CN119101160B - An anti-CD28 nanoantibody, a method for screening CD28 nanoantibodies based on a natural library, and applications thereof - Google Patents
An anti-CD28 nanoantibody, a method for screening CD28 nanoantibodies based on a natural library, and applications thereof Download PDFInfo
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- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
- C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
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Abstract
本发明涉及一种抗CD28纳米抗体、基于天然库筛选CD28纳米抗体的方法及其应用，属于生物技术领域。本发明的抗CD28纳米抗体，由重链构成，所述重链包括三个互补决定区，即CDR1、CDR2和CDR3；所述CDR1的氨基酸序列如SEQ ID NO:3所示；所述CDR2的氨基酸序列如SEQ ID NO:4所示；所述CDR3的氨基酸序列如SEQ ID NO:5所示。本发明通过羊驼纳米抗体天然库和噬菌体展示技术获得了靶向CD28的纳米抗体，能够特异性识别CD28，具有用于肿瘤治疗药物制备和肿瘤检测的应用前景。
The present invention relates to an anti-CD28 nano antibody, a method for screening CD28 nano antibodies based on a natural library and an application thereof, and belongs to the field of biotechnology. The anti-CD28 nano antibody of the present invention is composed of a heavy chain, and the heavy chain includes three complementary determining regions, namely CDR1, CDR2 and CDR3; the amino acid sequence of the CDR1 is shown in SEQ ID NO:3; the amino acid sequence of the CDR2 is shown in SEQ ID NO:4; the amino acid sequence of the CDR3 is shown in SEQ ID NO:5. The present invention obtains a nano antibody targeting CD28 through an alpaca nano antibody natural library and phage display technology, which can specifically recognize CD28 and has application prospects for the preparation of tumor therapeutic drugs and tumor detection.
Description
Technical Field
The invention relates to an anti-CD 28 nanobody, a method for screening the CD28 nanobody based on a natural library and application thereof, belonging to the technical field of biology.
Technical Field
The immune system is divided into nonspecific immunity and specific immunity. Specific immunity includes cellular immunity, in which activation of T lymphocytes is the core of the immune response, and humoral immunity. Inducing T cell activation, proliferation, and differentiation into effector T cells requires not only the T Cell Receptor (TCR) -CD3 complex to provide a first signal for the primary histocompatibility complex (major histocompatibility complex, MHC) -polypeptide recognition and binding of the target cell, but also a second signal from the co-stimulatory molecule. The first signal determines the specificity of T cell activation and the co-stimulatory signal determines the direction of T cell progression. In the absence of the second signal, the first signal alone is not able to induce an immune response in T cells, which would enter a non-responsive state or immune tolerance and even cause apoptosis, probably due to the small activation of MHC-polypeptides and the sustained internalization of the TCR-CD3 complex limiting TCR signaling. While participation of co-stimulatory complex B7-CD28 may prevent this from occurring. The most common antibodies used for T cell activation in vitro are anti-CD 3 antibodies and anti-CD 28 antibodies.
CD28, the first co-stimulatory receptor discovered, is an initiating member of the subfamily of co-stimulatory molecules characterized by extracellular variant immunoglobulin-like domains. The receptor CD28 on the surface of T cells binds to the B7 protein on the surface of Antigen Presenting Cells (APC) to generate a positive activation signal, activate T cells, and enhance immune response. Based on the importance of CD28 co-stimulation for T cell activation, antagonists and agonists can be developed for different clinical indications-in the treatment of autoimmune diseases and organ transplant rejection, the CD28/B7-1 (CD 80) or B7-2 (CD 86) pathway can be blocked, while in the treatment of tumors, prostate cancer, the ability to enhance T lymphocyte activation, proliferation is needed. At present, the development of clinical medicines mainly focuses on 3 aspects, namely CD28 associated target fusion protein medicines, monoclonal antibody medicines and multispecific antibody medicines.
Nanobodies, which are the smallest antibody molecules known to date to bind antigen, have a molecular weight of about 15kDa. Compared with the traditional antibody, the nano antibody has the advantages of small relative molecular mass, high affinity, high stability, good solubility, low immunogenicity, strong penetrating power, simple humanization, capability of being expressed in a large amount in escherichia coli and the like. The application of phage display technology to develop the nanometer antibody against human CD28 has good application prospect.
