CN110862455A - Polypeptides that can bind to CD47 and their applications - Google Patents

Abstract

The invention relates to a polypeptide capable of binding to human CD47 protein, which comprises 3 complementarity determining regions CDR1-3, wherein the sequence of CDR1 is or comprises one of the sequences shown in SEQ ID NO. 1-13, the sequence of CDR2 is or comprises one of the sequences shown in SEQ ID NO. 14-24, and the sequence of CDR3 is or comprises one of the sequences shown in SEQ ID NO. 25-34. Aiming at a tumor immunotherapy target of CD47, a nano antibody VHH specifically combined with CD47 is screened by preparing a CD47 protein, immunizing alpaca, utilizing a platform technology of phage library display nano monoclonal antibody and the like, and a humanized antibody is constructed; and simultaneously, identifying the binding of the humanized antibody and the cell surface CD47 protein by using a flow cytometry detection method. The invention provides a potential nano antibody new drug for clinical treatment of various cancers.

Description

Polypeptides that can bind to CD47 and their applications

technical field

The present invention relates to the field of biomedicine. More particularly, it relates to a CD47-binding polypeptide and uses thereof.

Background technique

CD47 is widely expressed on the cell surface of different tissues and is a member of the immunoglobulin superfamily. The CD47 protein can issue a "don't eat me" signal by binding to the ligand signal-regulating protein alpha (SIRPα) on macrophages. Tumor cells can escape the recognition and clearance of the body's immune system through a variety of ways, including the overexpression of the cell surface protein CD47. Studies have shown that using antibodies to block the CD47-SIRPα pathway can promote the phagocytosis of tumor cells, and targeting CD47 is a highly potential anti-tumor direction that can be used to treat patients with solid tumors and lymphomas.

In 1993, a novel natural antibody derived from the alpaca family was discovered. The antibody naturally lacks the light chain and only consists of the heavy chain, the heavy chain contains two constant regions (CH2 and CH3), a hinge region and a heavy chain variable region (Variableheavy chain domain, VHH, ie antigen binding site) , the relative molecular mass of the variable region of the heavy chain is about 13KDa, which is only 1/10 of that of conventional antibodies, and the molecular height and diameter are both at the nanometer level. It is the smallest functional antibody fragment currently available. Therefore, it is also called It is Nanobody (Nb). Nanomab has been widely used recently due to its high stability (not degraded at 90°C), high affinity, more than 80% homology with human antibodies, and low toxicity and immunogenicity. In the development of immunodiagnostic kits, imaging research and development of antibody drugs for the fields of tumor, inflammation, infectious diseases and neurological diseases. In addition, with the rapid development of the field of cell therapy, nanobodies are also widely used in antibody screening in the field of cell therapy.

The development trend of therapeutic antibody drugs is from mouse-derived, human-mouse chimeric, humanized to fully human, and then to nanobodies, which have received widespread attention in recent years, and the research and development of nanobodies has developed rapidly. The first nanobody drug was approved in September 2018. At present, there are many nanobody drugs in the clinical research stage at home and abroad. We expect to obtain high-affinity therapeutic nanobodies targeting CD47 by immunizing alpacas.

SUMMARY OF THE INVENTION

The present invention obtains the alpaca-derived nano-mAb and its VHH by immunizing the alpaca with the antigen, and is used for the treatment of solid tumor and lymphoma. Based on these studies, the present invention provides a CD47-binding polypeptide, comprising three complementarity determining regions CDR1-3, the CDR1 sequence is or includes one of the sequences shown in SEQ ID NOs: 1-13, and the CDR2 sequence is or includes SEQ ID NO: 1-13. One of the sequences shown in ID NO: 14-24, and the CDR3 sequence is or includes one of the sequences shown in SEQ ID NO: 25-34.

In a specific embodiment, the polypeptide further comprises four framework regions, FR1-4, which are staggered with the CDR1-3. For example, the FR1-4 sequence can be designed as shown in SEQ ID NOs: 35-38, but the scope of the present invention is not limited thereto, and the framework region FR1-4 sequence can also be humanized as SEQ ID NO: 39- 42 shown. The specific recognition and binding ability of an antibody is mainly determined by the sequence of the CDR region, and the FR sequence has little effect and can be designed according to the species, which is well known in the art. For example, FR region sequences of human, murine or alpaca origin can be designed to link the above CDRs to obtain a polypeptide or domain that can bind to human CD47.

