CN112062849B

CN112062849B - CD47 antagonists and uses thereof

Info

Publication number: CN112062849B
Application number: CN202010461205.5A
Authority: CN
Inventors: 宋德勇; 徐洪光; 韩静; 韩镇; 窦昌林; 曹发扬
Original assignee: Shandong Boan Biotechnology Co Ltd
Current assignee: Shandong Boan Biotechnology Co Ltd
Priority date: 2019-06-10
Filing date: 2020-05-27
Publication date: 2022-07-22
Anticipated expiration: 2040-05-27
Also published as: CN112062849A; CN112062848B; CN112062848A

Abstract

The present invention relates to antibodies or antigen-binding fragments thereof that bind to the CD47 antigen, and methods of making, compositions, and uses thereof. The antibody has good interaction with CD47 antigen, can be used for preparing anti-CD47 antibody medicines, and can be used for treating and/or preventing CD47 related diseases, such as cancers. It can also be used for immunological detection of CD47 antigen.

Description

CD47 antagonists and uses thereof

Technical Field

The invention relates to the technical field of biomedicine or biopharmaceutical, in particular to a human anti-CD47 antibody, and a coding sequence, a preparation method, a composition and application thereof.

Background

CD47, also known as Integrin Associated Protein (IAP), is a member of the immunoglobulin superfamily. CD47 is widely expressed on the Cell surface and interacts with Signal regulatory protein alpha (SIRP alpha), thrombospondin (TSP 1) and integrins (integrins) to mediate a series of reactions such as apoptosis, proliferation and immunity (Maes T, Joos GF, Brusselle GG. am J Respir Cell Mol biol. 2012; 47(3): 261-.

The human body requires 20-30trillion red blood cells in order to maintain efficient oxygen transport throughout the body. The life cycle of erythrocytes is as short as 120 days, with 100 hundred million erythrocytes produced per hour, and countless aged erythrocytes phagocytosed and eliminated by macrophages. CD47 is an important "self" marker on the cell surface and is an important signal for regulating macrophage phagocytosis. CD47 can bind to macrophage surface SIRP alpha, phosphorylate its ITIM, and subsequently recruit SHP-1 protein, resulting in a cascade of reactions that inhibit phagocytosis of macrophages. Young erythrocytes express higher CD47 releasing the "self, eat me" signal to macrophages, while aged erythrocytes CD47 are down regulated and eventually cleared by macrophages. The tumor cells have a series of schemes for avoiding the pursuit of the human immune system, including secreting immune suppression factors, reducing MHC I expression, and up-regulating PD-L1 to inhibit CD8⁺T cell activity. Smart tumor cells do not, of course, pass the perfect mask of CD 47. Different studies have shown that almost all tumor cells and tissues highly express CD47, 3-fold higher than corresponding normal cells and tissues. By means of the "self" signal of CD47, tumor cells effectively evade phagocytosis by macrophages. Tumor cells up-regulate the expression of CD47, thereby deceiving macrophages. Then the "eat me" signal is blocked by the CD47 antibody, causing the macrophages to exert phagocytosis. CD47 antibody treatment was by DC cells and CD8⁺T exerts a tumor killing effect. The DC cells phagocytose the tumor cells through the synergistic action of the CD47 antibody and the phagocytosis molecule, and present tumor-associated antigens to CD8⁺T, further develops CD8⁺T specific killing effect on tumor.

However, the CD47 antibody currently in clinical use or on the market still has deficiencies in terms of antibody affinity, immunogenicity, side effects, anti-tumor effects, etc., and therefore development of a candidate antibody for the target with better effect is urgently needed.

Disclosure of Invention

The invention provides an antibody capable of specifically binding to a CD47 antigen, and a preparation method and application thereof.

To achieve the above objects, the present invention provides an antibody or antigen-binding fragment thereof that binds to human CD 47. The antibody or antigen-binding fragment thereof of the present invention refers to an antibody or antigen-binding fragment that is free of the human or animal body, i.e., an isolated antibody or antigen-binding fragment thereof.

In one aspect of the invention, the antibody or antigen binding fragment thereof comprises light and heavy chain Complementarity Determining Regions (CDRs) selected from any one of the following sets of amino acid sequences:

1) light chain complementarity determining regions CDR1-CDR3 shown in SEQ ID NOs:11-13 and/or heavy chain complementarity determining regions CDR1-CDR3 shown in SEQ ID NOs: 14-16;

2) light chain complementarity determining regions CDR1-CDR3 shown in SEQ ID NOs:19-21 and/or heavy chain complementarity determining regions CDR1-CDR3 shown in SEQ ID NOs: 22-24;

3) the light chain complementarity determining regions CDR1-CDR3 shown in SEQ ID NOs:27-29 and/or the heavy chain complementarity determining regions CDR1-CDR3 shown in SEQ ID NOs: 30-32.

