An anti-CD28 nanoantibody, a method for screening CD28 nanoantibodies based on a natural library, and applications thereof

Abstract

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

[0001] The present invention relates to an anti-CD28 nano antibody, a method for screening CD28 nano antibodies based on a natural library and application thereof, and belongs to the field of biotechnology. Technical Background

[0002] The immune system is divided into nonspecific immunity and specific immunity. Specific immunity includes cellular immunity and humoral immunity. In cellular immunity, the activation of T lymphocytes is the core of the immune response. Inducing T cell activation, proliferation and differentiation into effector T cells requires not only the recognition and binding of the major histocompatibility complex (MHC)-peptide of the target cell by the T cell receptor (TCR)-CD3 complex to provide the first signal, but also the second signal provided by the co-stimulatory molecules. The first signal determines the specificity of T cell activation, and the co-stimulatory signal determines the direction of T cell movement. When there is only the first signal and the lack of the second signal, the immune response of T cells cannot be induced. T cells will enter an unresponsive state or immune tolerance, and even cause programmed cell death. The reason may be that the small amount of MHC-peptide activation and the continuous internalization of the TCR-CD3 complex limit the TCR signal. The participation of the co-stimulatory complex B7-CD28 can prevent such a situation from happening. The most common antibodies used for T cell activation in vitro are anti-CD3 antibodies and anti-CD28 antibodies.

[0003] As the first co-stimulatory receptor discovered, CD28 is the founding member of the co-stimulatory molecule subfamily characterized by an extracellular variant immunoglobulin-like domain. The receptor CD28 on the surface of T cells binds to the B7 protein on the surface of antigen presenting cells (APC) to produce a positive activation signal, activate T cells, and enhance the immune response. Based on the importance of CD28 co-stimulation for T cell activation, both antagonists and agonists can be developed for different clinical indications. In the treatment of autoimmune diseases and organ transplant rejection, the CD28 / B7-1 (CD80) or B7-2 (CD86) pathway can be blocked; and in the treatment of tumors and prostate cancer, it is necessary to enhance the activation and value-added ability of T lymphocytes. At present, clinical drug development is mainly focused on three aspects: CD28-associated target fusion protein drugs, monoclonal antibody drugs, and multi-specific antibody drugs.

[0004] Nanobodies are the smallest known antigen-binding antibody molecules, with a molecular weight of about 15kDa. Compared with traditional antibodies, nanobodies have the advantages of small molecular weight, high affinity, high stability, good solubility, low immunogenicity, strong penetration, simple humanization, and the ability to be expressed in large quantities in E. coli. The application of phage display technology to develop nanobodies against human CD28 has good application prospects.

[0005] Generally, the development of nanoantibodies is based on natural libraries, immune libraries, synthetic libraries or semi-synthetic libraries. Natural libraries can be used to screen any potential antigens. Relying on the high diversity of natural libraries, the immunization step can be avoided and highly specific antibodies can be directly screened. The immune library needs to prepare the antigen first, and then conduct multiple rounds of immunization to construct an immune library specific for a specific antigen. The overall process is time-consuming and costly. Synthetic and semi-synthetic libraries can also omit the animal immunization step, but the CDR region needs to be reasonably designed on a large scale to produce more CDR3 polymorphisms to meet the needs of library diversity. Summary of the invention

[0006] The purpose of the present invention is to provide an anti-CD28 nano antibody that can specifically recognize CD28, and also provide the application of the antibody. The present invention also provides a method for preparing the anti-CD28 nano antibody. The present invention develops nano antibodies through a natural library of alpaca nano antibodies, which has the advantages of lower cost and no antigenicity restriction of antigens compared to immune libraries, and can enrich specific nano antibodies without animal immunization, etc., which promotes the development of biomedical technology.