In a preferred embodiment, the polypeptide is a monoclonal antibody.

In a preferred embodiment, the polypeptide is VHH.

In a preferred embodiment, the polypeptide is an alpaca-derived VHH or a humanized VHH.

In a specific embodiment, the CDR sequence of the polypeptide is as follows:

1) The sequence of CDR2 is SEQ ID NO: 43, wherein X at position 4 represents isoleucine, lysine, arginine or threonine, and X at position 8 represents threonine or isoleucine amino acid; and

II) The sequences of CDR1 are selected from SEQ ID NOs: 1-4, and the sequences of CDR3 are selected from SEQ ID NOs: 25-27.

Preferably, the sequence of CDR1 is SEQ ID NO: 1, the sequence of CDR2 is SEQ ID NO: 14 and the sequence of CDR3 is SEQ ID NO: 25; or

The sequence of CDR1 is SEQ ID NO:2, the sequence of CDR2 is SEQ ID NO:15 and the sequence of CDR3 is SEQ ID NO:25; or

The sequence of CDR1 is SEQ ID NO:3, the sequence of CDR2 is SEQ ID NO:16 and the sequence of CDR3 is SEQ ID NO:25; or

The sequence of CDR1 is SEQ ID NO:3, the sequence of CDR2 is SEQ ID NO:16 and the sequence of CDR3 is SEQ ID NO:26; or

The sequence of CDR1 is SEQ ID NO:4, the sequence of CDR2 is SEQ ID NO:17 and the sequence of CDR3 is SEQ ID NO:27.

In another specific embodiment, the CDR sequence of the polypeptide is as follows:

1) the sequence of CDR2 is SEQ ID NO:44, wherein X at position 6 is arginine or threonine; and

II) The sequence of CDR1 is selected from SEQ ID NOs: 5-8; and the sequence of CD3 is selected from SEQ ID NOs: 28-30.

Preferably, the CDR sequence of the polypeptide is as follows:

The sequence of CDR1 is SEQ ID NO:5, the sequence of CDR2 is SEQ ID NO:18 and the sequence of CDR3 is SEQ ID NO:28; or

The sequence of CDR1 is SEQ ID NO:5, the sequence of CDR2 is SEQ ID NO:18 and the sequence of CDR3 is SEQ ID NO:29; or

The sequence of CDR1 is SEQ ID NO:6, the sequence of CDR2 is SEQ ID NO:18 and the sequence of CDR3 is SEQ ID NO:28; or

The sequence of CDR1 is SEQ ID NO:7, the sequence of CDR2 is SEQ ID NO:18 and the sequence of CDR3 is SEQ ID NO:28; or

The sequence of CDR1 is SEQ ID NO:8, the sequence of CDR2 is SEQ ID NO:19 and the sequence of CDR3 is SEQ ID NO:30.

In another specific embodiment, the CDR sequence of the polypeptide is as follows:

1) the sequence of CDR3 is SEQ ID NO: 31; and

II) The sequence of CDR1 is selected from SEQ ID NOs: 9 and 10; and the sequence of CD2 is selected from SEQ ID NOs: 20 and 21.

Preferably, the CDR sequence of the polypeptide is as follows:

The sequence of CDR1 is SEQ ID NO:9, the sequence of CDR2 is SEQ ID NO:20 and the sequence of CDR3 is SEQ ID NO:31; or

The sequence of CDR1 is SEQ ID NO:10, the sequence of CDR2 is SEQ ID NO:21 and the sequence of CDR3 is SEQ ID NO:31.

In another specific embodiment, the CDR sequence of the polypeptide is as follows:

The sequence of CDR1 is SEQ ID NO:11, the sequence of CDR2 is SEQ ID NO:22 and the sequence of CDR3 is SEQ ID NO:32; or

The sequence of CDR1 is SEQ ID NO:12, the sequence of CDR2 is SEQ ID NO:23 and the sequence of CDR3 is SEQ ID NO:33;

The sequence of CDR1 is SEQ ID NO:13, the sequence of CDR2 is SEQ ID NO:24 and the sequence of CDR3 is SEQ ID NO:34.

