CN110981959B

CN110981959B - CD47 single domain antibody, nucleotide sequence, expression vector and kit

Info

Publication number: CN110981959B
Application number: CN201911140013.8A
Authority: CN
Inventors: 张继帅; 李莹莹; 包朝乐萌; 栗红建; 蔡清华; 丁怡瑾; 蔡志波
Original assignee: Shenzhen Pregene Biopharma Co ltd
Current assignee: Shenzhen Prijin Biopharmaceutical Co ltd
Priority date: 2019-11-19
Filing date: 2019-11-19
Publication date: 2021-05-18
Anticipated expiration: 2039-11-19
Also published as: CN110981959A

Abstract

The invention discloses a CD47 single domain antibody, a nucleotide sequence, an expression vector and a kit, wherein the CD47 single domain antibody comprises at least one of ten antibodies of CD47-117, CD47-118, CD47-127, CD47-160, CD47-169, CD47-172, CD47-175, CD47-229, CD47-236 and CD 47-271. The CD47 single-domain antibody has high specific binding capacity and high affinity with the CD47 antigen.

Description

CD47 single domain antibody, nucleotide sequence, expression vector and kit

Technical Field

The invention relates to the technical field of genetic engineering and antibody medicines, in particular to a CD47 single-domain antibody, a nucleotide sequence, an expression vector and a kit.

Background

The CD47 antigen, also known as integrin-associated protein, is a member of the immunoglobulin superfamily and is expressed on almost all cells, but is significantly enhanced on tumor cells. CD47 was considered a tumor antigen for ovarian cancer in 1986, and CD47 was subsequently found to be expressed in a variety of tumors, including various leukemias, non-hodgkin's lymphomas, multiple myeloma, leiomyosarcoma, bladder cancer, breast cancer, colon cancer, brain cancer, liver cancer, prostate tumors, and virtually all solid tumors, with CD47 being expressed on these tumor cells at levels that are more than 3-fold higher than normal cells.

SIRP alpha (Signal regulatory protein alpha) is a glycosylated transmembrane protein belonging to a member of the immunoglobulin superfamily, and expresses the surfaces of macrophages, dendritic cells, neurons and neutrophils strictly in a limiting way, and plays an important role in Signal transduction among cells. The cytoplasmic domain of SIRP α includes two immunoreceptor tyrosine inhibitory motifs that regulate different functions of cells by recruiting and binding SHP-1, SHP-2. The extracellular region of sirpa includes three immunoglobulin domains, which bind primarily to CD47 and modulate specific biological functions.

The CD47 on the tumor cell and the SIRP alpha on the macrophage act to inhibit the elimination of the macrophage on the tumor cell and promote the growth and the metastasis of the tumor in vivo, the high expression CD47 is a general mechanism of the tumor cell to escape immune surveillance, and researches show that the blocking of a CD47-SIRP alpha channel by using an antibody can ensure that the tumor cell is phagocytized by the macrophage and various diseases related to the expression level of CD47 are treated in an improved way.

In the related art, the selection range for realizing the CD47 antigen binding is narrow, the binding capacity is not high, the affinity is low, the modification is not easy, and the stability is poor.

Disclosure of Invention

The main object of the present invention is to provide at least one polypeptide sequence aimed at solving at least one of the technical problems presented above with the CD47 antibody.

In order to achieve the purpose, the invention provides a CD47 single-domain antibody, which comprises at least one of ten antibodies of CD47-117, CD47-118, CD47-127, CD47-160, CD47-169, CD47-172, CD47-175, CD47-229, CD47-236 and CD 47-271; wherein the sequence of the CD47-117 is shown in SEQ ID NO: 1 is shown in the specification; the sequence of the CD47-118 is shown as SEQ ID NO: 2 is shown in the specification; the sequence of the CD47-127 is shown as SEQ ID NO: 3 is shown in the specification; the sequence of the CD47-160 is shown as SEQ ID NO: 4 is shown in the specification; the sequence of the CD47-169 is shown as SEQ ID NO: 5 is shown in the specification; the sequence of the CD47-172 is shown as SEQ ID NO: 6 is shown in the specification; the sequence of the CD47-175 is shown as SEQ ID NO: 7 is shown in the specification; the sequence of the CD47-229 is shown as SEQ ID NO: 8 is shown in the specification; the sequence of the CD47-236 is shown as SEQ ID NO: 9 is shown in the figure; the sequence of the CD47-271 is shown as SEQ ID NO: shown at 10.

