CN110862455A

CN110862455A - Polypeptide capable of binding CD47 and application thereof

Info

Publication number: CN110862455A
Application number: CN201911216842.XA
Authority: CN
Inventors: 吴喜林; 吴稚伟; 苏艾荣; 黄碧莲
Original assignee: Nanjing University
Current assignee: Nanjing University
Priority date: 2019-12-03
Filing date: 2019-12-03
Publication date: 2020-03-06
Anticipated expiration: 2039-12-03
Also published as: WO2021110181A3; WO2021110181A2; CN110862455B

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

Polypeptide capable of binding CD47 and application thereof

Technical Field

The invention relates to the field of biomedicine. More particularly, it relates to a polypeptide capable of binding to CD47 and its application.

Background

CD47 is widely expressed on the cell surface of different tissues and is a member of immunoglobulin superfamily, CD47 protein can emit 'self-eating' signal by combining with ligand signal regulatory protein α (SIRP α) on macrophage, tumor cells can escape from the recognition and elimination of the body immune system by various ways, wherein, the over-expression of cell surface protein CD47 is included, research shows that the blocking of CD47-SIRP α channel by using antibody can promote the tumor cells to be phagocytized, and the targeting CD47 is a very potential anti-tumor direction and can be used for treating patients with solid tumor and lymphoma.

In 1993, a novel natural antibody derived from alpacaceae was discovered. The antibody naturally lacks a light chain and consists only of a heavy chain comprising two constant regions (CH2 and CH3), a hinge region and a heavy chain variable region (VHH, i.e., antigen binding site) with a relative molecular mass of about 13KDa, which is only 1/10 of conventional antibodies, and is the smallest functional antibody fragment currently available, both in molecular height and diameter at the nanometer level, and thus is also known as a Nanobody (Nb). Because the nano monoclonal antibody has the characteristics of high stability (not degraded at 90 ℃), high affinity, homology of more than 80 percent with a human antibody, low toxicity and immunogenicity and the like, the nano monoclonal antibody is widely applied to the research and development of immunodiagnosis kits, the research and development of imaging, and the research and development of antibody drugs aiming at the fields of tumors, inflammations, infectious diseases, nervous system diseases and the like. In addition, with the rapid development of the cell therapy field, the nano antibody is also widely applied to antibody screening in the cell therapy field.

The development trend of therapeutic antibody drugs is from murine, human-murine chimeric, humanized to fully human, to recently focused nano-antibodies, and the development of nano-antibodies is rapidly progressing. The first nano-antibody drug was approved in 2018, 9 months. At present, a plurality of nano antibody medicines are in clinical research stage at home and abroad. We expect that by immunizing alpaca, high affinity therapeutic nanobodies targeting CD47 are obtained.

Disclosure of Invention

The alpaca source nanometer monoclonal antibody and the VHH thereof are obtained by immunizing alpaca with antigen and are used for treating solid tumors and lymphomas. Based on these studies, the present invention provides a polypeptide that binds to CD47, comprising 3 complementarity determining regions CDR1-3, the CDR1 sequence is or includes one of the sequences shown in SEQ ID NOS 1-13, the CDR2 sequence is or includes 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.

In a specific embodiment, the polypeptide further comprises 4 framework regions FR1-4, said FR1-4 being staggered with respect to said CDR 1-3. 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 shown in SEQ ID NOS: 39-42. The specific recognition and binding ability of an antibody is mainly determined by the CDR region sequences, and the FR sequences have little influence and can be designed according to species, which is well known in the art. For example, FR region sequences of human, murine or alpaca origin may be designed to link the above CDRs, resulting in a polypeptide or domain that binds human CD 47.

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 a VHH of alpaca origin or a humanized VHH.

In one embodiment, the CDR sequences of the polypeptides are as follows:

I) 43 in SEQ ID NO, wherein X at position 4 represents isoleucine, lysine, arginine or threonine, and X at position 8 represents threonine or isoleucine; and is

II) the sequence of CDR1 is selected from SEQ ID NOS: 1-4 and the sequence of CDR3 is 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 sequences of the polypeptides are as follows:

I) the sequence of CDR2 is SEQ ID NO. 44, where X at position 6 is arginine or threonine; and is

II) the sequence of CDR1 is selected from SEQ ID NOS: 5-8; and the sequence of CD3 is selected from SEQ ID NO 28-30.