The present invention also provides the application of the above-mentioned polypeptide in a tumor therapeutic drug.

The present invention also provides nucleic acid coding sequences of the above-mentioned polypeptides.

In one embodiment, the nucleic acid coding sequence is a DNA coding sequence or an RNA coding sequence.

In a specific embodiment, the nucleic acid coding sequence is present in gene expression frame.

The invention conducts nanobody drug development for solid tumors and lymphomas. The nanobody VHH that specifically binds to human CD47 is screened by preparing human CD47 protein, immunizing alpacas, and using phage library to display nanobody monoclonal antibody platform technology. Its CDR sequence, and constructed a humanized VHH-huFc1 (C47NB); at the same time, flow cytometry was used to evaluate the binding of the humanized antibody to the cell surface CD47 protein. The present invention provides a potential nanobody new drug for clinical treatment of tumors.

Description of drawings

Fig. 1 is the detection curve of antiserum titer one week after the 3rd and 4th immunization of alpacas with CD47;

Figure 2 is a flow cytometry result of the binding of serum CD47 to CD47 protein on the surface of multiple myeloma cells 8226 one week after the fourth immunization. Among them, the abscissa represents the antibody binding to the cell surface CD47 protein in serum, and the ordinate represents the commercially available direct-labeled antibody binding to the cell surface CD47 protein.

Fig. 3 is the electropherogram of PCR product amplified by CD47-VHH phage antibody library as template;

Fig. 4 is the panning identification of CD47-VHH phage antibody library, wherein, A is the ELISA detection statistics chart of the phage library after panning for CD47 protein; B is the second round ( ^2nd ) and the third round ( ^3rd ) panning After the phage antibody library, 40 and 46 clones were selected for phage ELISA detection statistics chart;

Fig. 5 is a statistical chart of ELISA detection of prokaryotically expressed VHH antibodies, each point represents a clone, the ordinate is the OD450 of human CD47/the OD450 of blank control, and the ratio greater than 5.0 is defined as positive;

Figures 6 and 7 are flow charts of the binding of 19 VHH antibodies prepared by the present invention to CD47 on the surface of PRMI8226 cells.

Detailed ways

1. Preparation of Immunogens

Based on the human CD47 protein sequence and gene sequence information on the NCBI website, we analyzed and designed the polypeptide CD47, which can effectively induce alpacas to produce specific antibodies against the cell surface CD47 protein, and connect His-tag (CD47-his) or Rabbit Fc (CD47-rFc) was used for subsequent purification and detection.

2. Alpaca immunization and acquisition of antiserum

Bactrian alpacas were primed with an emulsified mixture of 250 μg CD47-rFc protein and 250 μl Freund’s complete adjuvant, and boosted with CD47-rFc protein and 250 μl Freund’s incomplete adjuvant on days 14, 28, and 42 3 times, 1 week after the 2nd and 3rd immunization, blood was collected to detect the antiserum titer; 1 week after the 4th immunization, 200 ml of blood was collected for the construction of phage antibody library.

The antiserum titer was detected by ELISA. The detection plate was coated with CD47-his protein at a concentration of 0.5 μg/ml, and 100 μl of serially diluted antiserum or purified antibody was added to each well (the control was the alpaca serum before immunization), and the temperature was 37°C. Incubate for 1.5h, wash twice, add horseradish peroxidase-labeled Goat anti-Llamma IgG (H+L) secondary antibody diluted 1:10000 to each well, incubate at 37°C for 1h, wash 4-6 times, add 100 μl of TMB substrate was incubated at 37°C for 10 min, the reaction was stopped with 50 μl of 0.2M H ₂ SO ₄ , and the OD 450 nm was measured. The serum titer for ELISA detection was defined as the highest dilution at which the OD450 was more than 2.1 times that of the blank control and greater than 0.2.

The results are shown in Figure 1. The antiserum titers of 3 and 4 were 1.09×10 ⁶ and 3.28×10 ⁶ , respectively. It can be seen that this antigen can induce alpaca to produce high titer antiserum specific for human CD47 protein.

