] for 10min, adding 0.15ml of neutralizing solution [1M Tris-Cl (pH9.1) ] and mixing, and temporarily storing at 4 deg.C
5) And (3) measuring the titer: mixing 2ul, 0.2ul (2 ul after 10 times of stock solution diluted with LB) and 0.02ul (2 ul after 100 times of stock solution diluted with LB) with 0.2ml of ER2738 in middle logarithmic phase (OD600 ═ 0.5), incubating at room temperature for 5min, adding into preheated 3ml of top agar at 42 deg.C, and mixing. Quickly adding the mixture to an LB/IPTG/XGAl plate preheated at 37 ℃ and uniformly spreading. The plates were incubated overnight at 37 ℃ and the number of blue plaques on the plates was counted and the titer was calculated from the dilution factor.
6) Phage amplification: according to the proportion of 1:100, the overnight cultured ER2738 bacterial liquid is inoculated into 20ml of LB culture solution, 800ul of eluent is added at the same time, and the shaking culture is carried out at the temperature of 37 ℃ and the rpm of 250 for 4.5 hours. After centrifugation at 6000rpm for 10 minutes, the cells were removed, and 1/5 volumes of 2.5M NaCl/20% PEG8000 were added to the supernatant, which was then cooled in ice bath for 2 hours. Centrifugation at 10000rpm for 10min gave phage precipitate, the residue was removed and 0.2ml TBS solution was added to resuspend the precipitate and titer was determined as above.
7) Repeating the steps 2) -6) twice, adjusting the coating concentration of the CD70 protein to 30ug/ml in the first repetition, adjusting the coating concentration to 15ug/ml in the second repetition, increasing the washing times and keeping the rest unchanged.
(2) Monoclonal ELISA
1)0.5ug/ml human CD70 protein 4 ℃ overnight coated ELISA plate, blocking solution treatment for 2h, and PBS washing 4 times.
2) The strain ER2738 was cultured overnight with shaking and diluted 1: 100. And taking the plaque on the titer plate after the second repeated elutriation, adding the plaque into the diluted bacterial liquid, carrying out shaking culture at the temperature of 37 ℃ and the rpm of 250 for 4.5 hours, and centrifuging to remove the thallus.
3) Taking 50ul or 5ul of supernatant, taking TBST as a medium, supplementing 100ul, adding into an enzyme label plate, incubating for 1 hour, removing liquid, and washing with PBS for 4 times.
4) The HRP-labeled anti-M13 phage antibody was diluted with 1% nonfat dry milk in PBS, 100ul of the diluted antibody was added to the microplate, incubated for 1 hour, drained and washed 4 times with PBS.
5) Adding 100ul TMB developing solution, incubating at 37 deg.C for 10min or until yellow color is fully developed, adding 100ul 1M sulfuric acid to stop reaction, and reading OD450 on enzyme-labeling instrument. The results are shown in FIG. 1, the monoclonal ELISA reading value is obviously higher than that of the control, the result is positive, and the monoclonal ELISA reading value has the function of binding the human-derived CD70 protein at the ELISA level.
(3) Preparation and flow detection of human-derived CD70 antigen-binding polypeptide fusion protein
1) Phage genomic DNA was extracted from the ER2738 culture supernatant inoculated with monoclonal plaques and sequenced.
2) The chemical synthesis antigen-binding polypeptide fusion gene sequence is characterized in that a signal peptide of secreted luciferase, a dodecapeptide bound with CD70, a flexible connecting peptide, human IgG2 Fc and a coding sequence of His tag are connected and read in frame, the nucleic acid sequence is shown as SEQ ID NO.5, and the amino acid sequence of the coded fusion antibody is shown as SEQ ID NO. 6.
3) The above nucleic acid fragments were inserted into the MCS region of eukaryotic expression vector pLOE-purOR in the order (FIG. 2) to synthesize a fusion protein having a signal peptide at the N-terminus, an antigen-binding dodecapeptide at the middle segment, and Fc and His tags at the C-terminus.