The development of general nano antibodies is based on a natural library, an immune library, a synthetic library or a semisynthetic library, the natural library can be used for screening any potential antigens, the high diversity of the natural library is used for avoiding the immunization step, the antibodies with high specificity are directly screened, the immune library needs to prepare the antigens first, after multiple rounds of immunization, the immune library with the specificity aiming at the specific antigens is constructed, the whole time consumption is long, the cost is high, animal immunization links can be omitted from the synthetic library and the semisynthetic library, but the CDR regions need to be reasonably designed in a large-scale diversity manner to generate more CDR3 polymorphisms, and the requirements of library diversity are met.
Disclosure of Invention
The invention aims to provide an anti-CD 28 nano antibody which can specifically recognize CD28 and also provides application of the antibody. The invention also provides a preparation method of the anti-CD 28 nano antibody, the nano antibody is developed through the alpaca nano antibody natural library, compared with an immune library, the alpaca nano antibody has the advantages of lower cost, no antigenic limitation of antigen, capability of enriching and obtaining the specific nano antibody without animal immunity, and the like, and plays a role in promoting the development of biomedical technology.
The technical scheme for solving the technical problems is as follows:
In a first aspect of the invention, there is provided an anti-CD 28 nanobody consisting of a heavy chain comprising three complementarity determining regions, CDR1, CDR2 and CDR3;
The amino acid sequence of the CDR1 is shown in SEQ ID NO. 3;
The amino acid sequence of the CDR2 is shown as SEQ ID NO. 4;
the amino acid sequence of the CDR3 is shown in SEQ ID NO. 5.
Preferably, the amino acid sequence of the heavy chain antibody variable region is shown in SEQ ID NO. 2. The amino acid sequence of the CDR1 is 31-35 of the amino acid sequence shown in SEQ ID NO. 2, the CDR2 is 50-74 of the amino acid sequence shown in SEQ ID NO. 2, and the CDR3 is 107-123 of the amino acid sequence shown in SEQ ID NO. 2.
In a second aspect of the present invention there is provided a method of preparing anti-CD 28 nanobodies according to the first aspect, said method using phage display technology to enrich anti-CD 28 nanobodies from a natural library of alpaca nanobodies, comprising the steps of:
Separating Peripheral Blood Mononuclear Cells (PBMC) from non-immunized alpaca peripheral blood, taking the peripheral blood mononuclear cells as raw materials to extract total RNA, obtaining cDNA by reverse transcription PCR, amplifying gene fragments of alpaca nanobody VHH by taking the cDNA as a template, cloning target genes to phagemid vectors and transforming the target genes into competent cells to construct a natural alpaca nanobody library, packaging the constructed natural library with phage, carrying out first-generation sequencing on positive clones by panning and enriching anti-CD 28 phage and further selecting monoclonal clones, and carrying out sequencing analysis to select correct antibody sequences for expression of a lactation system.
Preferably, the monoclonal screening procedure is as follows:
The monoclonal culture after each round of panning is selected to the logarithmic phase, the expression of the inserted gene is induced by IPTG, the thalli are broken by the osmotic shock method to release the fusion protein to be tested in the periplasmic space, and the positive clone of the nanometer antibody for expressing CD28 is screened by ELISA.
In a third aspect of the invention, there is provided a nucleic acid encoding an anti-CD 28 nanobody as defined in the first aspect.
Preferably, the sequence of the nucleic acid is shown in SEQ ID NO. 1.
In a fourth aspect of the invention there is provided a mammalian system expression vector comprising a nucleic acid as described in the third aspect.
Preferably, the mammalian system expression vector is pcDNA3.4.
In a fifth aspect of the invention there is provided a host cell comprising an expression vector as described in the fourth aspect.
Preferably, the mammalian system expression host cell is CHO-K1.
In a sixth aspect of the invention there is provided the use of an anti-CD 28 nanobody according to the first aspect for the preparation of a product which binds to CD28 protein, or for the preparation of a medicament for tumour therapy or a tumour detection agent.
Compared with the prior art, the invention has the following technical effects:
1) According to the invention, the anti-CD 28 nano antibody is obtained by screening from the alpaca nano antibody natural library, so that an immune alpaca link is avoided, the use amount of antigen is reduced, the time and cost for antibody discovery are saved, and the antibody discovery efficiency is improved compared with the method of screening the nano antibody by animal immunity.