[0007] The technical solution of the present invention to solve the technical problem is as follows:

[0008] In a first aspect of the present invention, an anti-CD28 nanobody is provided, wherein the nanobody is composed of a heavy chain, wherein the heavy chain includes three complementarity determining regions, namely CDR1, CDR2 and CDR3;

[0009] The amino acid sequence of the CDR1 is shown in SEQ ID NO: 3;

[0010] The amino acid sequence of the CDR2 is shown in SEQ ID NO:4;

[0011] The amino acid sequence of the CDR3 is shown in SEQ ID NO:5.

[0012] Preferably, the amino acid sequence of the heavy chain antibody variable region is as shown in SEQ ID NO: 2. The amino acid sequence of the CDR1 is amino acid residues 31-35 in the amino acid sequence shown in SEQ ID NO: 2; the CDR2 is amino acid residues 50-74 in the amino acid sequence shown in SEQ ID NO: 2; and the CDR3 is amino acid residues 107-123 in the amino acid sequence shown in SEQ ID NO: 2.

[0013] In the second aspect of the present invention, a method for preparing the anti-CD28 nanobody as described in the first aspect is provided, wherein the method uses phage display technology to enrich anti-CD28 nanobodies from the natural library of alpaca nanobodies, comprising the following steps:

[0014] Peripheral blood mononuclear cells (PBMC) were isolated from the peripheral blood of unimmunized alpacas, and total RNA was extracted from PBMCs. cDNA was obtained by reverse transcription-PCR, and the gene fragment of alpaca nanoantibody VHH was amplified using cDNA as a template. The target gene was then cloned into a phagemid vector and transformed into competent cells to construct a natural library of alpaca nanoantibodies. The constructed natural library was phage packaged, anti-CD28 phages were enriched by panning, and single clones were further selected for screening. The positive clones were subjected to first-generation sequencing, and the correct antibody sequence was selected through sequencing analysis for mammalian system expression.

[0015] Preferably, the monoclonal screening process is as follows:

[0016] Single clones after each round of panning were selected and cultured to the logarithmic phase. The expression of the inserted gene was induced with IPTG. The bacteria were broken by osmotic shock to release the fusion protein to be tested in the periplasmic space. Positive clones expressing anti-CD28 nanoantibodies were screened by ELISA.

[0017] In the third aspect of the present invention, a nucleic acid encoding the anti-CD28 nanobody as described in the first aspect is provided.

[0018] Preferably, the sequence of the nucleic acid is as shown in SEQ ID NO:1.

[0019] In the fourth aspect of the present invention, a mammalian expression vector containing the nucleic acid according to the third aspect is provided.

[0020] Preferably, the mammalian system expression vector is pcDNA3.4.

[0021] In the fifth aspect of the present invention, a host cell containing the expression vector as described in the fourth aspect is provided.

[0022] Preferably, the mammalian expression host cell is CHO-K1.

[0023] In the sixth aspect of the present invention, there is provided the use of the anti-CD28 nanobody as described in the first aspect in the preparation of a product that binds to the CD28 protein, or in the preparation of a tumor therapeutic drug or a tumor detection reagent.

[0024] Compared with the prior art, the present invention has the following technical effects:

[0025] 1) The present invention obtains anti-CD28 nanoantibodies by screening from the natural library of alpaca nanoantibodies, thereby avoiding the step of immunizing alpacas. Compared with screening nanoantibodies by animal immunization, the amount of antigen used is reduced, the time and cost of antibody discovery are saved, and the efficiency of antibody discovery is improved.

[0026] 2) The present invention obtains a nano antibody targeting CD28 through phage display technology. The antibody can specifically recognize CD28 and has application prospects in the preparation of tumor therapeutic drugs and tumor detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The monoclonal screening results in Example 1. A is the monoclonal screening result of one plate after the first round of panning, B1-B3 are the monoclonal screening results of three plates after the second round of panning, and C1-C7 are the monoclonal screening results of seven plates after the third round of panning.

[0028] Figure 2 This is the statistical result of sequencing of positive clones in Example 1.

[0029] Figure 3 This is a map of the antibody mammalian system expression vector in Example 2.

[0030] Figure 4 This is the SDS-PAGE of the purified antibody in Example 2, wherein R represents reducing conditions, NR represents non-reducing conditions, and M represents a marker.