SEQ ID NO: 1-10 of the sequence is as follows:

SEQ ID NO：1：DVKLVESGGGLVQPGGSLRLSCAASGFTVSSYSMRW YRQVPGKERELIARITSTGQPIEYGESVKGRFTISRDNAKNTLYLEMNALKP EDTAVYYCWGAGYWGQGTQVTVSS；

SEQ ID NO：2：QVKLEESGGGLVQPGESLRLSCVASGFAFSSALMRW YRQAPGKERELVASITTAGGITGYADSVKGRFTISRDNAENTLYLQMNSLKP EDTAVYYCRAYGFQLDNWGQGTQVTVSS；

SEQ ID NO：3：HVQLEESGGGLVQPGGSLRLSCKASGLTFASYSMRW YRQAPGQERELVARLSSSGVPIEYVDSVKGRFTASRDDAKSTLYLQMNSLK PEDTAMYYCWIANYWGQGTQVTVSS；

SEQ ID NO：4：DVQLVESGGGSVQPGGSLTLSCAASGFTVSNYSMR WYRQAPGKERELIARITSTGVPIEYADSVKGRFTISRDNAKNTLYLEMNGL KPEDTAVYYCWGAAYWGQGTQVTVSS；

SEQ ID NO：5：GVQLVESGGGLVQPGESLRLSCVASGFAFSSALMRW YRQAPGKERELVASVTTTGGITGYADSVKGRFTISRDNAENTLYLRMNSLK PEDTAVYYCRAYGFGLDSWGQGTQVTVSS；

SEQ ID NO：6：QVKLEESGGGLVQPGGSLRLSCAASGLTFSSYSMRW YRQAPGQERELVARLTSSGEPIEYADSVKGRFTASRDNAKSTVYLQMNSLK PEDTAMYYCWIANYWGQGTQVTVSS；

SEQ ID NO：7：QVKLEESGGGLVQPGESLRLSCVASGFTFSDAHMRW YRQAPGKEREMVASISTTGSITIYADSVKGRFTISRDNDEKTVYLRMNSLKP EDTAAYYCRAYGFQIDSWGQGTQVTVSS；

SEQ ID NO：8：DVQLEESGGGLVQPGGSLRLSCAASGFTVSSYSMRW YRQAPGKERELVARITSTGEPIEYVESVKGRFTISRDNALNTLNLEMNDLKP EDTAVYYCWGAGYWGQGTQVTVSS；

SEQ ID NO：9：AVKLVESGGGLVQPGGSLRLSCAASGFTVSNNNLRW YRQAPGKERELVAMITSAGNTNYADAVKGRSTISRDNAKNTLYLEMYRLK PEDTAVYYCWGAAHWGRGTQVTVSS；

SEQ ID NO：10：GVKLVESGGGLVQPGGSLTLSCVASGFDFNSAHMR WYRQGPGKEREMVASISTTGGVTIYEDSVKGRFTISRDNADNTAYLRMNSL KPEDTAVYYCRAYGFGIDYWGQGTQVTVSS。

the amino acid sequence of the CD47 single-domain antibody comprises FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 regions which are sequentially composed, and is specifically shown in Table 1.

Table 1:

the invention also provides a nucleotide sequence encoding a CD47 single domain antibody as described above. The nucleotide sequence is shown as SEQ ID NO: 11 to 20. Specific nucleotide sequences are shown in table 2.

TABLE 2

The invention also provides an expression vector comprising the nucleotide as described above.

The invention also provides a kit comprising a CD47 single domain antibody as described above, or a nucleotide sequence as described above.

According to the technical scheme, the single domain antibody specifically binding to the CD47 protein is obtained by a gene engineering binding method, and the single domain antibody specifically binding to the CD47 protein has the advantages of high activity, high expression level, low production cost and easiness in modification.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 is a first round of PCR single domain antibody gene electrophoresis of the present invention;

FIG. 2 is a second round of PCR single domain antibody gene electrophoresis of the 750bp-500bp gene fragment and the 500bp gene fragment in FIG. 1;

FIGS. 3 to 9 show the purification of CD47 protein expression in examples of the present invention;

FIGS. 10 to 19 are graphs showing the binding activity of a single domain CD47 antibody to CD47 antigen in examples of the present invention;

FIGS. 20 to 29 are affinity diagrams for single domain CD47 antibodies in an example of the invention;

fig. 30-34 are graphs showing the binding of CD47 single domain antibodies to HL-60 cells in examples of the present invention.

The implementation, functional features and advantages of the objects of the present invention will be further explained with reference to the accompanying drawings.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention and the accompanying drawings, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. And the instruments, reagents, materials and the like referred to in the following examples are conventional instruments, reagents, materials and the like in the prior art and are commercially available in a normal way unless otherwise specified. Unless otherwise specified, the experimental methods, detection methods, and the like in the following examples are conventional experimental methods, detection methods, and the like in the prior art.

The invention provides a CD47 single-domain antibody, a nucleotide sequence, an expression vector and a kit. The following details are provided for the CD47 single domain antibody and the screening process thereof.