Preferably, the CDR sequences of the polypeptides are 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.

I) the sequence of CDR3 is SEQ ID NO. 31; and is

II) the sequence of CDR1 is selected from the group consisting of SEQ ID NOS: 9 and 10; and the sequence of CD2 is selected from SEQ ID NO:20 and 21.

Preferably, the CDR sequences of the polypeptides are 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.

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 invention also provides the application of the polypeptide in tumor treatment medicines.

The present invention also provides the nucleic acid encoding sequence of the polypeptide.

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 a gene expression cassette.

The invention carries out nano antibody drug development aiming at solid tumors and lymphomas, and the nano antibody VHH specifically combined with human CD47 is screened by preparing human CD47 protein, immune alpaca, platform technology utilizing phage library to display nano monoclonal antibody and the like, the CDR sequence is identified, and humanized VHH-huFc1(C47NB) is constructed; the binding of the humanized antibody to cell surface CD47 protein was also assessed using flow cytometry. The invention provides a potential nano-antibody new drug for clinical treatment of tumors.

Drawings

FIG. 1 is a graph showing the antiserum titer test curves of CD47 after one week of 3rd and 4 th immunization of alpaca;

figure 2 is a graph of flow results of serum binding to CD47 protein on the cell surface of multiple myeloma cells 8226 one week after the 4 th immunization with CD 47. Wherein the abscissa indicates that the antibody in serum binds to cell surface CD47 protein, and the ordinate indicates that the commercially available orthometric antibody binds to cell surface CD47 protein.

FIG. 3 is an electrophoretogram of PCR products amplified using a CD47-VHH phage antibody library as a template;

FIG. 4 shows the panning identification of the CD47-VHH phage antibody library, wherein A is the ELISA detection statistical chart after phage library panning against CD47 protein; b is a second wheel (2)^nd) And a third wheel (3)^rd) Respectively selecting 40 clones and 46 clones from the panned phage antibody library to carry out phage ELISA detection statistical chart;

FIG. 5 is a statistical ELISA assay for prokaryotically expressed VHH antibodies, each dot representing a clone, with OD450 for human CD 47/OD 450 for the blank on the ordinate, a positive ratio greater than 5.0 being defined;

figures 6 and 7 are flow charts of the binding of 19 VHH antibodies prepared according to the invention to PRMI8226 cell surface CD 47.

Detailed Description

1. Preparation of immunogens

According to the sequence and gene sequence information of human CD47 protein on NCBI website, polypeptide CD47 capable of effectively inducing alpaca to generate specific antibody aiming at cell surface CD47 protein is analyzed and designed, and His-tag (CD47-His) or rabbit Fc (CD47-rFc) is connected at the C terminal for subsequent purification and detection.

2. Preparation of alpaca immune and antiserum

Priming alpaca doublet with emulsified mixture of 250 μ g CD47-rFc protein and 250 μ l Freund's complete adjuvant, boosting 3 times with CD47-rFc protein and 250 μ l Freund's incomplete adjuvant on 14, 28, and 42 days, collecting blood to detect antiserum titer 1 week after 2nd and 3rd immunization; after 1 week of the 4 th immunization, 200ml of blood was collected for the construction of phage antibody library.

Antiserum titers were measured by ELISA, assay plates were coated with CD47-his protein at a concentration of 0.5 μ g/ml, and 100 μ l of either the antiserum or purified antibody (control was pre-immune alpaca serum) was added to each well in a gradient, incubated at 37 ℃ for 1.5h, washed 2 times, and 1: 10000 diluted second antibody of horse radish peroxidase labeled Goat anti-Llama IgG (H + L) is incubated for 1H at 37 ℃, after washing for 4-6 times, 100 mu L of TMB substrate is added, incubation is carried out for 10min at 37 ℃, and 50 mu L of 0.2M H is added₂SO₄The reaction was stopped and the OD450 nm was measured. ELISA for serum titer determinationIs the highest dilution factor at OD450 that is more than 2.1 times that of the blank and greater than 0.2.