In one aspect of the invention, the antibody or antigen-binding fragment thereof light chain comprises a light chain variable region (VL) comprising the light chain variable region amino acid sequence set forth in SEQ ID NO:9 or SEQ ID NO:16 or SEQ ID NO:23 or a light chain variable region comprising an amino acid sequence having at least 80%, 85% or 90% sequence identity to the light chain variable region amino acid sequence set forth above; the antibody or antigen-binding fragment heavy chain comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO:10 or SEQ ID NO:17 or SEQ ID NO:24 or an amino acid sequence having at least 80%, 85% or 90% sequence identity to the above heavy chain variable region amino acid sequence. Preferably, the antibody or antigen-binding fragment comprises an amino acid sequence comprising a sequence selected from any one of the following groups:

1) a light chain variable region comprising the amino acid sequence shown in SEQ ID NO. 9 and/or a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO. 10;

2) a light chain variable region comprising the amino acid sequence shown in SEQ ID NO. 17 and/or a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO. 18;

3) the variable region of the light chain having the amino acid sequence shown in SEQ ID NO. 25 and/or the variable region of the heavy chain comprising the amino acid sequence shown in SEQ ID NO. 26.

The invention also provides nucleic acid sequences encoding antibodies or antigen-binding fragments thereof that bind to human CD 47.

In one aspect of the invention, the nucleic acid sequence encoding an antibody or antigen-binding fragment thereof that binds human CD47 is a nucleic acid encoding an antibody or antigen-binding fragment thereof that comprises the amino acid sequences of any one of the following groups:

2) the light chain complementarity determining regions CDR1-CDR3 shown in SEQ ID NOs:19-21 and/or the heavy chain complementarity determining regions CDR1-CDR3 shown in SEQ ID NOs: 22-24;

3) light chain complementarity determining regions CDR1-CDR3 shown in SEQ ID NOs:27-29 and/or heavy chain complementarity determining regions CDR1-CDR3 shown in SEQ ID NOs: 30-32.

4) A light chain variable region comprising an amino acid sequence having at least 80%, 85% or 90% sequence identity to the amino acid sequence set forth in SEQ ID NO 9 or SEQ ID NO 17 or SEQ ID NO 25 and/or a heavy chain variable region comprising an amino acid sequence having at least 80%, 85% or 90% sequence identity to the amino acid sequence set forth in SEQ ID NO 10 or SEQ ID NO 18 or SEQ ID NO 26;

5) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 9 and/or a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 10;

6) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 17 and/or a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 18;

7) the variable region of the light chain having the amino acid sequence shown in SEQ ID NO. 25 and/or the variable region of the heavy chain comprising the amino acid sequence shown in SEQ ID NO. 26.

The invention also provides a vector comprising a nucleotide sequence encoding an antibody or antigen-binding fragment thereof that specifically binds CD47, preferably the vector is an expression vector. The expression vector of the present invention includes, but is not limited to, viral vectors such as adenoviral vectors, retroviral vectors, adeno-associated viral vectors, etc.; non-viral vectors, such as plasmids, transposon vectors, and the like.

In one aspect of the invention, the expression vector having a nucleotide sequence encoding an antibody or antigen-binding fragment thereof that binds CD47 is a plasmid vector. Preferably, the plasmid vector containing a nucleotide sequence encoding an antibody or antigen-binding fragment thereof that specifically binds to human CD47 is a pcdna3.4(Life Technology) vector.

The invention also provides a host cell for expressing an antibody or antigen-binding fragment thereof that binds to human CD47, the host cell comprising an expression vector encoding the antibody or antigen-binding fragment thereof that specifically binds to human CD47 or a nucleic acid encoding the antibody or antigen-binding fragment thereof that specifically binds to human CD 47.

In one aspect of the invention, host cells expressing an antibody or antigen-binding fragment thereof that binds human CD47 include, but are not limited to, mammalian cells, insect cells, plant cells, fungal cells, bacterial cells. Preferably, the host cell for expressing the antibody or antigen-binding fragment thereof that specifically binds to human CD47 is HEK 293.

The invention also relates to an antibody detection kit, which comprises any one of the antibodies or antibody fragments thereof which are combined with the CGRP antigen. In one aspect of the invention, the test kit comprises an antibody or antigen-binding fragment thereof comprising an amino acid sequence from any one of the following groups:

7) the variable region of the light chain of the amino acid sequence shown in SEQ ID NO. 25 and/or the variable region of the heavy chain comprising the amino acid sequence shown in SEQ ID NO. 26.

In one aspect of the invention, the antibody detection kit further comprises a detection reagent for detecting the CGRP antigen-antibody reaction, a negative control and a positive control.