[0031] Figure 5 This is the SEC-HPLC of the purified antibody in Example 2, wherein A is 214 nM and B is 280 nM.

[0032] Figure 6 The antibody in Example 3 binds to the hCD28 recombinant protein.

[0033] Figure 7 This is the SPR affinity test between the antibody and hCD28 in Example 4, wherein A is ANb-17-CD28-3LP-3, B is the positive control antibody Anti-Human CD28 Theralizumab (TGN1412), and C is the negative control antibody Anti-HELIgG1hFc.

[0034] Figure 8 The antibody in Example 5 binds to the CHO-K1hCD28 overexpressing cell line. DETAILED DESCRIPTION

[0035] The present invention is further described in detail below with reference to the examples. However, the present 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 unless otherwise specified.

[0036] Example 1 Screening of anti-CD28 nanobodies from a natural library of alpaca nanobodies

[0037] Phage display technology was used to screen anti-CD28 nanobodies from the natural library of alpaca nanobodies.

[0038] Using the liquid phase panning method, three 30 μL Dynabeads TM M-280 streptavidin magnetic beads (Thermo Fisher Scientific, 11206D) were placed in a 1.5 mL EP tube, washed once with 1 mL 0.05% PBST, and then 1 mL of 5% NON-fat Powdered Milk (Sangon Biotech, A600669-0250) was added, and the mixture was blocked at 25°C for 1 hour. 2E+12 C.FU of phages were taken, 1 mL of 5% NON-fat Powdered Milk was added, and the mixture was blocked at 25°C for 1 hour. Another portion of 250 μL magnetic beads was placed in a 1.5 mL EP tube, 1 mL PBS, 15 μg biotin-labeled hCD28 / His recombinant protein (Biointron, B22406301) were added, and the mixture was incubated at 25°C for 1 hour, washed 5 times with 1 mL 0.05% PBST, and 1 mL of 5% NON-fat Powdered Milk was added, and the mixture was blocked at 25°C for 1 hour. Remove the blocking solution from the 30 μL magnetic beads, and incubate the blocked phage with three portions of 30 μL blank magnetic beads for 0.5 hours to remove background interference. Add the background-removed phage supernatant to the blocked 250 μL magnetic beads and incubate at 25°C for 1 hour. Remove the phage supernatant that is not bound to the magnetic beads, wash the remaining magnetic beads 15 times with 1 mL of 0.05% PBST, resuspend them, and add them to the logarithmic phase bacterial solution of SS320 for amplification for the next round of panning.

[0039] After three rounds of pressure selection, 968 monoclones in 11 plates (88 monoclones per plate, 1 plate for LPR1 in the first round, 3 plates for LPR2 in the second round, and 7 plates for LPR3 in the third round) were selected for monoclonal screening. The sample preparation process was as follows: 600 μL of 2×YT dual-antibody culture medium containing tetracycline and carbenicillin was added to a 96-well deep-well plate, and a monoclonal clone was inoculated. The culture was cultured at 220 rpm and 37°C for 3 h, and 100 μL of bacterial solution was aspirated from each well to a new 96-well plate for storage, and then 100 μL of 2×YT dual-antibody culture medium and IPTG were added to the original 96-well plate, and expression was induced overnight at 30°C. The supernatant was discarded by centrifugation the next day, and the bacteria were broken with TES buffer (30 mM Tris-HCl, 0.5 M sucrose, 0.5 mM EDTA, pH = 8.0) to release the fusion protein to be tested in the periplasmic space.