Example 1 anti-CD 47 antigen-specific antibody library construction

It should be noted that the CD47 antigen selected in this example was purchased from Beijing Yi Qiao Shen, Cat No. 12283-H08H. The QIAGEN Kit is QIAamp RNA Blood Mini Kit (50) from Beijing Yaada Biotechnology, Inc., cat # 52304. The cDNA synthesis Kit is MiniBESTAgarose Gel DNAextraction Kit Ver.4.0 of TAKARA company.

1. And (4) performing an immunization experiment.

1) Selecting healthy alpaca as an immune subject;

2) 2mgCD47 antigen is mixed with equal volume of Freund's adjuvant, and a healthy alpaca is immunized for 6 times;

3) after the 4 th immunization experiment, the alpaca serum is sampled and the antigen immunization titer is measured;

4) continuing the immune experiment until the measured immune titer of the alpaca serum reaches more than 1 ten thousand times, taking 100ml of alpaca whole blood, and separating lymphocytes;

5) lysing the lymphocytes in the step 4), extracting RNA in the lysed lymphocytes by using a QIAGEN kit, and purifying the RNA; wherein, the anticoagulant adopted in the QIAGEN kit comprises one of citrate, heparin and EDTA.

2. Phage antibody libraries were constructed and screened for the specific antigen CD47 single domain antibodies.

1) Peripheral blood of alpaca immunized against CD47 antigen in an immunization experiment is used as a starting material, and RNA purified in the immunization experiment is reversely transcribed into cDNA by utilizing a cDNA synthesis kit to construct a cDNA library.

2) The target gene is cloned by adopting nested PCR.

a. Primers were designed based on the light chain-deleted antibody heavy chain variable region VHH gene fragment in the CD47 antigen, and in this example, two sets of primers were designed to amplify the light chain-deleted antibody heavy chain variable region VHH gene fragment.

b. First round PCR.

PCRFd 5' primer:

primer 1, nucleotide sequence as shown in SEQ ID NO: 21, and: CGCCATCAAGGTACCAG TTGA

And 2, the nucleotide sequence of the primer is shown as SEQ ID NO: 22, in which: CGGGATCCCAGGTACAG CTGGTGGAGTCTGGGGGAG

PCR Bd 3' primer:

and 3, the nucleotide sequence of the primer is shown as SEQ ID NO: 23, in the following steps: CCGCTCGAGTACTTCAT TCGT TCC TGAGGAGACGGT

The first round of PCR amplification can obtain a common heavy chain antibody gene fragment of more than 800bp, a heavy chain antibody gene fragment of 750bp-500bp which is a deletion light chain and a VHH target gene fragment of 500 bp. And screening the gene segments of 750bp to 500bp and the gene segments of 500bp by gel electrophoresis.

The electrophoresis results are shown in FIG. 1. Wherein, the common antibody is amplified to obtain two bright bands shown by a gel electrophoresis 1 runway in figure 1, and the basic group quantity is mainly distributed in 500 bp-750 bp; the primer 1 and the primer 2 are used in a matched mode, a bright band shown in a gel electrophoresis 2 track in the picture 1 is obtained through amplification, and the base quantity of the bright band is mainly distributed at about 500 bp; the primer 2 and the primer 3 are used in a matched mode, a bright band shown in a gel electrophoresis 3 runway in the picture 1 is obtained through amplification, and the base quantity of the bright band is mainly distributed at about 500 bp.

c. Cutting the gel to recover the segment containing the target gene. Cutting and recovering a No. 1 band with the base amount between 500bp and 750bp in the electrophoresis, wherein the No. 1 band comprises a common antibody gene, a heavy chain antibody gene without a light chain and a target gene; and cutting and recovering the No. 2 band and the No. 3 band with the base amount of 500bp in the electrophoresis, wherein the No. 2 band and the No. 3 band are bands with target genes.

d. Second round PCR.

Constructing primers:

and primer 4, the nucleotide sequence is shown as SEQ ID NO: 24, showing: CCGCTCGAGTGAGGAGA CGGTGACCTGG

Primer 5, nucleotide sequence shown as SEQ ID NO: 25, and: CGGGATCCGAGGTACAG CTGGTGGAGTCTGGGGGAG

The second round of PCR amplification and gel electrophoresis can screen out the VHH target gene (i.e. 500bp gene fragment) containing the heavy chain antibody variable region.

The electrophoresis results are shown in FIG. 2. Wherein, the left bright band is a labeled DNA molecule, the right bright band is obtained by using a primer 4 and a primer 5 for pairing and amplification, and the gene base amount is 500bp (namely the target gene).

3) Constructing a target gene library.

And carrying out enzyme digestion on the obtained VHH target gene fragment and the pHEN6 phage display vector plasmid by using BamHI endonuclease and XhoI endonuclease, and connecting the VHH target gene fragment to a pHEN6 vector by using ligase after enzyme digestion to construct a recombinant plasmid. The constructed recombinant plasmid is electrically transformed into a competent cell, so that the VHH target gene segment and the pHEN6 vector can be expressed in a fusion manner.