According to another aspect of the present invention, there is also provided a composition comprising an antibody or antigen-binding fragment thereof that binds human CD47 as described above, and a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier includes one or more of the following: pharmaceutically acceptable solvent, dispersant, additive, plasticizer and medicinal auxiliary material.

According to another aspect of the present invention, there is also provided the use of the antibody or antigen-binding fragment thereof that binds to human CD47 described above for the prevention and/or treatment of CD 47-related diseases and for the preparation of a medicament for the prevention and/or treatment of CD 47-related diseases. Wherein the CD 47-associated disorder is cancer.

The invention also provides the application of the antibody or the antigen binding fragment thereof for binding to the human CD47 in preparing a diagnostic reagent for CD47 related diseases.

Drawings

Figure 1 shows the mouse serum titers after the sextupley.

FIG. 2 shows the binding sensitivity of flow cytometry to detect anti-CD47 antibody to Raji cells.

Figure 3 shows that CD47 antibody blocks the binding of CD47 protein to SIRP.

Figure 4 shows the hemagglutination activity of CD47 antibody on human RBCs.

FIG. 5 shows the effect of anti-CD47 antibodies on macrophage phagocytosis

FIG. 6 shows the pharmacodynamic experiment of anti-CD47 antibody in B-hCD 47/SIRPa humanized mouse MC38 colon cancer animal model (tumor volume change after administration)

Details of the invention

The inventor successfully obtains a class of anti-CD47 antibodies through extensive and intensive research and a large number of screens, and experimental results show that the CD47 antibody obtained by the invention can effectively block the interaction between CD47 and a receptor thereof, and surprisingly, the CD47 antibody optimized by the invention can effectively block the combination between CD47 and the receptor thereof, and has the characteristics of high antibody affinity, low immunogenicity, no coagulation side effect, high macrophage tumor phagocytosis induction, high tumor inhibitory activity and the like through identification. The present invention has been completed on the basis of this finding.

The antibodies of the invention are designed with engineered CDRs and with portions of the antibodies (all or part of the framework, hinge and constant regions) that are identical or substantially identical (substantially human) to the framework and constant regions derived from human genomic sequences, of human origin. Fully human framework, hinge and constant regions are those of human germline sequences as well as sequences with naturally occurring somatic mutations and those with engineered mutations. The antibodies of the invention may comprise a framework, hinge or constant region derived from a fully human framework, hinge or constant region comprising one or more amino acid substitutions, deletions or additions therein. Furthermore, the antibodies of the invention are preferably substantially non-immunogenic in humans.

The antibodies of the invention are IgG-type antibodies and have "heavy" chains and "light" chains that are cross-linked via intra-and inter-chain disulfide bonds. Each heavy chain comprises an N-terminal heavy chain variable region HCVR (or VH) and a heavy chain constant region ("HCCR"). Each light chain comprises a light chain variable region LCVR (or VL) and a light chain constant region ("LCCR").

The HCVR and LCVR regions can be further subdivided into hypervariable regions, referred to as complementarity determining regions ("CDRs"), interspersed with more conserved regions, referred to as framework regions ("FRs"). Each HCVR and LCVR consists of three CDRs and four FRs, arranged in the following order from amino-terminus to carboxy-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR 4. Herein, the three CDRs of the heavy chain are referred to as "HCDR 1, HCDR2 and HCDR 3" and the three CDRs of the light chain are referred to as "LCDR 1, LCDR2 and LCDR 3". The CDRs contain most of the residues that form specific interactions with the antigen.

The term "encoded nucleic acid" may be a nucleic acid that includes the encoding sequence, and may also include additional coding and/or non-coding sequences.

The invention also relates to nucleic acids which hybridize to the sequences described above and which have at least 50%, preferably at least 70%, more preferably at least 80%, identity between the two sequences. The invention relates in particular to nucleic acids which hybridize under stringent conditions with the nucleic acids according to the invention. In the present invention, "stringent conditions" mean: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 XSSC, 0.1% SDS,60 ℃; or (2) denaturant is added during hybridization, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42 ℃ and the like; or (3) hybridization only when the identity between two sequences is at least 90% or more, preferably 95% or more. Furthermore, the hybridizable nucleic acid encodes a polypeptide having the same biological function and activity as the mature polypeptide.

Once the sequence of interest has been obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods. The biomolecules (nucleic acids, proteins, etc.) to which the present invention relates include biomolecules in an isolated form.

At present, the DNA sequence encoding the protein of the present invention (or its fragment, or its derivative) can be obtained completely by chemical synthesis. The DNA sequence may then be introduced into various existing DNA molecules (e.g., vectors) and cells known in the art. Furthermore, mutations can also be introduced into the protein sequences of the invention by chemical synthesis.

The invention also relates to a vector comprising a suitable DNA sequence as described above and a suitable promoter or control sequence. These vectors may be used to transform an appropriate host cell so that it can express the protein.