2) The invention obtains the nano antibody targeting CD28 through phage display technology, and the antibody can specifically recognize CD28, thereby having application prospects of tumor therapeutic drug preparation and tumor detection.
Drawings
FIG. 1 shows the results of the monoclonal screening in example 1. Wherein A is the 1-plate monoclonal screening result after the first round of panning, B1-B3 is the 3-plate monoclonal screening result after the second round of panning, and C1-C7 is the 7-plate monoclonal screening result after the third round of panning.
FIG. 2 shows the sequencing statistics of positive clones in example 1.
FIG. 3 is a map of an expression vector of the antibody nursing system in example 2.
FIG. 4 is SDS-PAGE of purified antibodies of example 2, wherein R is reducing conditions, NR is non-reducing conditions, and M is marker.
FIG. 5 is SEC-HPLC of the purified antibody of example 2 wherein A is 214nM and B is 280nM.
FIG. 6 shows the binding of the antibodies of example 3 to hCD28 recombinant protein.
FIG. 7 shows the SPR affinity assay of the antibodies of example 4 for hCD28, wherein A is ANb-17-CD28-3LP-3, B is the positive control antibody Anti-Human CD28Theralizumab (TGN 1412), and C is the negative control antibody Anti-HEL IgG1hFc.
FIG. 8 shows the binding of the antibodies of example 5 to CHO-K1hCD28 overexpressing cell lines.
Detailed Description
The present invention will be described in further detail with reference to examples. The invention is not limited to the examples given. The methods used are conventional methods unless otherwise specified, and the reagents and materials used are commercially available products unless otherwise specified.
Example 1 screening of anti-CD 28 nanobodies from alpaca nanobody Natural library
Phage display technology was applied to screen anti-CD 28 nanobodies from the alpaca nanobody natural library.
Three 30. Mu.L Dynabeads TM M-280 streptavidin beads (Thermo FisherScientific, 11206D) were pipetted into a 1.5mL EP tube using liquid phase panning, 1mL of 5% NON-fat Powdered Milk (Sangon Biotech, A600669-0250) were added after one wash with 1mL of 0.05% PBST and blocked at 25℃for 1 hour. 2E+12C.F.U.phage was taken and blocked for 1 hour at 25℃with 1mL of 5% NON-fat Powdered Milk. Another 250. Mu.L of magnetic beads was placed in a 1.5mL EP tube, 1mL of PBS was added, 15. Mu.g of biotin-labeled hCD28/His recombinant protein (Biointron, B22406301), incubated at 25℃for 1 hour, 1mL of 0.05% PBST was washed 5 times, and 1mL of 5% NON-fat Powdered Milk was added and blocked at 25℃for 1 hour. The blocking solution in 30. Mu.L of magnetic beads was removed, and the blocked phage were incubated with 3 parts of 30. Mu.L of blank magnetic beads for 0.5 hour, respectively, in order to remove background interference. The phage supernatant from which the background was removed was added to the blocked 250. Mu.L magnetic beads and incubated at 25℃for 1 hour. Phage supernatant not bound to beads was removed and the remaining beads were washed 15 times with 1mL of 0.05% PBST, resuspended and added to the log phase bacterial fluid of SS320 for amplification for the next round of panning.
After three rounds of pressurized panning, 11 plates were selected for a total of 968 monoclonal (88 monoclonal per plate, 1 plate for the first round of LPR1, 3 plate for the second round of LPR2, and 7 plate for the third round of LPR 3) for monoclonal screening, and sample preparation was performed by adding 600. Mu.L of 2 XYT dual-antibody medium containing tetracycline and carbenicillin to 96 well deep-well plates, inoculating the monoclonal, culturing at 220rpm,37℃for 3 hours, sucking 100. Mu.L of bacterial liquid per well into a new 96 well plate for storage, and supplementing 100. Mu.L of 2 XYT dual-antibody medium and IPTG into the original 96 well plate for 30℃for induction expression overnight. The supernatant was discarded by the next day centrifugation, and the cells were disrupted with TES buffer (30 mM Tris-HCl,0.5M sucrose, 0.5mM EDTA, pH=8.0) to release the fusion protein to be assayed in the periplasmic space.