As shown in FIG. 1, the antiserum titers of 3-and 4-immunization were 1.09X 10, respectively⁶And 3.28X 10⁶. It can be seen that the antigen can induce alpaca to produce high-titer antiserum specific to human CD47 protein.

To further verify whether the high titer alpaca antisera was able to effectively bind to cell surface CD47 protein, flow cytometry assays were performed. And (3) respectively incubating antiserum and preimmune serum with different dilution concentrations with human multiple myeloma RPMI8226 cells for 60min, washing the cells, adding a fluorescent secondary antibody Alexa Fluor 488 coat anti Alpaca IgG (H + L), incubating at 4 ℃ for 30min, washing the cells, and detecting on a machine. Flow cytometry results showed that alpaca antiserum immunized with CD47 can bind to cell surface CD47 protein (fig. 2). In summary, the CD47 protein induces high titers of antisera, which have the ability to bind to cell surface CD47 protein and can be used for flow assays.

Construction and panning of VHH phage library

Collecting 200ml of immunized alpaca peripheral blood, separating by using lymphocyte separation liquid (GE Ficoll-Paque Plus) to obtain alpaca PBMC, extracting RNA according to a TRIzol operation manual, inverting by using oligo (dT) into cDNA, cloning the VHH gene of the alpaca to phagemid plasmid through technologies such as primer amplification, molecular cloning and the like, and transforming TG1 bacteria to obtain the VHH phage library. To further identify whether the CD47-VHH phage library was successfully constructed, the VHH target gene of immune CD47 alpaca was amplified by PCR, and it was found that the target band was 500bp and the size was as expected (FIG. 3), indicating that the VHH gene was contained in the CD47-VHH phage antibody library. Selecting 33 clones for sequencing, wherein sequencing results show that the diversity of the sequenced sequence is 93.9%; the alignment results show that the most of the different sequences are in the CDR binding region. Through detection, the library capacity of the constructed CD47-VHH phage antibody library is 1.35 multiplied by 10⁹The positive rate was 100%, the sequence Diversity (Diversity) was 93.9%, and the effective insertion rate (In frame rate) was greater than 95%.

The phage antibody library was recovered from VHH-phagemid transformed bacteria with the help of M13KO7 helper phage and precipitated with PEG/NaCl. The phage antibody library was enriched three times with CD47-His protein coated with 50. mu.g/ml. And (3) carrying out elution, transformation, plate coating and monoclonal picking on the enriched phage, carrying out binding identification on the phage and CD47 protein ELISA, sequencing the clone with the binding reading value of more than 1.0, cloning to an expression vector phv13, and transforming SS320 cells to express to produce the nano monoclonal antibody.

The panned library was tested for binding to CD47 protein. The phage ELISA results showed that the binding reading values of the CD47-VHH phage library and CD47 protein before enrichment were 0.33, and the reading values of the phage library after one, two and three rounds of enrichment were 0.49, 1.73 and 3.34 respectively (FIG. 4A). To further verify the positive phage rate of binding to CD47-VHH protein in the enriched library, 40 and 46 clones were selected from the enriched library of rounds 2 and 3, respectively, for single phage ELISA detection. The results showed that 42.5% of the individual phage clones were positive in the library round 2 and 89% of the phage clones were positive in the library round 3, and the mean reading for binding was around 3.0 (FIG. 4B), and the high binding CD47-VHH phage library was successfully enriched by CD47 protein panning.

Construction of VHH prokaryotic expression library and VHH expression

PCR amplification of the enriched 2nd-CD47-VHH and 3rd-cCD47-VHH phage antibody libraries after the two and three rounds of panning described above; obtaining and purifying all VHH gene fragments in an antibody library, cloning the VHH gene fragments to a prokaryotic expression vector, converting an SS320 strain, and constructing a prokaryotic expression antibody library of the VHH; coating a plate with the prokaryotic expression antibody library, culturing overnight, randomly selecting 600 monoclonal colonies the next day, inducing expression of antibody supernatant by IPTG, and carrying out ELISA binding detection on the antibody supernatant and CD47 protein.