The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples are: escherichia coli, streptomyces; bacterial cells of salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf 9; CHO, COS7, 293 cells, etc.

Transformation of a host cell with recombinant DNA may be carried out using conventional techniques well known to those skilled in the art. When the host is prokaryotic, e.g., E.coli, competent cells capable of DNA uptake can be harvested after exponential growth phase using CaCl₂Methods of treatment, the steps used are well known in the art. Another method is to use MgCl₂. If desired, transformation can also be carried out by electroporation. When the host is a eukaryote, the following DNA transfection methods may be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

The obtained transformant can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention. The medium used in the culture may be selected from various conventional media depending on the host cell used. The culturing is performed under conditions suitable for the growth of the host cell. After the host cells have been grown to an appropriate cell density, the selected promoter is induced by an appropriate method (e.g., temperature shift or chemical induction) and the cells are cultured for an additional period of time.

The recombinant polypeptide in the above method may be expressed intracellularly or on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (such as salt precipitation), centrifugation, cell lysis by osmosis, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, High Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques, and combinations thereof.

The invention also provides a composition. Preferably, the composition is a pharmaceutical composition comprising the above antibody or an active fragment thereof or a fusion protein thereof, and a pharmaceutically acceptable carrier. Generally, these materials can be formulated in a non-toxic, inert, and pharmaceutically acceptable carrier medium. The formulated pharmaceutical compositions may be administered by conventional routes including, but not limited to: intratumoral, intraperitoneal, intravenous, or intramuscular injection.

The antibody has wide biological application value and clinical application value, and the application relates to the diagnosis and treatment of diseases related to CD47, basic medical research, biological research and other fields. One preferred application is for clinical diagnosis and targeted therapy against CD 47.

Detailed Description

The invention may be better understood by reference to the following examples. However, it is to be understood that the following examples are for illustrative purposes only and are not to be construed as limiting the scope of the present invention in any way.

EXAMPLE 1 Generation of anti-CD47 monoclonal antibodies

1.1 immunization protocol

A BoAn-hMab1 human safe antibody transgenic mouse was immunized after emulsification with CD47-His (ACROBIOSystems, Cat. No. CD7-H5227) or CD47-mFc (ACROBIOSystems, Cat. No. CD7-H52A5) with Freund's adjuvant. Freund's complete adjuvant was used for the first immunization, and Freund's incomplete adjuvant was used for the second to sixth immunizations. This time 20 mice were co-immunized and the serum titers were measured by Elisa. Coating protein CD47-His (ACROBIOSystems, catalog number CD7-H5227) with CBS coating solution (pH9.6 carbonic acid solution) at a concentration of 1ug/ml, 100 ul/well 4 ℃ overnight; sealing with 3% skimmed milk powder at 37 deg.C for 1 hr; serum was diluted to 100X, 500X, 2500X, 12500X with PBST, 100ul per well. Incubating at 37 ℃ for 1 h; then HRP-goat anti-human antibody (474- & lt- & gt 1006 & lt/EN & gt, KPL) is added, the mixture is incubated for 1h at 37 ℃, after 10min of color development, the mixture is stopped by 2M concentrated sulfuric acid, and OD450 is read on a microplate reader. 9 mice with higher serum titers were selected for boosting, and 3 days later, the mice were sacrificed and spleens were removed for subsequent experiments. Murine serum titers are shown in FIG. 1.

1.2 phage library creation

Get 10⁷Centrifuging 500g of spleen cells of an immunized mouse for 5min, removing a culture medium, adding 50 mu L of PBS to resuspend the cells, adding 1mL of Trizol, fully blowing the cells, adding 200 mu L of chloroform, vigorously shaking and uniformly mixing for 1min, standing for 10-20min, centrifuging at 4 ℃ at 12000rpm for 20min, taking an upper aqueous solution into a new EP tube, taking out about 550 mu L of supernatant from each tube, adding equal volume of isopropanol, uniformly mixing, standing at-20 ℃ for 20min, centrifuging at 4 ℃ at 12000rpm for 20min, pouring out the solution to remove the aqueous solution completely as much as possible, adding 1mL of 75% ethanol, centrifuging at 4 ℃ at 12000rpm for 5min, pouring out the solution to remove the solution as much as possible, if the solution remains, instantly centrifuging, and sucking the solution away by using a 10-microliter pipette, standing at room temperature for 1-2min, and dissolving with DEPC.