In order to further verify whether the high titer of alpaca antiserum can effectively bind to the CD47 protein on the cell surface, flow cytometry was performed. Antiserum and pre-immune serum at different dilution concentrations were incubated with human multiple myeloma cells RPMI8226 cells for 60min respectively. After washing the cells, fluorescent secondary antibody Alexa Fluor 488Goat antiAlpaca IgG (H+L) was added, and the cells were washed after incubating at 4°C for 30min. , check on the machine. Flow cytometry results showed that CD47-immunized alpaca antiserum could bind to cell surface CD47 protein (Figure 2). In summary, CD47 protein induces high titer antiserum, and the antiserum has the ability to bind to cell surface CD47 protein, which can be used for flow detection.

3. VHH phage library construction and panning

200 ml of peripheral blood of immunized alpaca was collected, and the PBMC of alpaca was obtained by using lymphocyte separation medium (GE Ficoll-Paque Plus) to separate and obtain the PBMC of alpaca. According to the TRIzol operation manual, RNA was extracted, and oligo (dT) was used to invert it into cDNA, and the primers were used. Amplification, molecular cloning and other techniques, clone the VHH gene of alpaca into phagemid plasmid, transform TG1 bacteria, and obtain VHH phage library. In order to further identify whether the CD47-VHH phage library was successfully constructed, the VHH target gene of the immunized CD47 alpaca was amplified by PCR. It can be seen that the target band is 500bp and the size is in line with expectations (Figure 3), indicating that the CD47-VHH phage antibody library It contains the VHH gene. Thirty-three clones were selected for sequencing, and the sequencing results showed that the sequence diversity was 93.9%; the comparison results showed that most of the difference sequences were in the CDR binding region. After testing, the constructed CD47-VHH phage antibody library has a capacity of 1.35×10 ⁹ , the positive rate is 100%, the sequence diversity (Diversity) is 93.9%, and the effective insertion rate (In frame rate) is greater than 95%.

With the help of M13KO7 helper phage, bacteria transformed with VHH-phagemid were subjected to recovery of the phage antibody library and precipitation with PEG/NaCl. The phage antibody library was enriched three times with CD47-His protein coated with 50 μg/ml. The enriched phages were eluted, transformed, plated, and single clones were picked to identify the binding of phages to CD47 protein ELISA. The clones with a binding reading > 1.0 were sequenced and cloned into the expression vector phv13, transformed into SS320 cells for expression Production of Nanomab.

The panned library was detected for binding to CD47 protein. The results of phage ELISA showed that the binding read value of CD47-VHH phage library and CD47 protein before enrichment was 0.33, and the read value of phage library after one round, two rounds and three rounds of enrichment were 0.49, 1.73, 3.34 ( Figure 4A). In order to further verify the positive rate of phage binding to CD47-VHH protein in the enriched library, 40 and 46 clones were selected from the enriched library in the second and third rounds for single phage ELISA detection. The results showed that in the second round library, 42.5% of the single phage clones were positive, and 89% of the phage clones in the third round library were positive, and the average reading value of binding was around 3.0 (Fig. 4B). Panning by CD47 protein A high-binding CD47-VHH phage library was successfully enriched.

4. Construction of VHH prokaryotic expression library and VHH expression

PCR amplification was performed on the 2nd-CD47-VHH and 3rd-cCD47-VHH phage antibody libraries after the second and third rounds of panning and enrichment; the gene fragments of all VHHs in the antibody library were obtained and purified, and the gene fragments of VHH were cloned To the prokaryotic expression vector, transform the SS320 strain, and construct the prokaryotic expression antibody library of VHH; spread the prokaryotic expression antibody library on the plate, cultivate it overnight, and randomly select 600 monoclonal colonies the next day, use IPTG to induce the expression of the antibody supernatant. The serum was detected by ELISA binding to CD47 protein.

The results showed that there was bacterial supernatant binding to CD47 protein, while not binding to blank control, and the read value of CD47 binding/blank control read value was greater than 5.0 (Figure 5 and Table 1). These sequences were sequenced and compared, and repeated sequences were eliminated, and finally 52 VHH antibody sequences were obtained. Further experiments confirmed that 19 of the 52 VHH antibodies could bind to the cell surface CD47 protein (SEQ ID NOs: 1-19).