The results show that there was bacterial supernatant that bound CD47 protein while not binding to the blank, and that CD47 bound reads/blank reads greater than 5.0 (figure 5 and table 1). Sequencing and comparing the sequences, and removing repeated sequences to finally obtain 52 VHH antibody sequences. Further experiments demonstrated that 19 of these 52 VHH antibodies could bind to cell surface CD47 protein (SEQ ID NOS: 1-19).

Binding values and sequences of the 119 VHH antibodies to CD47 protein

5. Detection of binding of VHH antibodies to tumor cells by lost cell assay

Mixing VHH antibody and PRMI8226 cells, incubating at 100 μ l/sample, and 4 ℃ for 1 h; washing twice with 0.5% PBSF, adding secondary Alexa Fluor 488 coat anti human IgG, and keeping the temperature at 4 ℃ for 30 min; after washing twice with 0.5% PBSF, the machine is used for detection. MOCK is PBS control; neg group is negative control, namely prokaryotic expression supernatant control without antibody; positive control, i.e. a positive antibody control that binds to CD47 membrane protein. As a result, as shown in fig. 6 and 7, flow assay showed that 19 VHH antibodies were all able to bind to PRMI8226 cells, with higher binding of IAP-114, 118, 121, 129, 132, 140 and 148. Similar results were obtained using humanized VHH antibodies. Therefore, the VHH antibody has the capacity of being combined with tumor cells in a targeted mode, and meanwhile, the phagocytosis of macrophages is promoted through blocking CD47 molecules on the surfaces of the tumor cells, so that the effect of treating or inhibiting the growth of tumors is achieved, and therefore the 19 VHH antibodies have the potential to become novel antibody medicines for treating the tumors.

Since 19 VHHs recognize CD47 molecules on the surface of tumor cells, 19 VHH antibody sequences can also be applied in the treatment of tumors by CAR (Chimeric Antigen Receptor, consisting of VHH sequence fused to third or fourth generation CD28-4-1BB-CD3zeta molecule sequence) cells. In addition, since 19 VHHs recognize CD47 molecules on the surface of tumor cells, VHHs can be used for ADC (Antibody-drug conjugate) therapy by coupling drugs or for molecular image diagnosis depending on antibodies by coupling isotopes, and the like.

Provides a potential new nano-drug for clinical treatment of tumors.

6. In vivo experiments using humanized VHH loaded AAV viral vectors

Adeno-associated virus (AAV) is derived from non-pathogenic wild adeno-associated virus, and is considered one of the most promising gene transfer vectors due to its high safety, wide host cell range (dividing and non-dividing cells), low immunogenicity, and long time for expressing foreign genes in vivo, and is widely used in gene therapy and vaccine research worldwide.

AAV Helper-Free viral packaging system was purchased from Cell Biolabs, San Diego USA. Inserting the DNA coding sequence of the VHH into the pAAV-MCS plasmid by a molecular cloning technology; after the successful construction is proved by sequencing, the constructed plasmid pAAV-Ab and pHelper and pAAV-DJ plasmids are used for co-transfecting AAV-293T cells by using a PEI transfection reagent according to the mass ratio of 1:1: 1. Supernatants were collected at 48, 72, 96 and 120 hours post transfection and concentrated with 5xPEG8000(sigma) and finally purified with 1.37g/ml cesium chloride. Purified AAV was dissolved in PBS, identified and stored at-80 ℃ after packaging.

Multiple myeloma model mice were subjected to AAV-VVH (1X 10)¹¹gc/100. mu.l) were injected intramuscularly, and AAV-GFP was used as a control group. The result shows that AAV-VVH has therapeutic effect on multiple myeloma.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Sequence listing