After extraction of RNA, cDNA was obtained by reverse transcription using a Kit (Transcriptor First Strang cDNA Synthesis Kit cat # 04897030001), and the Phage library was constructed by referring to Carlos F. Barbas III, Phage display: the method described in A laboratory Manual was performed by obtaining the variable regions of the heavy and light chains from cDNA by PCR, performing overlap extension PCR on the variable regions of the heavy and light chains to obtain ScFv, subjecting the gel-purified ScFv to digestion reaction at 50 ℃ with Sfi I (NEB cat # R0123L) for 5 hours, and then purifying the solution again, wherein 50. mu.l of the purified ScFv fragment was ligated with pCOMB3x vector which was also linearized with Sfi I.

The purified ligation product was electroporated at 1800V into competent cells of Escherichia coli TG1 (Lucigen cat # 60502-2), resuspended in 5ml Recovery Medium (Lucigen cat # 80026-1), the cell suspension was placed in a shaker at 37 ℃ for 1 hour at 250rpm, 10. mu.l was taken for reservoir volume calculation, 70ml of 2YT-AG (2YT Medium was supplemented with 2% glucose aqueous solution at final concentration and 100ug/ml ampicillin) Medium at 37 ℃, further incubated at 250rpm for 1.5 hours, 1ml of UV600 was taken for testing, and the cells contained 2.5 x 10 per ml of bacteria at 0.3OD⁸For each clone calculation, VCSM13 was added to the vial at 20-fold. Standing at 37 deg.C for 30min, shaking at 37 deg.C and 200rpm for 1 hr. Transferring the shaken bacterial solution into a centrifuge tube, centrifuging at 4000rpm for 10min, removing supernatant, mixing the precipitate with 100ml of 2YT-AK (2YT culture medium added with 100ug/ml ampicillin and 70ug/ml kanamycin antibiotic) culture medium, transferring into a culture bottle, and culturing at 30 deg.C and 250rpm in a shaker overnight.

Transferring 100ml of bacterial liquid expressed by 2YT-AK culture medium overnight into a 50ml centrifuge tube at 10000rpm for 5min to remove bacterial precipitates, carefully transferring supernatant into a new centrifuge tube, adding 4% of PEG8000 and 3% of NaCl into the supernatant, mixing uniformly, and incubating on ice for 1 h. The incubated supernatant was centrifuged at 13000rpm4 ℃ for 35min, the supernatant was discarded and the tube was inverted on absorbent paper. Add 3ml 2YT medium to each library basis weight suspension phage precipitation, mixing. The resuspended phage was centrifuged at 6000g for 1min to remove bacterial debris. The supernatant was filtered through a 0.2um filter into a new 2ml centrifuge tube using a syringe, added with DMSO to a final concentration of 7%, mixed well and stored at-80 ℃.

Phage library CD47Q 06, library 6 x 10, established with mouse accession number CD47Q 06⁸(ii) a Phage library CD47Q19, library volume 1.1 x 10, established with mouse accession number CD47Q19⁹。

1.3 phage library screening

1.3.1 plate screening

Plates were coated with CD47-His protein (ACROBIOSystems, CD7-H5227) at 1. mu.g/well, left overnight at 4 ℃, plates were blocked the next day by 2% BSA for 1H, phage pool (2E12) was added for 2H incubation, and CD 47-specifically bound phage were eluted with an elusion Buffer (pH 2.2) after washing 4-10 times.

1.3.2 magnetic bead screening

CD47-mFc protein (ACROBIOSystems, CD7-H52A5) was biotinylated according to the conventional procedure (molar ratio of input CD47-mFc protein to biotin 1:2), and then bound to Thermo magnetic beads (Invitrogen Dynabeads M-280Streptavidin, 00355871), and incubated with phage library, and after washing 4-10 times, CD 47-specifically bound phage were eluted with an Elution Buffer (pH 2.2).

Phage obtained by each round of plate screening and magnetic bead screening elution are mixed together, and the next round of panning is continued after amplification, so that the CD47Q1.2.9.12.19mix library is obtained by the method. The selected clone was tested for its binding to CD47 and blocking of CD47/SIRP alpha binding by E.coli epitope scFv, Elisa, and the clone remained bound and had blocking activity.

Candidate clones obtained by screening and sources are shown in the following table

Table 1 candidate CD47 antibody source table

Cloning	Source bank	Mouse of origin	Elutriation method
				CD47QMIX-136-IgG4	CD47Q1.2.9.12.19mix	CD47Q1.2.9.12.19	Plate + magnetic bead screening
CD47QMIX-189-IgG4	CD47Q1.2.9.12.19mix	CD47Q1.2.9.12.19	Plate + magnetic bead screening
				CD47Q6-155-IgG4	CD47Q6	CD47Q6	Plate + magnetic bead screening
CD47Q19-210-IgG4	CD47Q19	CD47Q19	Plate + magnetic bead screening

Example 2 molecular construction and production of blocking antibodies

Clones CD47QMIx-136\189, CD47Q6-155 and CD47Q19-210 (hereinafter referred to as clone "136/189/155/210") were sequenced by Invitrogen Biotechnology Inc., and the sequences are shown in Table 2.