4) Sterile plasmid without endotoxin was prepared, CHO-K1 cells were transfected according to the transfection reagent requirements, and the supernatant was collected.
5) SDS-PAGE detects the expression of the antibody, and as shown in FIG. 3, the fusion antigen-binding polypeptide is successfully expressed.
6) Flow assay for cell binding capacity
① wild-type CHO-K1 cells and CHO-K1 cells overexpressing human CD70 were extensively digested with 0.25% pancreatin and single cell suspensions were prepared by gentle pipetting with PBS.
② 10ml PBS washed cells 1 times, 1000rpm centrifugation for 5min, then 1ml PBS suspension cells, cell count.
③ is 2.5 × 105The cells were collected by centrifugation in a 1.5ml centrifuge tube.
④ mu.l CHO-K1 cell culture supernatant over-expressing human CD70 antigen-binding polypeptide fusion protein was added, mixed well and incubated at room temperature for 30 min.
⑤ cells were harvested by centrifugation and washed 1 time with 1ml PBS.
⑥ mu.l of 20-fold diluted secondary antibody (2.5. mu.l/2.5X 10) against human IgGFc fragment from goat labeled with APC was added5Optionally binding with human IgG) and reacting at room temperature in the dark for 20 min.
⑦ cells were washed 1 time with 1ml PBS, centrifuged at 1000rpm for 8min and the supernatant removed.
⑧, adding 200 μ l PBS to re-suspend into single cell suspension, detecting on a flow cytometer, as shown in FIG. 4, compared with wild type CHO-K1 cell strain which does not express CD70, the CHO-K1 cell strain which expresses CD70 with high expression has obviously enhanced signal, the histogram shifts to the right, and the antibody can be combined with CD70 on the cell surface.
(4) Preparation of human-derived CD70 antigen-binding polypeptide fusion protein by CHO-K1 cell
1) Large quantities of positive plasmids were prepared by transfection of three T25 flasks of CHO-K1 cells.
2) 72 hours after transfection, the supernatant (total 12ml) was collected while cells were trypsinized and collected by centrifugation.
3) Cells were suspended in 5ml cold PBS. The ultrasonic power is set to 25% of the maximum power, the work is carried out for 2s, the pause is carried out for 6s, and the total time is 3 min. Cells were disrupted by sonication in an ice bath and combined with cell culture supernatant after completion. A cocktail protease inhibitor was added.
4) Taking 4ml of Ni-NTA agarose gel, adding the gel into a screening plate of a purification column, and filling a gravity purification column.
5) The column was equilibrated with 10ml of 20mM Tris-HCl buffer (pH 8.0)/500mM NaCl.
6) The combined solution was applied to a purification column at low temperature, and the flow rate was controlled at 3 s/drop.
7) The column was washed with 10ml of 20mM Tris-HCl buffer (pH 8.0)/500mM NaCl solution containing 30mM imidazole.
8) The antigen-binding peptide fusion protein was eluted with 4ml of 20mM Tris-HCl buffer (pH 8.0)/500mM NaCl solution containing 300mM imidazole.
9) Adding into deionized water-wetted ultrafiltration tube with cut-off of 10KD, cooling PBS to 10ml, and centrifuging at 4 deg.C and 3000rpm for 10min until the liquid surface is at the upper edge of V-shaped ultrafiltration membrane. The filtrate was aspirated off, 10ml of cold PBS was added to the inner column, and the column was centrifuged until the liquid surface was on the upper edge of the V-shaped ultrafiltration membrane.
10) The upper column was aspirated, the ultrafiltration membrane was washed with 0.4ml of cold PBS, and the combined fluids amounted to about 1.5 ml.