[0040] The detection process of monoclonal was as follows: 1 μg / mL hCD28 / His recombinant protein and 1 μg / mL BSA were coated on ELISA plates (Corning, 3590) respectively, and incubated at 4°C overnight. The next day, the plates were washed three times with 0.1% PBST, blocked with 3% BSA (Sangon, A500023) for 1 hour, washed three times with 0.1% PBST, and the prepared samples were added at 100 μL / well, incubated at 25°C for 1 hour, washed five times with 0.1% PBST, and Mouse anti-flag, HRP, mAb (Sigma, A8592) were added, diluted 1:10000, and incubated at 25°C for 1 hour. 10μg / mL purified antibody Anti-Human CD28Theralizumab (TGN1412) was used as a positive control, Anti-HELIgG1hFc was used as a negative control (Biointron, B117901), and GoatAnti-Human IgG-Fc, HRP (Sigma, A0170) was used as a control antibody secondary antibody, diluted 1:10000. 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 value at a wavelength of 450nm was read with an enzyme reader. We defined the clones with 3×blank (CD28)<OD450<2×blank (BSA) as positive clones. The test results are as follows Figure 1 shown.

[0041] The results showed that 89 of the 968 monoclonal clones recognized CD28. These 89 positive clones were sequenced and analyzed to obtain 9 unique sequences, among which Unique 5 was the dominant enriched clone (e.g. Figure 2 shown).

[0042] The dominant monoclone corresponding to Unique5 is ANb-17-CD28-3LP-3, and the DNA sequence of its heavy chain variable region is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 2.

[0043] In the amino acid sequence shown in SEQ ID NO: 2, amino acid residues 31-35 (ie, SEQ ID NO: 3) are heavy chain CDR1, amino acid residues 50-74 (ie, SEQ ID NO: 4) are heavy chain CDR2, and amino acid residues 107-123 (ie, SEQ ID NO: 5) are heavy chain CDR3.

[0044] Example 2 Expression and purification of anti-CD28 nanobodies in mammalian system

[0045] The mammalian expression vector pcDNA3.4 of the ANb-17-CD28-3LP-3 nanobody in Example 1 was constructed. Figure 3 Then the plasmid was prepared by this method. CHO-K1 cells were selected as the host cells for antibody expression, and the expression volume was 40 mL. The supernatant after expression was purified using a Protein A affinity chromatography column.

[0046] The specific operation is as follows: take 5.5mL of CHO-K1 cells with a density of 7.2E+6 cells / mL and have been passed for 4 generations, centrifuge at 1000rpm for 5min, and discard the supernatant; add 1.4mL of electroporation solution to the centrifuge tube, mix well, and then add the plasmid; take 1.0mL of the mixed solution in the previous step and add it to the electroporation cup, and then put it into the electroporation instrument at 540V-580V for electroporation; prepare two shaking flasks in advance, each with 20mL of DMEM culture medium, divide the electroporated cells evenly into the two shaking flasks, and let them stand at room temperature for 30min; put the shaking flasks into the CO2 incubator The cells were cultured in a 37°C incubator at a rotation speed of 250 rpm and a controlled CO2 concentration of 5.0%. After 24 hours, 2 mL of feed was added and culture was continued for 4 days. The ProteinA affinity chromatography column was balanced with 1×PBS. After the balance was completed, the sample (i.e., cell culture supernatant) was prepared. The sample flow rate was set to 1.0 mL / min. After the sample was loaded, impurities were washed with 1×PBS, and the antibodies bound to the affinity column were eluted with sodium acetate buffer (pH=3.4). Finally, the high-concentration protein was sucked into a dialysis bag and dialyzed in a beaker filled with 1×PBS to obtain a high-purity expressed antibody. The purity was measured by SDS-PAGE and SEC-HPLC, and the results were as follows: Figure 4 and Figure 5 As shown, the purity of the antibodies reached more than 95%, which is relatively high.