The obtained transformant bacterial liquid is coated on an LB/Amp plate and cultured overnight at 32 ℃. The PCR method of the next day colony verifies the connection efficiency of the antibody. If the connection efficiency of the antibody is less than or equal to 90%, repeating the experiment until the connection efficiency of the antibody is greater than 90%; if the antibody connection efficiency is higher than 90%, the target gene and the vector are well connected, the expression of the recombinant plasmid is good, and the colony amplification culture experiment can be continued.

4) And (5) performing amplification culture.

a. Coating the electro-transformation bacterial liquid on an LB/Amp plate, and culturing overnight at 32 ℃;

b. then washing with 2YT medium to wash off other components in the original culture environment, and adding the bacterial solution: amplifying and culturing phage expressing target genes on a 2YT culture medium at a ratio of 1: 1000;

c. adding helper phage M13K07(Invitrogen) for infection, and culturing overnight;

d. centrifuging and collecting the supernatant infected with the helper phage M13K 07;

e. adding 20% PEG-2.5M NaCl into the supernatant, mixing, centrifuging again, collecting precipitate, adding PBS and glycerol, resuspending, and storing at-80 deg.C.

EXAMPLE 2 screening of specific phages

1) And (5) preparing in the early stage. 5 x10 each of the CD47 negative control cell line and the engineered CD47 positive cell line were prepared separately⁶CPBSH or 2% BSA-PBS was used to incubate on the drum for 2 hours at room temperature and centrifuged for use.

2) And (4) screening. Phage (5 x 10)¹¹～1*10¹²) Adding into negative cells, quantifying to 0.5ml with CPBS or 2% BSA-PBS, and incubating on a rotary drum at room temperature for 2 hours; taking supernatant, adding into positive cells, quantifying to 0.5ml with CPBS or 2% BSA-PBS, and incubating for 2 hours at room temperature on a rotary drum; so that the outer shell of the phage specifically secretes the CD47 antibody, which binds to the CD47 antigen; wash 5 times with PBST, wash 3 times with PBS, centrifuge, discard supernatant to wash away unbound phage.

3) And (4) measuring the titer. The cell-bound phage was resuspended, 2ml of TG1 was taken in the logarithmic growth phase, infected, left to stand at 37 ℃ for 30min, and the titer was determined.

4) And (5) amplification and purification. Amplifying and purifying the amplified phage.

The above experiment was repeated three times, and the phage in step 4) was used as the phage to be added in the next step 2).

The screening results were as follows:

in this example, phage were screened 3 times and the titer of phage eluted from each screening test was measured, as shown in the table above: with the increase of the number of screening rounds, the concentration of the coating solution is gradually decreased, but the eluted phages are increased, namely the phages specific to the CD47 are enriched.

EXAMPLE 3 screening of specific Positive monoclonal antibodies

1) Screening the recombinant plasmid with high expression efficiency.

PCR-amplifying the specific CD47 antibody gene fragment obtained in example 2 to obtain a PCR product with restriction enzyme BbsI and BamHI sites; treating the PCR product and the pSJF2 vector with restriction enzymes BbsI and BamHI respectively; the digested gene fragments are connected by T4 ligase, and are recombined to obtain a recombinant plasmid sdAb-pSJF2 which can be efficiently expressed in Escherichia coli.

2) Screening for antibodies to CD47 positive clones.

Immediately picking single colony from the agar plate for growing colony, inoculating in 96-hole deep-hole culture plate containing Amp 2YT liquid culture medium, and culturing for 4 hr; inoculating single clones on numbered LB solid plates containing Amp and separated by small grids in a one-to-one correspondence manner; adding IPTG to the deep-hole culture plate to the final concentration of 0.5mM for induction, and culturing overnight; collecting the supernatant of the expressed protein, performing ELISA (enzyme-Linked immuno sorbent assay) by using CD47 antigen, and selecting colonies with OD (optical density) values 5 times larger than that of a negative control as selected CD47 positive clones; the positive clones were cultured in a small amount for seed preservation and subjected to DNA sequencing to identify the gene sequence of the anti-CD 47 single domain antibody, resulting in the gene sequences as shown in Table 2 above.

Example 4 expression and purification of Single Domain CD47 antibody in host E.coli

1) And (4) expression and purification.

Inoculating the positive clone bacterial liquid in the embodiment 3) into 5ml of LB culture solution containing ampicillin, and culturing overnight by shaking at 37 ℃; transferring 2mL of overnight culture into 200mL of LB culture solution containing ampicillin, carrying out shake culture at 37 ℃, and carrying out 240 r/min of shake culture until OD value reaches 0.4-0.6; adding 0.5-1.0 mM IPTG, continuously culturing overnight, then centrifuging, and collecting bacteria; bacteria are lysed by a hypertonic method, centrifuged, and soluble single domain antibody protein in supernatant is collected; obtaining the protein with the purity of more than 95 percent by Ni + ion affinity chromatography.