(5) Purified human CD70 antigen binding polypeptide fusion protein titer detection
1) Concentration of fusion protein by Bradford method
The Quick Start of Bio-Rad was usedTMThe Bradford 1x Dye Reagent (cat # 5000205) kit was used to test the test samples at four concentrations, 2, 5, 10 and 20. The absorbance was read at 595nm after completion of the kit procedure.
2) The plate was coated with 100ul of human CD70 at 2ug/ml under 4 ℃ overnight.
3) Wash plate 3 times with 0.05% PBST, block with 200ul 2% skim milk for 1 hour at room temperature, wash plate 3 times with 0.05% PBST.
4) The purified protein was diluted to 1ng/ul, 5ng/ul, 25ng/ul, 125ng/ul, 625ng/ul with 1% nonfat dry milk in PBS, 100ul of the diluted purified protein was added to the ELISA plate, and 1% nonfat dry milk in PBS was added to the control wells.
5) Incubate at room temperature for 2 hours, shaking several times during this period. The liquid was removed and washed 3 times with 0.05% PBST.
6) Secondary antibody of HRP-labeled rabbit anti-human IgG Fc fragment was added and incubated at room temperature for 1 hour. The liquid was removed and washed 3 times with 0.05% PBST.
7) 100ul of TMB substrate was added and incubated at 37 ℃ for about 10 minutes until the yellow color was fully developed.
8) The reaction was stopped by adding 100ul 1M sulfuric acid and the OD450 read on a microplate reader. The results are shown in fig. 5, where the purified fusion antigen-binding polypeptide exhibits a typical antigen-antibody binding concentration-dependent profile in vitro with antigen.
Sequence listing
<110> Zhejiang Landun pharmaceuticals Co., Ltd
<120> human CD70 antigen binding polypeptide
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<210>2
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Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
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Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly
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Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn
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Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp
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Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro
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Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu
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Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
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Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
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Ser Leu Ser Pro Gly Lys
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Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
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Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val
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Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu
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Leu Ser Pro Gly Lys
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ggtggcggat cgccagcacc acctgtggca ggaccgtcag tcttcctctt ccccccaaaa 180
cccaaggaca ccctcatgat ctcccggacc cctgaggtca cgtgcgtggt ggtggacgtg 240
agccacgaag accccgaggt ccagttcaac tggtacgtgg acggcgtgga ggtgcataat 300
gccaagacaa agccacggga ggagcagttc aacagcacgt tccgtgtggt cagcgtcctc 360
accgttgtgc accaggactg gctgaacggc aaggagtaca agtgcaaggt ctccaacaaa 420
ggcctcccag cccccatcga gaaaaccatc tccaaaacca aagggcagcc ccgagaacca 480
caggtgtaca ccctgccccc atcccgggag gagatgacca agaaccaggt cagcctgacc 540
tgcctggtca aaggcttcta ccccagcgac atcgccgtgg agtgggagag caatgggcag 600
ccggagaaca actacaagac cacacctccc atgctggact ccgacggctc cttcttcctc 660
tacagcaagc tcaccgtgga caagagcagg tggcagcagg ggaacgtctt ctcatgctcc 720
gtgatgcatg aggctctgca caaccactac acgcagaaga gcctctccct gtctccgggt 780
aaacaccacc atcaccacca ttga 804
<210>6
<211>267
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>6
Met Gly Val Lys Val Leu Phe Ala Leu Ile Cys Ile Ala Val Ala Glu
1 5 10 15
Ser Phe Lys Gln Asp Ala Trp Glu Ala Val Asp Ile Arg Gly Gly Gly
20 25 30
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Pro Ala Pro Pro
35 40 45
Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
50 55 60
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
65 70 75 80
Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val
85 90 95
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser
100 105 110
Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu
115120 125
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala
130 135 140
Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro
145 150 155 160
Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln
165 170 175
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
180 185 190
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
195 200 205
Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
210 215 220
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
225 230 235 240
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
245 250 255
Leu Ser Pro Gly Lys His His His His His His
260 265
Claims (9)
1. The human CD70 antigen-binding peptide is characterized in that the human CD70 antigen-binding peptide is of a single-chain structure, and the amino acid sequence of the human CD70 antigen-binding peptide is shown as SEQ ID NO. 1.