TABLE 2 candidate cloned amino acid sequences

The N glycosylation sites of clone 155, clone 189 and clone 210 are found by sequence analysis, and the clones 155.1, 189.1 and 210.1 are obtained by designing primers and mutating the N glycosylation sites. Clone 155.1 was sequenced and the heavy chain was in the germline gene IGHV4-34 family and the light chain was in the germline gene IGKV4-1 family. The amino acid sequences of each clone are shown in Table 3 below:

TABLE 3 candidate clone amino acid sequences

The antibody variable region gene was amplified by conventional molecular biology PCR (2. Phanta Max Master Mix manufacturer: Vazyme cat # P515-AA lot # TE211G8), the signal peptide was ligated to the variable region gene by overlap extension PCR, the antibody heavy chain gene was ligated to the vector pCDNA3.4(Life Technology) carrying the antibody Fc sequence and the antibody light chain gene was ligated to the vector pCDNA3.4 carrying the antibody light chain constant region sequence, respectively, by homologous recombination (Clon express II One Step Cloning Kit manufacturer: Vazyme cat # C112-01 lot # TE211L 8). After plasmids are extracted from sequenced positive clones, the positive clones are cotransfected and enter HEK293 cells to be cultured in a shaker at 37 ℃ and 8% CO2 and 125rpm, after the transient expression is carried out for 7 days, the supernatant is purified by Protein A affinity chromatography to obtain CD47 antibody, and the concentration of the antibody is determined by UV280 combined with theoretical extinction coefficient.

Heavy chain constant region amino acid sequence:

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK-

light chain constant region amino acid sequence:

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC-

control antibody production: the amino acid sequence of CD47 antibody hu5F9-G4 of FortySeven is determined by an IMGT database and a patent (WO 2011/143624A3), a vector pCDNA3.4 is inserted after the whole gene synthesis and is expressed by HEK293 cells, and the produced antibody is named as CD47-FS-IgG 4. The antibody sequences are as follows:

CD47-FS-IgG4 heavy chain sequence:

QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYNMHWVRQAPGQRLEWMGTIYPGNDDTSYNQKFKDRVTITADTSASTAYMELSSLRSEDTAVYYCARGGYRAMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK-

CD47-FS-IgG4 light chain sequence:

DIVMTQSPLSLPVTPGEPASISCRSSQSIVYSNGNTYLGWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC-

example 3 characterization of candidate antibodies

3.1Forte Bio detection of affinity of candidate antibodies to CD47 protein

Antibody binding kinetics were measured using an OctetRED 96 instrument based on biofilm interference technology (Biolayer interference BLI). Antibodies (10. mu.g/mL) were coupled to PROA Biosensors at a loading height of 1nM, CD47 was serially diluted 2-fold in PBST, starting at 25nM and set at 0 concentration, Association time set at 300s, and Association time set at 300 s. After completion of the detection, the binding constant (kon) and dissociation constant (kdis) were calculated using Curve Fitting of 1:1Model, and the equilibrium dissociation constant (kD) was calculated as the ratio kD/ka. The results are shown in Table 4, and the affinity of the four candidate antibodies is compared with that of the control antibody, and the KD of each candidate antibody is higher than that of the control antibody except that the KD of the CD47Q19-210.1-IgG4 is similar to that of the control antibody.

TABLE 4 CD47 antibody affinity assays

Loading Sample ID	KD(M)	kon(1/Ms)	kdis(1/s)
				CD47-FS-IgG4	8.86E-09	4.97E+05	4.40E-03
CD47QMIX-136-IgG4	2.15E-09	6.31E+05	1.36E-03
				CD47Q6-155.1-IgG4	3.64E-09	6.99E+05	2.54E-03
CD47QMIX-189.1-IgG4	5.30E-09	6.40E+05	3.40E-03
				CD47Q19-210.1-IgG4	8.81E-09	6.24E+05	5.50E-03

3.2 flow assay of binding of candidate antibodies to CD47 cells

To a 96-well round bottom plate, 50. mu.L of Raji cells were added in a cell count of 5E4 cells/well, each antibody was diluted in a gradient by FACS buffer (sterile PBS, 0.2% BSA), added to the 96-well round bottom plate in a proportion of 50. mu.L/well, and incubated at 4 ℃ for 1 hour. After centrifugation at 2000rpm for 3min, the supernatant was discarded, washed 2 times with FACS buffer, 100. mu.L/well fluorescent secondary antibody (Southern Biotech, 2040-09) was added to a final concentration of 1. mu.g/mL, incubated at 4 ℃ for 1h, centrifuged at 2000rpm for 3min, discarded, washed 2 times with FACS buffer, resuspended at 100. mu.L/well FACS buffer, and examined by flow cytometry (Exson, Novocyte 2060). The results are shown in fig. 2, and the four candidate antibodies all have stronger binding capacity to Raji cells than the control antibody.