The purification results are shown in FIGS. 3 to 9. In FIGS. 3 to 5, the Marker is a protein molecule standard, CD47-117, CD47-118, CD47-127, CD47-160, (CD47-)169, CD47-172, CD47-175, (CD47-)229, (CD47-)236 and (CD47-)271 are the sequences of CD47 genes of SEQ ID NO: 100-109 total protein crude extraction samples after lysis, which shows that the positive clone bacteria screened in the example 3) can induce and express the CD47 antibody.

In fig. 6 to 9, the elution sample is purified by a nickel column.

Lanes 160(100), lanes 127(100), lanes 118(100), lanes 117(100), lanes 169(100), lanes 172(100), lanes 175(100), lanes 229(100), lanes 236(100), and lanes 271(100) are the samples remaining after passing through the nickel-removal column with an eluent containing 100 mM imidazole, which is described after one elution: the proteins in the (CD47-)117, (CD47-)118 and (CD47-)127 samples were substantially eluted; (CD47-)160, (CD47-) 169) little protein was eluted; most of the proteins in the samples (CD47-)172, (CD47-)175, (CD47-)229, (CD47-)236 and (CD47-) 271) were eluted.

Strip 160(200), strip 169(200), strip 172(200), strip 175(200), strip 229(200), strip 236(200), and strip 271(200) are samples remaining after passing through a nickel-removing column with an eluent containing 200 mM imidazole, after passing through two elutions: the protein in the (CD47-)160 and (CD47-)169 samples is basically eluted, and the purity of the eluted target protein is higher; very little protein was eluted in the samples (CD47-)172, (CD47-)175, (CD47-)229, (CD47-)236 and (CD47-) 271.

Strip 172(400), strip 175(400), strip 229(400), strip 236(400), and strip 271(400) are samples remaining after passing through a nickel-removal column with an eluent containing 400 mmol of imidazole, after three elutions: the proteins on the nickel column in the (CD47-)172, (CD47-)175, (CD47-)229, (CD47-)236 and (CD47-)271 samples are basically eluted, and the purity of the eluted target proteins is higher.

Example 5 determination of binding Activity of Single Domain CD47 antibody with CD47 antigen

1) And (6) coating. At 0.05M Na₂CO₃·NaHCO₃(pH9.5) diluting the CD47 antigen to 2. mu.g/ml; adding 100 μ l of diluted CD47 antigen protein into each reaction micropore of a 96-well plate, and incubating overnight at 4 ℃; the next day, the reaction micropore inner coating solution is discarded, and the plate is washed with PBS for three times; add 300. mu.l of 2% BSA (or 1% CPBS) to the blocked 96-well plate and incubate for 2 hours at 37 ℃.

2) Antigen-antibody binding. Add 100. mu.l of purified CD47 single domain antibody at various dilution concentrations to the reaction wells of each 96-well plate, incubate 1 hour at 37 ℃ and wash the plate three times with 0.05% PBST; add 100. mu.l 5000-fold diluted anti-His antibody (HRP) to each reaction well, incubate 1 hr at 37 ℃, wash plate three times with 0.05% PBST; mu.l TMB was added to each reaction well and left to stand at room temperature for 10min in the dark.

3) The reaction was terminated. The reaction was stopped by adding 50. mu.l of 2M H2SO4 to each reaction well.

4) And (6) judging the result. OD values of the samples at a wavelength of 450nm were measured by a microplate reader, and the results shown in FIGS. 10 to 19, which are concentration gradients (ELISA) of binding of CD47 protein to CD47 single domain antibodies purified from different gene fragments, were obtained. As seen from the figure, even the concentration of the CD47 single-domain antibody combined with the CD47 antigen is 32ng/ml, the antibody still has good binding activity, which indicates that the antibody binding activity is excellent and superior to that of the prior art.

Example 6 Single Domain antibody affinity detection of CD47

1) And (4) measuring the affinity. Capturing Human CD47/Biotinylated with SA probe in PBST solution and reacting well with the sample; the solid phase conjugate obtained from the above reaction was analyzed by dissociation in PBST buffer, and the results were analyzed by data analysis software to obtain the binding rate, dissociation rate and affinity constant, and the specific results are shown in fig. 20 to 29.

2) And (6) analyzing results. The four curves in the figure are the results of antibody measurement with different concentration gradients (1 st line for antibody with a concentration of 73.5nM, 2 nd line for antibody with a concentration of 147.1nM, 3 rd line for antibody with a concentration of 294.1nM, 4 th line for antibody with a concentration of 588.2 nM), with the abscissa as time axis (0-900 seconds) and the ordinate as the Response value (Response) read by the machine during the experiment. According to the experimental design, the antigen-antibody binding process is carried out 1200 seconds earlier, and the dissociation process is carried out 1200 seconds to 2400 seconds, which are shown by the cut line in the figure. The binding constant Kon (1/Ms) was obtained from the change of the machine-read value with time during the binding. The dissociation constant Kdis (1/s) can be obtained from the change of the reading value with time during the dissociation process. According to the formula: the affinity constant KD ═ dissociation constant Kdis/binding constant Kon, and the affinity constant KD of CD47 antibody determined using different concentrations can be obtained. The detection results are consistent under each concentration gradient, and the experimental results are reliable (refer to the fitted curve in the figure and the table below, R2> 0.95). The results show that: the affinity constant of each antibody is less than 1x10-9(nM grade), is superior to that of most existing antibodies, 1x10-8 and above, and is superior to that of the prior art.