2. A human CD70 antigen-binding peptide, wherein the human CD70 antigen-binding peptide comprises a signal peptide and an antigen-binding site, and the amino acid sequence of the antigen-binding peptide is shown as SEQ ID NO. 2.
3. A human CD70 antigen-binding peptide, wherein the human CD70 antigen-binding peptide comprises a signal peptide, an antigen-binding site and a human Fc region, and the amino acid sequence of the human CD70 antigen-binding peptide is shown as SEQ ID NO. 3.
4. A human CD70 antigen-binding peptide, wherein the human CD70 antigen-binding peptide comprises a signal peptide, an antigen-binding site, a flexible connecting peptide and a human Fc region, and the amino acid sequence of the human CD70 antigen-binding peptide is shown as SEQ ID NO. 4.
5. A nucleic acid molecule comprising a sequence encoding the human CD70 antigen binding peptide of claim 1.
6. A vector comprising the nucleic acid molecule of claim 5.
7. A host cell comprising the nucleic acid molecule of claim 5 or the vector of claim 6.
8. A kit comprising the human CD70 antigen binding peptide of any one of claims 1-4.
9. Use of the human CD70 antigen binding peptide of any one of claims 1-4 in the preparation of a medicament for anti-cancer or cancer detection.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20210380707A1 (en) * | 2020-06-04 | 2021-12-09 | Crispr Therapeutics Ag | Anti-cd70 antibodies and uses thereof |
| CN111548390B (en) * | 2020-06-17 | 2022-09-06 | 清华大学深圳国际研究生院 | CD133 antagonistic polypeptide, derivative and application thereof |
| CN116120451A (en) * | 2021-06-17 | 2023-05-16 | 南京蓝盾生物科技有限公司 | anti-CD 70 internalizing antibodies, antibody conjugates and uses thereof |
| CN113292652A (en) * | 2021-06-17 | 2021-08-24 | 南京蓝盾生物科技有限公司 | anti-CD 70 antibodies with enhanced ADCC effect and uses thereof |
| CN114933636B (en) * | 2022-06-24 | 2023-05-09 | 浙江省人民医院 | L1 CAM-targeted polypeptide and application thereof |
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|---|---|---|---|---|
| CN109021106A (en) * | 2018-08-30 | 2018-12-18 | 浙江蓝盾药业有限公司 | A kind of humanization CD70 antibody LD70 and the preparation method and application thereof |
| CN109476734A (en) * | 2015-07-31 | 2019-03-15 | 安进研发（慕尼黑）股份有限公司 | The antibody construct of CD70 and CD3 |
| CN109880802A (en) * | 2018-11-30 | 2019-06-14 | 北京美康基免生物科技有限公司 | A kind of immunocyte of the dual Chimeric antigen receptor gene modification based on CD19 and CD70 and its application |
| WO2019152705A1 (en) * | 2018-02-01 | 2019-08-08 | Pfizer Inc. | Antibodies specific for cd70 and their uses |
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| CN109476734A (en) * | 2015-07-31 | 2019-03-15 | 安进研发（慕尼黑）股份有限公司 | The antibody construct of CD70 and CD3 |
| WO2019152705A1 (en) * | 2018-02-01 | 2019-08-08 | Pfizer Inc. | Antibodies specific for cd70 and their uses |
| CN109021106A (en) * | 2018-08-30 | 2018-12-18 | 浙江蓝盾药业有限公司 | A kind of humanization CD70 antibody LD70 and the preparation method and application thereof |
| CN109880802A (en) * | 2018-11-30 | 2019-06-14 | 北京美康基免生物科技有限公司 | A kind of immunocyte of the dual Chimeric antigen receptor gene modification based on CD19 and CD70 and its application |
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| Title |
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