3.3Elisa detection antibody blocking the binding of CD47 protein to SIRP

Diluting SIRP protein (Sino Biological, catalog number: 11612-H08H) to 0.4 μ g/mL with pH9.6 CBS, coating enzyme-labeled plate (Suzhou beaver, 40301), 100 μ L/well, and incubating overnight at 4 deg.C; after washing the plate, sealing the plate for 1h by using 3% skimmed milk powder at 37 ℃; after washing the plate, PBST (PBS + 0.05% Tween20) is added to dilute antibodies with different concentrations (9.52pM, 4.76pM, 2.38pM, 1.19pM, 0.595pM, 0.2975pM, 0.14875pM and 0.074375pM) and 50 mu L/hole respectively, then CD47-Fc protein (final concentration is 0.03 mu g/mL) marked with biotin (1:5) is added to 50 mu L/hole, and the plate is incubated for 1h at 37 ℃; after washing the plates, PBST diluted STREP/HRP (R) was added&D, catalog number: 890803), 100. mu.L/well, incubated at 37 ℃ for 1 h. Washing plate, adding TMB (Beijing Meike Wander organism, catalog number: 1001) for developing, adding 50 μ L2M H per well after 10min₂SO₄The development was stopped and OD450 was read on the microplate reader. As shown in fig. 3, the blocking effect of each of the four candidate antibodies was significantly better than that of the control antibody.

3.4 hemagglutination assay for antibody Activity

The clinical side effects of antibodies to CD47 are a very problematic issue.

Centrifuging 2% human RBC (red blood cell, Borxi technology) at 1500rpm for 3min, washing with DPBS, adding DPBS again to prepare 8% RBC suspension, preparing samples with different concentrations (0, 0.01, 0.03, 0.08, 0.23, 0.7, 2.1, 6.2, 18.5, 55.6, 166.7, 500 μ g/ml) of antibody to be detected by PBS buffer, mixing with human RBC in volume of 50 μ L +50 μm L, placing in 96 shallow well plate, standing at 4 deg.C for 2h, taking out, and observing agglutination.

As shown in FIG. 4, CD47-FS showed significant hemagglutination at 6.2, 2.1, 0.7, 0.23, 0.08. mu.g/mL, 136, 155.1, 189.1 and 210.1 did not show significant hemagglutination, and 4 candidate antibodies were expected to cause no hemagglutination clinically in humans, thus having higher safety compared to CD 47-FS.

3.5FACS assay for macrophage-induced phagocytosis

Macrophage-induced phagocytosis assays were performed by flow cytometry, entrusted to the family endocoroceraceae, and 45 μ L of M1 macrophages and 45 μ L of CFSE-labeled tumor cells (macrophages: tumor cells ═ 1:1) were added to each well of a 96-well U-cell culture plate. 10 μ L of each test drug was added to the corresponding well, so that the working concentrations of the test drugs were 20 μ g/mL, 5 μ g/mL, 1.25 μ g/mL, 0.31 μ g/mL, 0.08 μ g/mL, 0.02 μ g/mL.

Two compound holes are designed for each drug concentration, and the mixture is placed at 37 ℃ and 5% CO after being mixed evenly₂Incubate for 2 hours in an incubator.

(Positive control antibody only selected for 1 concentration experiment) heavy suspension cells, adding 100u L FACS buffer washing cells twice. Add 80. mu.L FACS buffer to resuspend the cells per well and 20. mu.L human Fc blocking reagent. After mixing, the mixture is incubated for 15 minutes at 4 ℃ in the dark. Add 5. mu.L of APC anti-human CD206 to each well, mix gently, incubate at 4 ℃ for 30min in the dark. The plate was centrifuged at 1200rpm for 5 minutes at 4 ℃ in a centrifuge. The supernatant was discarded, 200. mu.L of pre-cooled FACS buffer was added to the plate and the cells resuspended, 1200rpm, and centrifuged for 5 minutes at 4 ℃. And repeating the steps once. Add 200. mu.L FACS buffer to each tube to resuspend the cells, add 10. mu.L 7-AAD, mix gently, and incubate at room temperature (25 ℃) for 5 minutes in the dark. The sample was analyzed by flow cytometry. The resulting electronic data were analyzed using Flowjo10 or Kaluza software.

According to the data analyzed by the flow scattergram, the macrophage phagocytosis index of each drug treatment group was calculated according to the following formula.

The specific results are shown in FIG. 5, and overall, the candidate antibody CD47Q6-155.1-IgG4 has a similar phagocytosis index to the control antibody, and the phagocytosis index is higher than that of the control antibody at a concentration of 0.31. mu.g/mL.