The monoclonal assay was performed by coating 1. Mu.g/mLhCD/His recombinant protein and 1. Mu.g/mLBSA, respectively, on an ELISA plate (Corning, 3590), overnight at 4 ℃,3 times the next day with 0.1% PBST, 3 times 3% BSA (Sangon, A500023) blocked for 1 hour, 3 times with 0.1% PBST, adding a sample prepared in advance, incubating for 1 hour at 100. Mu.L/well, 25 ℃,5 times with 0.1% PBST, adding Mouse-flag, HRP, mAb (Sigma, A8592), diluting with 1:10000, incubating for 1 hour at 25 ℃.10 μg/mL purified antibody Anti-Human CD28Theralizumab (TGN 1412) was used as positive control, anti-HEL IgG1hFc as negative control (Biointron, B117901), control antibody secondary antibody was diluted 1:10000 using GoatAnti-Human IgG-Fc, HRP (Sigma, A0170). After washing 5 times with 0.1% PBST, TMB (Makewonderbio, 1001) was added to develop color at room temperature in the dark, and finally the color development was stopped with stop solution (Beyotime, P0215), the absorbance at 450nm was read by a microplate reader, and we defined clones with 3 Xblank (CD 28) < OD450<2 Xblank (BSA) as positive clones. The detection results are shown in FIG. 1.
The results indicated 89 positive clones recognizing CD28 out of 968 monoclonal. Sequencing analysis was performed on these 89 positive clones to yield 9 Unique sequences, of which Unique 5 was the dominant rich clone (as shown in FIG. 2).
The corresponding dominant monoclonal of Unique5 is ANb-17-CD28-3LP-3, the DNA sequence of the heavy chain variable region is shown as SEQ ID NO. 1, and the amino acid sequence is shown as SEQ ID NO. 2.
In the amino acid sequence shown as SEQ ID NO. 2, amino acid residues 31-35 (i.e., SEQ ID NO. 3) are heavy chain CDR1, amino acid residues 50-74 (i.e., SEQ ID NO. 4) are heavy chain CDR2, and amino acid residues 107-123 (i.e., SEQ ID NO. 5) are heavy chain CDR3.
EXAMPLE 2 mammalian System expression and purification of anti-CD 28 nanobodies
The expression vector pcDNA3.4 of the mammalian system for ANb-17-CD28-3LP-3 nanobody of example 1 was constructed and the map is shown in FIG. 3. Then, a plasmid was prepared therefrom. The expression host cell of the antibody was selected from CHO-K1 cells with an expression volume of 40mL, and the supernatant after expression was purified using a ProteinA affinity column.
The preparation method comprises the following steps of taking 5.5mL of CHO-K1 cells with the density of 7.2E+6cells/mL and 4 generations, centrifuging at 1000rpm for 5min, discarding supernatant, adding 1.4mL of electrotransfer liquid into a centrifuge tube, uniformly mixing, adding plasmids, taking 1.0mL of the mixed liquid obtained in the previous step, adding the mixed liquid into an electric shock cup, then placing the electric shock cup into an electrotransfer instrument for electrotransfer at 540V-580V, preparing two shake flasks in advance, each shake flask is filled with 20mLDMEM culture medium, uniformly dividing the electric shock cells into the two shake flasks, standing at room temperature for 30min, placing the shake flasks into a CO 2 incubator for culture at 37 ℃ at the rotating speed of 250rpm and controlling the concentration of 5.0% CO 2, continuously culturing for 4 days after 24h, balancing a ProteinA affinity chromatography column by 1X PBS, preparing a sample (namely cell culture supernatant), setting the sample loading flow rate to be 1.0mL/min, washing the sample by 1X PBS, and finally placing the sample into a dialysis solution with high-purity antibody-eluting buffer solution (PBS=3) in a dialysis cup, and finally obtaining the antibody with high-purity antibody. The purity of the antibody is measured by SDS-PAGE and SEC-HPLC, and the results are shown in figures 4 and 5, respectively, and the purity of the antibody is higher than 95%.