<110> Source daolong (Suzhou) medical science and technology, Inc

<120> polypeptide capable of binding to CD47 and use thereof

<160>44

<170>SIPOSequenceListing 1.0

<210>1

<211>8

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>1

Gly Arg Asn Leu Asn Glu Asn Gly

1 5

<210>2

<211>8

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>2

Gly Arg Thr Phe Ser Glu Thr Gly

1 5

<210>3

<211>8

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>3

Gly Arg Asn Leu Ser Glu Asn Gly

1 5

<210>4

<211>7

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>4

Gln Thr Ile Ser Glu Asn Gly

1 5

<210>5

<211>8

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>5

Gly Gly Ile Phe Gly Pro Ile Ala

1 5

<210>6

<211>8

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>6

Gly Gly Ile Phe Gly Ser Ile Ala

1 5

<210>7

<211>8

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>7

Gly Gly Ile Phe Gly Thr Ile Ala

1 5

<210>8

<211>8

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>8

Gly Gly Ile Phe Gly Phe Asn Ala

1 5

<210>9

<211>8

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>9

Gly Leu Thr Phe Ser Glu Ser Gly

1 5

<210>10

<211>8

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>10

Gly Leu Pro Phe Ser Glu Thr Gly

1 5

<210>11

<211>8

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>11

Gly Ser Asp Phe Glu Phe Tyr Ala

1 5

<210>12

<211>8

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>12

Gly Arg Ile Val Ser Ile Asn Val

1 5

<210>13

<211>8

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>13

Gly Arg Thr Ile Ser Thr Tyr Arg

1 5

<210>14

<211>8

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>14

Ile Asn Trp Ile Gly Gly Asn Thr

1 5

<210>15

<211>8

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>15

Ile Asn Trp Lys Gly Gly Asn Ile

1 5

<210>16

<211>8

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>16

Ile Asn Trp Arg Gly Gly Asn Thr

1 5

<210>17

<211>8

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>17

Ile Asn Trp Thr Gly Gly Asn Thr

1 5

<210>18

<211>7

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>18

Ile Ser Gly Gly Gly Arg Thr

1 5

<210>19

<211>7

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>19

Ile Ser Gly Gly Gly Ser Thr

1 5

<210>20

<211>8

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>20

Ile Ser Trp Arg Gly Gly Asn Thr

15

<210>21

<211>8

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>21

Ile Ser Trp Thr Gly Asp Tyr Thr

1 5

<210>22

<211>7

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>22

Ile Thr Arg Ala Leu Tyr Thr

1 5

<210>23

<211>7

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>23

Ile Thr Ser Asp Gly Ala Thr

1 5

<210>24

<211>8

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>24

Ile Thr Trp Val Val Gly Arg Thr

1 5

<210>25

<211>17

<212>PRT

<213> Artificial sequence (artificacial 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 (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 (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 (Artificial Sequence)

<400>28

Trp Ile Gly Gly Tyr

1 5

<210>29

<211>5

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>29

Trp Phe Gly Gly Tyr

1 5

<210>30

<211>5

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>30

Trp Ile Arg Gly Tyr

1 5

<210>31

<211>17

<212>PRT

<213> Artificial Sequence (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 (Artificial Sequence)

<400>32

Asn Val Gly Gly Pro Tyr

1 5

<210>33

<211>15

<212>PRT

<213> Artificial Sequence (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 (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 (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 (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 (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 (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 (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 (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 (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 (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 (Artificial Sequence)

<400>43

Ile Asn Trp Xaa Gly Gly Asn Xaa

1 5

<210>44

<211>7

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>44

Ile Ser Gly Gly Gly Xaa Thr

1 5

Claims

1. A polypeptide that binds CD47, comprising 3 complementarity determining regions CDR1-3, the CDR1 sequence is or includes one of the sequences shown in SEQ ID NOS 1-13, the CDR2 sequence is or includes 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 of claim 1, wherein said polypeptide further comprises 4 framework regions FR1-4, said FR1-4 being sequentially staggered from said CDR 1-3.

3. The polypeptide of claim 2, wherein the polypeptide is a monoclonal antibody.

4. The polypeptide of claim 2, wherein the polypeptide is a VHH.

5. The polypeptide of claim 4, wherein the polypeptide is a VHH of alpaca origin or a humanized VHH.

6. Use of the polypeptide of any one of claims 1-5 for detecting cell surface CD 47.

7. Use of a polypeptide according to any one of claims 1-5 for the manufacture of a medicament for the treatment of a tumor.

8. Use of a polypeptide according to any one of claims 1 to 5 in the preparation of a CAT T cell therapeutic.

9. Use of a nucleic acid encoding sequence for a polypeptide according to any of claims 1-5 in gene therapy.

10. A reagent for detecting CD47 on the surface of a cell, comprising the polypeptide of any one of claims 1-5.