Table 1 Binding values and sequences of 19 VHH antibodies to CD47 protein

5. Detection of the binding of VHH antibody to tumor cells by drain cell method

The VHH antibody was mixed with PRMI 8226 cells and incubated, 100 μl/sample, at 4 °C for 1 h; after washing twice with 0.5% PBSF, the secondary antibody Alexa Fluor 488goat anti human IgG was added, at 4 °C for 30 min; after washing twice with 0.5% PBSF, On-board detection. MOCK is the PBS control; the neg group is the negative control, that is, the prokaryotic expression supernatant control without antibody; the positive is the positive control, that is, the positive antibody control that binds to the CD47 membrane protein. The results are shown in Figures 6 and 7. Flow detection showed that all 19 VHH antibodies could bind to PRMI8226 cells, among which IAP-114, 118, 121, 129, 132, 140 and 148 had higher binding capacity. Similar results were obtained using humanized VHH antibodies. It can be seen that the above VHH antibodies have the ability to target and bind to tumor cells, and at the same time, it may promote the phagocytosis of macrophages by blocking the CD47 molecules on the surface of tumor cells, thereby achieving the effect of treating or inhibiting tumor growth. Therefore, these 19 VHH antibodies Antibodies have the potential to become novel antibody drugs for the treatment of tumors.

Because 19 VHHs can recognize CD47 molecules on the surface of tumor cells, 19 VHH antibody sequences can also be applied to CAR (Chimeric Antigen Receptor, antigen chimeric receptors, which are fused by VHH sequences of the third or fourth generation CD28-4- 1BB-CD3zeta molecular sequence composition) cell therapy for tumor therapy. In addition, because 19 VHHs can recognize CD47 molecules on the surface of tumor cells, VHHs can be used for ADC (Antibody-drug conjugate, antibody-drug conjugates) treatment by conjugating drugs or isotopes for antibody-dependent molecular imaging diagnosis.

Provide potential nano-drugs for clinical treatment of tumors.

6. In vivo experiments using AAV viral vector-loaded humanized VHH

Adeno-associated virus vectors (AAVs) are derived from non-pathogenic wild-type adeno-associated viruses, and due to their good safety, wide host cell range (dividing and non-dividing cells), low immunogenicity, and long in vivo expression of exogenous genes It is regarded as one of the most promising gene transfer vectors and has been widely used in gene therapy and vaccine research worldwide.

The AAV Helper-Free Viral Packaging System was purchased from Cell Biolabs, San Diego USA. The DNA coding sequence of the above VHH was inserted into the pAAV-MCS plasmid by molecular cloning technology; after the successful construction was proved by sequencing, the constructed plasmid pAAV-Ab and pHelper and pAAV-DJ plasmids were in a mass ratio of 1:1:1. AAV-293T cells were co-transfected using PEI transfection reagent. Supernatants were collected at 48, 72, 96 and 120 hours after transfection, concentrated with 5xPEG8000 (sigma) and finally purified with 1.37 g/ml cesium chloride. Purified AAV was dissolved in PBS, identified and aliquoted and stored at -80°C.

Multiple myeloma model mice were injected intramuscularly with AAV-VVH (1×10 ¹¹ gc/100 μl), and AAV-GFP was used as a control group. The results show that AAV-VVH has a therapeutic effect on multiple myeloma.

The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the protection of the present invention. within the range.

sequence listing

<110> Yuandaolong (Suzhou) Medical Technology Co., Ltd.