Example 7 binding of CD47 Single Domain antibodies to HL-60 cells

1) Flow cytometric binding. Adopting 20 mu g/ml CD47 nanometer antibody protein to combine with HL-60 cells; anti-HIS antibody staining and flow cytometry analysis using a BD FACSCalibur flow cytometer gave the results shown in figures 30 to 34.

2) And (6) analyzing results. As can be seen from fig. 30 to 34, the CD47 nanobody can bind to the positive cell (i.e., HL-60 cell line) with a binding rate of > 99%, which indicates that the nanobody claimed in the embodiment group of the present invention has very good binding ability to CD47 antigen expressed on the surface of cell membrane, and is superior to the prior art.

It is understood that the cell screening method is adopted to replace the traditional solid phase screening method in the CD47 single domain antibody screening method of the embodiment, so that the target antigen can maintain the natural conformation, and the screening of the antibody of the membrane antigen or the epitope on the cell surface is more beneficial. The internalization type antibody aiming at the membrane antigen is obtained by screening in the embodiment of the invention, and has very positive effect on treating tumors.

Finally, the CD47 specific monoclonal nano-antibody claimed in the embodiment group can be used for identification of CD47 positive cells, positive cell screening, positive rate detection, development of antibody drugs targeting CD47 positive cells, ADC complex drugs, CAR-T cell drugs and other biological drugs related to expression of CD47 protein.

The above description is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention, and all modifications and equivalents of the present invention, which are made by the contents of the present specification and the accompanying drawings, or directly/indirectly applied to other related technical fields, are included in the scope of the present invention.

SEQUENCE LISTING

<110> Shenzhen Puruijin biopharmaceutical industry Co., Ltd

<120> CD47 single-domain antibody, nucleotide sequence, expression vector and kit

<130>

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<170> PatentIn version 3.3

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Gln Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Leu Thr Phe Ser Ser Tyr

20 25 30

Ser Met Arg Trp Tyr Arg Gln Ala Pro Gly Gln Glu Arg Glu Leu Val

35 40 45

Ala Arg Leu Thr Ser Ser Gly Glu Pro Ile Glu Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ala Ser Arg Asp Asn Ala Lys Ser Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Trp Ile Ala Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

100 105 110

<210> 7

<211> 116

<212> PRT

<213> Artificial Synthesis

<400> 7

Gln Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Glu

1 5 10 15

Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Asp Ala

20 25 30

His Met Arg Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Met Val

35 40 45

Ala Ser Ile Ser Thr Thr Gly Ser Ile Thr Ile Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Asp Glu Lys Thr Val Tyr

65 70 75 80

Leu Arg Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Ala Tyr Tyr Cys

85 90 95

Arg Ala Tyr Gly Phe Gln Ile Asp Ser Trp Gly Gln Gly Thr Gln Val

100 105 110

Thr Val Ser Ser

115

<210> 8

<211> 112

<212> PRT

<213> Artificial Synthesis

<400> 8

Asp Val Gln Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Ser Tyr

20 25 30

Ser Met Arg Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val

35 40 45

Ala Arg Ile Thr Ser Thr Gly Glu Pro Ile Glu Tyr Val Glu Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Leu Asn Thr Leu Asn

65 70 75 80

Leu Glu Met Asn Asp Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Trp Gly Ala Gly Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

100 105 110

<210> 9

<211> 111

<212> PRT

<213> Artificial Synthesis

<400> 9

Ala Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Asn Asn

20 25 30

Asn Leu Arg Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val

35 40 45

Ala Met Ile Thr Ser Ala Gly Asn Thr Asn Tyr Ala Asp Ala Val Lys

50 55 60

Gly Arg Ser Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu

65 70 75 80

Glu Met Tyr Arg Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Trp

85 90 95

Gly Ala Ala His Trp Gly Arg Gly Thr Gln Val Thr Val Ser Ser

100 105 110

<210> 10

<211> 116

<212> PRT

<213> Artificial Synthesis

<400> 10

Gly Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Thr Leu Ser Cys Val Ala Ser Gly Phe Asp Phe Asn Ser Ala

20 25 30

His Met Arg Trp Tyr Arg Gln Gly Pro Gly Lys Glu Arg Glu Met Val

35 40 45

Ala Ser Ile Ser Thr Thr Gly Gly Val Thr Ile Tyr Glu Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Asp Asn Thr Ala Tyr