[0047] Example 3 Detection of the binding ability of the antibody of Example 2 with hCD28 / His recombinant protein

[0048] 1 μg / mL hCD28 / His recombinant protein was coated on the ELISA plate, overnight at 4°C, washed 3 times with 0.1% PBST, blocked for 1 hour with 3% BSA, washed 3 times with 0.1% PBST, diluted the purified ANb-17-CD28-3LP-3 antibody and control antibody in Example 2 to 100 nM as the first well concentration, 4-fold gradient dilution, the last well was blank, incubated at 25°C for 1 hour, washed 5 times with 0.1% PBST, used HRP-labeled Goat anti-human IgG-Fc, 1:10000 as the secondary antibody, incubated at 25°C for 0.5 hour, washed 5 times with 0.1% PBST. Add 100 μL TMB to each well and react at room temperature in the dark for 3-5 minutes before stopping the color development, and read the absorbance value at 450 nm wavelength with an ELISA reader. The positive control was Anti-Human CD28 (Theralizumab, TGN1412), and the negative control was Anti-HEL, human IgG1Fc. Figure 6 As shown: ANb-17-CD28-3LP-3 can bind to hCD28 / His with an EC50 of 0.37 nM.

[0049] Example 4 SPR affinity detection of the antibody of Example 2 and hCD28 / His

[0050] 2μg / mL of ANb-17-CD28-3LP-3 purified antibody was injected into the experimental channel at a flow rate of 10μL / min for 60s, and the capture amount was about 145-2000RU. The hCD28 / His recombinant protein was diluted 2 times starting from 200nM using HBS-EP+Buffer running buffer. The diluted hCD28 / His recombinant protein was injected into the experimental channel and the reference channel at a flow rate of 30μL / min in turn, and the binding and dissociation times were corresponding. The binding and dissociation steps were all performed in the running buffer. ProteinAChip (Cytiva, 29127556) needs to be regenerated with 10mM Gly-HCl (pH=1.5) at a flow rate of 30μL / min for 30s to wash away the undissociated analytes. The KD value of the sample was calculated using Biacore 8K analysis software. The reference channel (Fc1) was used for background subtraction, Fitting model 1:1, and the results are as follows. Figure 7 As shown: ANb-17-CD28-3LP-3 antibody has binding activity with hCD28 / His recombinant protein with an affinity of 1.90E-07M.

[0051] Example 5 Detection of the binding ability of the antibody of Example 2 to the CHO-K1hCD28 overexpressing cell line

[0052] CHO-K1Human CD28 cells (Biointron, B21927403) were collected at a density of 3E+06 cells / mL. 100 nM antibody was added to the first well, and 4-fold gradient dilution was performed. 50 μL / well cell suspension (1.50E+5 cells / well) and 50 μL of Lanb-17-CD28-3LP-3 antibody were added to a 96-well V-bottom plate. The cells were incubated at 4°C for 1 h, washed twice with MACS buffer, centrifuged at 400 g for 5 min, and Alexa Fluor was added. 647AffiniPure Goat Anti-Human IgG, Fcγfragment specific (Jackson Immuno, 109-605-190), 1:1000, incubate at 4°C for 0.5 h, wash twice with MACS buffer, centrifuge at 400 g for 5 min, resuspend cells in 100 μL MACS buffer, detect using flow cytometry, generate FACS binding curves, and the results are shown in the figure. Figure 8 As shown: ANb-17-CD28-3LP-3 has binding activity to the CHO-K1hCD28 overexpressing cell line, and its EC50 value is 2.52nM.

[0053] The above are only embodiments of the present invention, and are not intended to limit the patent scope of the present invention. Any equivalent transformations made using the contents of the present invention specification, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. An anti-CD28 nanobody, characterized in that: The nanobody is composed of a heavy chain, which 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.

2. The anti-CD28 nanobody according to claim 1, characterized in that The amino acid sequence of the heavy chain antibody variable region is shown in SEQ ID NO:

2.

3. A nucleic acid encoding an anti-CD28 nanobody as claimed in claim 1 or 2.

4. The nucleic acid according to claim 3, characterized in that The sequence of the nucleic acid is shown in SEQ ID NO:

1.

5. A mammalian expression vector containing the nucleic acid according to claim 3.

6. A host cell containing the mammalian system expression vector according to claim 5.

7. Use of the anti-CD28 nanobody according to claim 1 or 2 in the preparation of a CD28 detection reagent, or in the preparation of a tumor detection reagent.

8. Use of the anti-CD28 nanobody as claimed in claim 1 or 2 in the preparation of a product that binds to CD28 protein.

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