<120> Polypeptides that can bind to CD47 and their applications

<160> 44

<170> SIPOSequenceListing 1.0

<210> 1

<211> 8

<212> PRT

<213> Artificial Sequence

<400> 1

Gly Arg Asn Leu Asn Glu Asn Gly

1 5

<210> 2

<211> 8

<212> PRT

<213> Artificial Sequence

<400> 2

Gly Arg Thr Phe Ser Glu Thr Gly

1 5

<210> 3

<211> 8

<212> PRT

<213> Artificial Sequence

<400> 3

Gly Arg Asn Leu Ser Glu Asn Gly

1 5

<210> 4

<211> 7

<212> PRT

<213> Artificial Sequence

<400> 4

Gln Thr Ile Ser Glu Asn Gly

1 5

<210> 5

<211> 8

<212> PRT

<213> Artificial Sequence

<400> 5

Gly Gly Ile Phe Gly Pro Ile Ala

1 5

<210> 6

<211> 8

<212> PRT

<213> Artificial Sequence

<400> 6

Gly Gly Ile Phe Gly Ser Ile Ala

1 5

<210> 7

<211> 8

<212> PRT

<213> Artificial Sequence

<400> 7

Gly Gly Ile Phe Gly Thr Ile Ala

1 5

<210> 8

<211> 8

<212> PRT

<213> Artificial Sequence

<400> 8

Gly Gly Ile Phe Gly Phe Asn Ala

1 5

<210> 9

<211> 8

<212> PRT

<213> Artificial Sequence

<400> 9

Gly Leu Thr Phe Ser Glu Ser Gly

1 5

<210> 10

<211> 8

<212> PRT

<213> Artificial Sequence

<400> 10

Gly Leu Pro Phe Ser Glu Thr Gly

1 5

<210> 11

<211> 8

<212> PRT

<213> Artificial Sequence

<400> 11

Gly Ser Asp Phe Glu Phe Tyr Ala

1 5

<210> 12

<211> 8

<212> PRT

<213> Artificial Sequence

<400> 12

Gly Arg Ile Val Ser Ile Asn Val

1 5

<210> 13

<211> 8

<212> PRT

<213> Artificial Sequence

<400> 13

Gly Arg Thr Ile Ser Thr Tyr Arg

1 5

<210> 14

<211> 8

<212> PRT

<213> Artificial Sequence

<400> 14

Ile Asn Trp Ile Gly Gly Asn Thr

1 5

<210> 15

<211> 8

<212> PRT

<213> Artificial Sequence

<400> 15

Ile Asn Trp Lys Gly Gly Asn Ile

1 5

<210> 16

<211> 8

<212> PRT

<213> Artificial Sequence

<400> 16

Ile Asn Trp Arg Gly Gly Asn Thr

1 5

<210> 17

<211> 8

<212> PRT

<213> Artificial Sequence

<400> 17

Ile Asn Trp Thr Gly Gly Asn Thr

1 5

<210> 18

<211> 7

<212> PRT

<213> Artificial Sequence

<400> 18

Ile Ser Gly Gly Gly Arg Thr

1 5

<210> 19

<211> 7

<212> PRT

<213> Artificial Sequence

<400> 19

Ile Ser Gly Gly Gly Ser Thr

1 5

<210> 20

<211> 8

<212> PRT

<213> Artificial Sequence

<400> 20

Ile Ser Trp Arg Gly Gly Asn Thr

1 5

<210> 21

<211> 8

<212> PRT

<213> Artificial Sequence

<400> 21

Ile Ser Trp Thr Gly Asp Tyr Thr

1 5

<210> 22

<211> 7

<212> PRT

<213> Artificial Sequence

<400> 22

Ile Thr Arg Ala Leu Tyr Thr

1 5

<210> 23

<211> 7

<212> PRT

<213> Artificial Sequence

<400> 23

Ile Thr Ser Asp Gly Ala Thr

1 5

<210> 24

<211> 8

<212> PRT

<213> Artificial Sequence

<400> 24

Ile Thr Trp Val Val Gly Arg Thr

1 5

<210> 25

<211> 17

<212> PRT

<213> Artificial Sequence

<400> 25

Ala Ala Ser Asp Leu Ser Gly Ser Phe Gly Ser Glu Thr Trp Tyr His

1 5 10 15

Tyr

<210> 26

<211> 17

<212> PRT

<213> Artificial Sequence

<400> 26

Val Ala Ser Asp Leu Ser Gly Ser Phe Gly Ser Glu Thr Trp Tyr His

1 5 10 15

Tyr

<210> 27

<211> 17

<212> PRT

<213> Artificial Sequence

<400> 27

Ala Ala Ser Ser Leu Ser Gly Ser Phe Gly Ser Glu Thr Trp Tyr His

1 5 10 15

Tyr

<210> 28

<211> 5

<212> PRT

<213> Artificial Sequence

<400> 28

Trp Ile Gly Gly Tyr

1 5

<210> 29

<211> 5

<212> PRT

<213> Artificial Sequence

<400> 29

Trp Phe Gly Gly Tyr

1 5

<210> 30

<211> 5

<212> PRT

<213> Artificial Sequence

<400> 30

Trp Ile Arg Gly Tyr

1 5

<210> 31

<211> 17

<212> PRT

<213> Artificial Sequence

<400> 31

Ala Ala Ser Asp Leu Ser Gly Ser Phe Gly Ser Glu Thr Trp Phe His