Example 4 pharmacodynamic Studies of anti-CD47 antibodies in the B-hCD47/SIRP α humanized mouse MC38-hCD47 Colon cancer model

In vivo efficacy study of anti-CD47 antibody is carried out by inoculating B-hCD47/SIRP humanized mice of Beijing Baioecker diagram to mouse colon cancer MC38 cells to establish a mouse colon cancer animal model. Mouse colorectal cancer MC38 cells were obtained from Chorispora Biotech, Inc., and cultured in Dulbecco's Modified Eagle's Medium containing 10% inactivated fetal bovine serum at 37 ℃ in a 5% CO2 incubator. MC38 cells were genetically modified by Beijing Baioeoxi chart Gene biotechnology, Inc., to overexpress humanized CD47 and knock out murine CD47, which was named MC38-hCD47 cells. PBS resuspended MC38-hCD47 tumor cells at 1X 10⁶The cells were inoculated subcutaneously on the right side of B-hSIRPa/hCD47 humanized mice at a concentration of 0.1mL, at a volume of 0.1 mL. When the mean tumor volume reaches about 100-³In the process, mice with moderate individual tumor volume are selected and grouped, animals are randomly distributed into 8 experimental groups according to the tumor volume, each group comprises 5 animals, and the administration is started on the same day. The result of the inhibition of the antibody on the tumor volume increase of the mice is shown in FIG. 6, and the inhibition effect of the candidate antibody CD47Q6-155.1-IgG4 on the tumor is better than that of the control antibody.

Sequence listing

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Claims

1. An antibody or antigen-binding fragment thereof that binds to CD47, wherein the antibody or antigen-binding fragment thereof comprises light and heavy chain Complementarity Determining Regions (CDRs) selected from any one of the following sets of amino acid sequences: light chain complementarity determining regions CDR1-CDR3 shown in SEQ ID NOs:11-13 and heavy chain complementarity determining regions CDR1-CDR3 shown in SEQ ID NOs: 14-16; alternatively, the light chain complementarity determining regions CDR1-CDR3 shown in SEQ ID NOs:19-21 and the heavy chain complementarity determining regions CDR1-CDR3 shown in SEQ ID NOs: 22-24; alternatively, the light chain complementarity determining regions CDR1-CDR3 shown in SEQ ID NOs:27-29 and the heavy chain complementarity determining region CDR1-CDR3 shown in SEQ ID NOs: 30-32.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable region comprising an amino acid sequence having at least 80%, 85%, or 90% sequence identity to the amino acid sequence set forth in SEQ ID No. 9, and a heavy chain variable region comprising an amino acid sequence having at least 80%, 85%, or 90% sequence identity to the amino acid sequence set forth in SEQ ID No. 10; alternatively, the antibody or antigen-binding fragment thereof comprises a light chain variable region comprising an amino acid sequence having at least 80%, 85%, or 90% sequence identity to the amino acid sequence set forth in SEQ ID NO. 17, and a heavy chain variable region comprising an amino acid sequence having at least 80%, 85%, or 90% sequence identity to the amino acid sequence set forth in SEQ ID NO. 18; alternatively, the antibody or antigen-binding fragment thereof comprises a light chain variable region comprising an amino acid sequence having at least 80%, 85%, or 90% sequence identity to the amino acid sequence set forth in SEQ ID No. 25 and a heavy chain variable region comprising an amino acid sequence having at least 80%, 85%, or 90% sequence identity to the amino acid sequence set forth in SEQ ID No. 26.

3. The antibody or antigen-binding fragment thereof according to claim 2, characterized in that it comprises an amino acid sequence selected from any one of the following groups: the variable region of the light chain of the amino acid sequence shown in SEQ ID NO. 9 and the variable region of the heavy chain of the amino acid sequence shown in SEQ ID NO. 10; or, the variable region of the light chain of the amino acid sequence shown in SEQ ID NO. 17 and the variable region of the heavy chain of the amino acid sequence shown in SEQ ID NO. 18; alternatively, the variable region of the light chain of the amino acid sequence shown in SEQ ID NO. 25 and the variable region of the heavy chain of the amino acid sequence shown in SEQ ID NO. 26.

4. An isolated nucleic acid encoding the antibody or antigen-binding fragment thereof of any one of claims 1-3.

5. A vector comprising the nucleic acid of claim 4.

6. The vector of claim 5, wherein the vector is an expression vector.

7. A host cell comprising the nucleic acid of claim 4 or the vector of claim 5 or 6, said host cell not comprising a plant cell, a mammalian embryonic stem cell.

8. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-3 for the manufacture of a medicament for the prevention and/or treatment of a CD 47-associated cancer, which is colon cancer.

9. A test kit comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3.

10. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-3, and a pharmaceutically acceptable carrier therefor.