65 70 75 80

Leu Arg Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Arg Ala Tyr Gly Phe Gly Ile Asp Tyr Trp Gly Gln Gly Thr Gln Val

100 105 110

Thr Val Ser Ser

115

<210> 11

<211> 336

<212> DNA

<213> Artificial Synthesis

<400> 11

gatgtgaagc tggtggagtc tgggggaggc ttggtgcagc ctggggggtc tctgagactc 60

tcctgtgcag cctctggatt caccgtcagt agctactcca tgagatggta ccgccaggtt 120

ccaggaaagg agcgcgagtt gatcgcaaga attactagta ctggtcaacc tatagaatat 180

ggcgagtccg tgaagggccg attcaccatc tccagagaca atgccaagaa cacgctgtat 240

ctggaaatga acgcactgaa acctgaggac acggccgtgt attactgttg gggcgctggg 300

tactggggcc aggggaccca ggtcaccgtc tcctca 336

<210> 12

<211> 348

<212> DNA

<213> Artificial Synthesis

<400> 12

caggtgaagc tggaggagtc tgggggaggc ttggtgcagc caggggagtc tctgagactc 60

tcctgcgtag cctctggatt cgccttcagt agcgcactca tgaggtggta ccgccaggct 120

ccagggaagg agcgcgagtt ggtcgcatct attactaccg ctggtggtat cacaggttat 180

gcagacagcg tgaagggccg attcaccatc tccagagaca atgccgagaa cacgctgtat 240

ctgcaaatga acagcctgaa acctgaggac acggccgtat attactgtag ggcttacggg 300

tttcaacttg acaactgggg ccaggggacc caggtcaccg tctcctca 348

<210> 13

<211> 336

<212> DNA

<213> Artificial Synthesis

<400> 13

catgtgcagc tggaggagtc tgggggaggc ctggtgcagc ctggggggtc tctgagactc 60

tcctgtaaag cctctggatt gaccttcgct agttattcta tgagatggta ccgccaggct 120

ccagggcagg agcgcgaact tgtcgcacga ttgtctagta gcggtgttcc tatagagtat 180

gtggactccg tgaagggccg attcaccgcc tccagagacg atgccaagag cacgctatat 240

ttgcaaatga acagcctgaa acctgaggac acggccatgt attactgctg gattgcgaat 300

tactggggcc aggggaccca ggtcaccgtc tcctca 336

<210> 14

<211> 336

<212> DNA

<213> Artificial Synthesis

<400> 14

gatgtgcagc tggtggagtc tgggggaggc tcggtgcagc ctggggggtc tctgacactc 60

tcctgtgcag cctctggatt caccgtcagt aactactcca tgagatggta ccgccaggct 120

ccagggaagg agcgcgagtt gatcgcaaga attactagta ctggtgtacc tatagaatat 180

gcagactctg tgaagggccg attcaccatc tccagagaca atgccaaaaa cacgctgtat 240

ctggaaatga acggcctgaa acctgaggac acggccgtct attactgttg gggcgctgcg 300

tactggggcc aggggaccca ggtcaccgtc tcctca 336

<210> 15

<211> 348

<212> DNA

<213> Artificial Synthesis

<400> 15

ggggtgcagc tggtggagtc tgggggaggc ttggtgcagc ctggggagtc tctgagactc 60

tcctgcgtag cctctggatt cgccttcagt agcgcactca tgaggtggta ccgccaggct 120

ccagggaagg agcgcgagtt ggtcgcatct gttactacta ctggtggtat cacaggttat 180

gcagactccg tgaagggccg attcaccatc tccagagaca atgccgagaa cacgctgtat 240

ctgcgaatga acagcctgaa acctgaggac acggccgtgt attactgtag ggcttacggg 300

tttggacttg actcctgggg ccaggggacc caggtcaccg tctcctca 348

<210> 16

<211> 336

<212> DNA

<213> Artificial Synthesis

<400> 16

caggtgaagc tggaggagtc tgggggaggc ctggtgcagc ctggggggtc tctgagactc 60

tcctgtgcag cctctggatt gaccttcagt agttattcta tgagatggta ccgccaggct 120

ccagggcagg agcgcgaact tgtcgcacgt cttactagta gcggtgagcc tatagagtat 180

gcagactccg tgaagggccg attcaccgcc tccagagaca atgccaagag cacggtatat 240

ttgcaaatga acagcctgaa acctgaggac acggccatgt attactgctg gattgcgaac 300

tactggggcc aggggaccca ggtcaccgtc tcctca 336

<210> 17

<211> 348

<212> DNA

<213> Artificial Synthesis

<400> 17

caggtgaagc tggaggagtc tgggggaggc ttggtgcagc ctggggaatc tctgagactc 60

tcctgcgtag cctctggatt caccttcagt gacgcacaca tgaggtggta ccgccaggct 120

ccagggaagg agcgcgagat ggtcgcatct atttctacta ctggtagtat cacaatttat 180

gcagactccg tgaagggccg attcaccatc tccagagaca atgacgagaa aacggtgtat 240

ctgcgaatga atagtctgaa acctgaggac acggccgcgt attactgtag ggcttacggg 300