Example 3 detection of binding Capacity of the antibody of example 2 to hCD28/His recombinant protein
1 Μg/mL hCD28/His recombinant protein was coated onto an ELISA plate, incubated at 4℃overnight, 3 times with 0.1% PBST, 3% BSA blocked for 1 hour, 3 times with 0.1% PBST, and the ANb-17-CD28-3LP-3 purified antibody and control antibody of example 2 diluted to 100nM as first Kong Nongdu, 4-fold gradient dilution, end wells blank, 1 hour incubation at 25℃and 5 times with 0.1% PBST, 5 times with HRP-labeled Goat anti-human IgG-Fc,1:10000 as secondary antibody, 0.5 hours incubation at 25℃and 5 times with 0.1% PBST. After 100 mu LTMB is added to each hole and reacts at room temperature for 3-5 minutes in a dark place, the color development is stopped, and the absorbance at the wavelength of 450nm is read by an enzyme-labeled instrument. Wherein the positive control is Anti-Human CD28 (Theralizumab, TGN 1412), and the negative control is Anti-HEL, human IgG1Fc. As a result, ANb-17-CD28-3LP-3 was able to bind hCD28/His with an EC50 of 0.37nM, as shown in FIG. 6.
Example 4 SPR affinity detection of example 2 antibodies with hCD28/His
The ANb-17-CD28-3LP-3 purified antibody, 2. Mu.g/mL, was injected into the experimental channel at a flow rate of 10. Mu.L/min for 60s, with a capture of approximately 145-2000RU, respectively. hCD28/His recombinant protein was diluted 2-fold with HBS-EP+buffer running from 200 nM. The diluted hCD28/His recombinant protein is sequentially injected into the experimental channel and the reference channel at the flow rate of 30 mu L/min, and is combined and dissociated for corresponding time. The binding dissociation steps were all performed in running buffer. ProteinAChip (Cytiva, 29127556) required regeneration with 10mM Gly-HCl (pH=1.5) at a flow rate of 30. Mu.L/min for 30s, washing away undissociated analytes. The KD values of the samples were calculated using Biacore 8K analysis software. The reference channel (Fc 1) was used for background subtraction, and the binding model 1:1, results are shown in FIG. 7, ANb-17-CD28-3LP-3 antibody binding activity to hCD28/His recombinant protein with affinity of 1.90E-07M.
Example 5 detection of binding Capacity of the example 2 antibody to CHO-K1hCD28 overexpressing cell line
CHO-K1Human CD28 cells (Biointron, B21927403) were collected at 3E+06cells/mL density, 100nM antibody in the first well, 4-fold gradient diluted, 50. Mu.L/well cell suspension (1.50E+5cell/well) and 50. Mu. LANb-17-CD28-3LP-3 antibody were added to 96-well V-bottom plate, incubated at 4℃for 1h, MACS buffer washed 2 times, centrifuged at 400g for 5min, alexa was added647AffiniPure Goat Anti-Human IgG, fcγ FRAGMENT SPECIFIC (Jackson Immuno, 109-605-190), 1:1000,4℃incubation for 0.5h, MACS buffer washing 2 times, 400g centrifugation for 5min, 100. Mu.L MACS buffer resuspension of cells, and detection using a flow cytometer, gave a FACS binding curve, which resulted in ANb-17-CD28-3LP-3 having binding activity with CHO-K1hCD28 over-expression cell line with an EC50 value of 2.52nM, as shown in FIG. 8.
The foregoing is merely exemplary embodiments of the present invention and are not intended to limit the scope of the present invention, and all equivalent modifications made by the present invention or direct or indirect application in other related technical fields are included in the scope of the present invention.
Claims (8)
1. An anti-CD 28 nanobody, wherein said nanobody is comprised of a heavy chain comprising three complementarity determining regions, CDR1, CDR2, and CDR3;
The amino acid sequence of the CDR1 is shown in SEQ ID NO. 3;
The amino acid sequence of the CDR2 is shown as SEQ ID NO. 4;
the amino acid sequence of the CDR3 is shown in SEQ ID NO. 5.
2. The anti-CD 28 nanobody of claim 1, wherein the amino acid sequence of the heavy chain antibody variable region is as shown in SEQ ID No. 2.
3. Nucleic acid encoding the anti-CD 28 nanobody of claim 1 or 2.
4. The nucleic acid of claim 3, wherein the sequence of the nucleic acid is set forth in SEQ ID NO. 1.
5. A mammalian expression vector comprising the nucleic acid of claim 3.
6. A host cell comprising the expression vector of claim 5.
7. Use of an anti-CD 28 nanobody according to claim 1 or 2 for the preparation of a CD28 detection reagent or for the preparation of a tumor detection reagent.
8. Use of an anti-CD 28 nanobody according to claim 1 or 2 for the preparation of a product that binds to CD28 protein.
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