1 5 10 15

Tyr

<210> 32

<211> 6

<212> PRT

<213> Artificial Sequence

<400> 32

Asn Val Gly Gly Pro Tyr

1 5

<210> 33

<211> 15

<212> PRT

<213> Artificial Sequence

<400> 33

Asn Thr Asn Leu Leu Ala Asp Tyr Glu Leu Arg Tyr Arg Asp Tyr

1 5 10 15

<210> 34

<211> 16

<212> PRT

<213> Artificial Sequence

<400> 34

Ala Ala Asp Asn Gly Pro Arg Ala Tyr Lys Ser Glu Ala Phe Glu Tyr

1 5 10 15

<210> 35

<211> 25

<212> PRT

<213> Artificial Sequence

<400> 35

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Thr Gly Gly

1 5 10 15

Ser Leu Thr Leu Ser Cys Ala Ala Ser

20 25

<210> 36

<211> 17

<212> PRT

<213> Artificial Sequence

<400> 36

Leu Gly Trp Phe Arg Gln Ala Thr Gly Lys Glu Arg Glu Phe Val Gly

1 5 10 15

Ser

<210> 37

<211> 38

<212> PRT

<213> Artificial Sequence

<400> 37

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

1 5 10 15

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

20 25 30

Thr Ala Val Tyr Tyr Cys

35

<210> 38

<211> 20

<212> PRT

<213> Artificial Sequence

<400> 38

Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Glu Pro Lys Thr Pro

1 5 10 15

Lys Pro Gln Pro

20

<210> 39

<211> 25

<212> PRT

<213> Artificial Sequence

<400> 39

Gln Val Arg Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Glu

1 5 10 15

Thr Leu Arg Leu Ser Cys Thr Ala Ser

20 25

<210> 40

<211> 17

<212> PRT

<213> Artificial Sequence

<400> 40

Met Gly Trp Tyr Arg Gln Gly Pro Gly Asn Glu Cys Glu Met Val Ala

1 5 10 15

Tyr

<210> 41

<211> 36

<212> PRT

<213> Artificial Sequence

<400> 41

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

1 5 10 15

His Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Gly

20 25 30

Val Tyr Tyr Cys

35

<210> 42

<211> 10

<212> PRT

<213> Artificial Sequence

<400> 42

Gly Gln Gly Thr Arg Val Thr Val Ser Ser

1 5 10

<210> 43

<211> 8

<212> PRT

<213> Artificial Sequence

<400> 43

Ile Asn Trp Xaa Gly Gly Asn Xaa

1 5

<210> 44

<211> 7

<212> PRT

<213> Artificial Sequence

<400> 44

Ile Ser Gly Gly Gly Xaa Thr

1 5

Claims (10)

1. a polypeptide that can bind CD47, is characterized in that, comprises 3 complementarity determining regions CDR1-3, CDR1 sequence is or comprises one of the sequences shown in SEQ ID NO:1-13, CDR2 sequence is or comprises SEQ ID NO : one of the sequences shown in SEQ ID NOs: 14-24, and the CDR3 sequence is or includes one of the sequences shown in SEQ ID NOs: 25-34. 2 . The polypeptide according to claim 1 , wherein the polypeptide further comprises four framework regions, FR1-4, and the FR1-4 and the CDR1-3 are staggered in sequence. 3 . 3. The polypeptide according to claim 2, wherein the polypeptide is a monoclonal antibody. 4. The polypeptide according to claim 2, wherein the polypeptide is VHH. 5 . The polypeptide according to claim 4 , wherein the polypeptide is an alpaca-derived VHH or a humanized VHH. 6 . 6. Use of the polypeptide of any one of claims 1-5 in detecting cell surface CD47. 7. Use of the polypeptide according to any one of claims 1-5 in the preparation of a tumor therapeutic drug. 8. Use of the polypeptide of any one of claims 1-5 in the preparation of a CAT T cell therapeutic agent. 9. Use of the nucleic acid coding sequence of the polypeptide of any one of claims 1-5 in gene therapy. 10 . A reagent for detecting CD47 on a cell surface, comprising the polypeptide according to claim 1 . 11 .

Images