tttcaaattg actcctgggg ccaggggacc caggtcaccg tctcctca 348

<210> 18

<211> 336

<212> DNA

<213> Artificial Synthesis

<400> 18

gatgtgcagc tggaggagtc tgggggaggc ttggtgcagc ctggggggtc tctgagactc 60

tcctgtgcag cctctggatt caccgtcagt agctactcca tgagatggta ccgccaggct 120

ccagggaagg agcgcgagtt ggtcgcaaga atcactagta ctggtgaacc tatagagtat 180

gtagaatccg tgaagggccg attcaccatc tccagagaca atgccttgaa cacgctgaat 240

ctggaaatga acgacctgaa acctgaggac acggccgtgt attactgttg gggcgctggg 300

tactggggcc aggggaccca ggtcaccgtc tcctca 336

<210> 19

<211> 333

<212> DNA

<213> Artificial Synthesis

<400> 19

gctgtgaagc tggtggagtc tgggggaggc ttggtgcagc ctggggggtc tctgagactc 60

tcctgtgcag cctctggatt caccgtcagt aataacaact tgaggtggta ccgacaggct 120

ccaggaaaag agcgcgagtt ggttgcgatg atcactagcg ctggtaacac aaactatgca 180

gacgccgtga agggccgatc caccatctcc agagacaatg ccaagaacac gttgtatctg 240

gaaatgtata ggctgaaacc tgaggacacg gccgtgtatt actgttgggg tgcggcccac 300

tggggccggg ggacccaggt caccgtctcc tca 333

<210> 20

<211> 348

<212> DNA

<213> Artificial Synthesis

<400> 20

ggtgtgaagc tggtggagtc tgggggaggc ttggtgcagc ctggggggtc tctgacgctc 60

tcctgcgtag cctctggatt cgacttcaat agcgcacaca tgaggtggta ccgccagggt 120

ccagggaagg agcgcgagat ggtcgcatct atttctacta ctggtggagt cacaatttat 180

gaagactccg tgaagggccg attcaccatc tccagagaca atgccgacaa tacggcgtat 240

ctgcgaatga acagcctgaa acctgaggac acggccgtgt attactgtag ggcttacggg 300

tttggaattg actattgggg ccaggggacc caggtcaccg tctcctca 348

<210> 21

<211> 21

<212> DNA

<213> Artificial Synthesis

<400> 21

cgccatcaag gtaccagttg a 21

<210> 22

<211> 36

<212> DNA

<213> Artificial Synthesis

<400> 22

cgggatccca ggtacagctg gtggagtctg ggggag 36

<210> 23

<211> 36

<212> DNA

<213> Artificial Synthesis

<400> 23

ccgctcgagt acttcattcg ttcctgagga gacggt 36

<210> 24

<211> 28

<212> DNA

<213> Artificial Synthesis

<400> 24

ccgctcgagt gaggagacgg tgacctgg 28

<210> 25

<211> 36

<212> DNA

<213> Artificial Synthesis

<400> 25

cgggatccga ggtacagctg gtggagtctg ggggag 36

Claims

A CD47 single domain antibody comprising at least one of CD47-117, CD47-118, CD47-127, CD47-160, CD47-169, CD47-172, CD47-175, CD47-229, CD47-236, and CD47-271 antibodies; wherein the sequence of the CD47-117 is shown in SEQ ID NO: 1 is shown in the specification; the sequence of the CD47-118 is shown as SEQ ID NO: 2 is shown in the specification; the sequence of the CD47-127 is shown as SEQ ID NO: 3 is shown in the specification; the sequence of the CD47-160 is shown as SEQ ID NO: 4 is shown in the specification; the sequence of the CD47-169 is shown as SEQ ID NO: 5 is shown in the specification; the sequence of the CD47-172 is shown as SEQ ID NO: 6 is shown in the specification; the sequence of the CD47-175 is shown as SEQ ID NO: 7 is shown in the specification; the sequence of the CD47-229 is shown as SEQ ID NO: 8 is shown in the specification; the sequence of the CD47-236 is shown as SEQ ID NO: 9 is shown in the figure; the sequence of the CD47-271 is shown as SEQ ID NO: shown at 10.
2. A nucleotide sequence encoding the CD47 single domain antibody of claim 1.
3. The nucleotide sequence of claim 2, wherein the nucleotide sequence is as set forth in SEQ ID NO: 11 to 20.
4. An expression vector comprising the nucleotide of claim 2 or 3.
5. A kit comprising a CD47 single domain antibody according to claim 1, or a nucleotide sequence according to claim 2 or 3.