WO2019157843A1 - Cd47单域抗体及其用途 - Google Patents

Cd47单域抗体及其用途 Download PDF

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WO2019157843A1
WO2019157843A1 PCT/CN2018/116006 CN2018116006W WO2019157843A1 WO 2019157843 A1 WO2019157843 A1 WO 2019157843A1 CN 2018116006 W CN2018116006 W CN 2018116006W WO 2019157843 A1 WO2019157843 A1 WO 2019157843A1
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single domain
domain antibody
antibody
seq
fusion protein
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PCT/CN2018/116006
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English (en)
French (fr)
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万亚坤
沈晓宁
朱敏
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上海洛启生物医药技术有限公司
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Priority to US16/763,052 priority Critical patent/US11427635B2/en
Priority to EP18906655.8A priority patent/EP3753953A4/en
Publication of WO2019157843A1 publication Critical patent/WO2019157843A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • the invention belongs to the field of biomedical or biopharmaceutical technology, and relates to a blocking type single domain antibody and a derivative protein thereof for the extracellular segment of the integrin-related protein (CD47) molecule. Also disclosed are coding sequences thereof, related methods of preparation, and uses thereof, particularly in the treatment and/or prevention, or diagnosis of CD47-associated diseases, such as tumors.
  • CD47 integrin-related protein
  • SIRPa-Fc has a molecular weight of about 80 kDa, which has better penetrability and tissue distribution than 150 kDa of antibody molecules; SIRPa-Fc has much lower affinity for red blood cells than Hu5F9-G4, indicating that it may be better. Security.
  • the terminal variable region (VHH), the hinge region and two constant regions (CH1, CH2), the variable region (VHH) is called a single domain antibody (nanobody).
  • the single domain antibody has a molecular weight of only about 15kDa, and its nanometer molecular size and unique structure give it superior characteristics to traditional antibodies, such as high stability, good water solubility, simple humanization, high targeting, and wearability. Strong and so on. Due to its special structural properties, single-domain antibodies have the advantages of traditional antibodies and small molecule drugs, and almost completely overcome the shortcomings of traditional antibody development cycle, low stability and harsh storage conditions. Single-domain antibodies with a molecular weight of only 1/10 of conventional antibodies are becoming an emerging force in the diagnosis and treatment of next-generation antibodies. Therefore, the development of CD47 therapeutic antibody drugs using single domain antibody technology has broad prospects.
  • the technical problem to be solved by the present invention is to provide a single domain antibody against the extracellular domain of CD47, which can effectively block the interaction of CD47 with its ligand SIRPa, and has good binding activity, blocking activity, affinity and Stability, can effectively enhance the phagocytosis of tumor cells by macrophages, and shows very significant anti-tumor activity in human lymphoma model and human ovarian cancer model.
  • the antibody does not cause human red blood cell agglutination not only in vitro, but also excellent safety in cynomolgus monkeys.
  • the invention also provides the coding sequence, preparation method and use in the diagnosis and treatment of the single domain antibody and its derivative.
  • a CDR region of a complementarity determining region of an anti-CD47 single domain antibody VHH chain wherein the CDR1 of the complementarity determining region of the VHH chain is represented by SEQ ID NO. , CDR2 shown in SEQ ID NO.: 6, CDR3 shown in SEQ ID NO.: 7.
  • said CDR1, CDR2 and CDR3 are separated by framework regions FR1, FR2, FR3 and FR4 of the VHH chain.
  • a VHH chain of an anti-CD47 single domain antibody comprising a framework region FR and a complementarity determining region CDR according to the first aspect of the present invention, wherein the framework region FR is
  • VHH chain of the anti-CD47 single domain antibody is set forth in SEQ ID NO.: 8 or 14.
  • the invention provides an anti-CD47 single domain antibody which is a single domain antibody directed against a CD47 epitope and which has the amino acid sequence set forth in SEQ ID NO.: 8 or SEQ ID NO.: VHH chain.
  • an anti-CD47 single domain antibody Fc fusion protein is provided, the structure of the fusion protein from N-terminus to C-terminus is as shown in Formula Ia or Ib:
  • A is the anti-CD47 single domain antibody of claim 3;
  • B is an Fc fragment of IgG
  • L is a no or flexible joint.
  • the flexible linker is a peptide linker.
  • the peptide linker has from 1 to 50 amino acids, preferably from 1 to 20 amino acids.
  • the peptide linker has the structure of (GGGGS)n, wherein n is a positive integer from 1 to 5.
  • the Fc fragment of the IgG comprises an Fc fragment of human IgG.
  • the Fc fragment of the IgG is selected from the group consisting of IgGl, IgG2, IgG3, an Fc fragment of IgG4, or a combination thereof.
  • the Fc fragment of the IgG is hIgG4PE.
  • amino acid sequence of the Fc fragment is SEQ ID NO.: 18.
  • amino acid sequence of the fusion protein is shown in SEQ ID NO.: 16.
  • the fusion protein is a single domain antibody Fc fusion protein directed against a CD47 epitope.
  • the present invention provides a polynucleotide encoding a protein selected from the group consisting of the CDR region of the anti-CD47 single domain antibody VHH chain of the first aspect of the invention, the second of the present invention The VHH chain of the anti-CD47 single domain antibody, the anti-CD47 single domain antibody of the third aspect of the invention, or the anti-CD47 single domain antibody Fc fusion protein of the fourth aspect of the invention.
  • the polynucleotide has a nucleotide sequence as shown in SEQ ID NO.: 9, 15, or 17.
  • the polynucleotide comprises DNA or RNA.
  • an expression vector comprising the polynucleotide of the fifth aspect of the invention is provided.
  • the invention provides a host cell comprising the expression vector of the sixth aspect of the invention, or the polynucleotide of the fifth aspect of the invention integrated in the genome.
  • the host cell comprises a prokaryotic cell or a eukaryotic cell.
  • the host cell is selected from the group consisting of mammalian cells, E. coli, yeast cells, phage, or a combination thereof.
  • the prokaryotic cell is selected from the group consisting of Escherichia coli, Bacillus subtilis, Lactobacillus, Streptomyces, Proteus, or a combination thereof.
  • the eukaryotic cell is selected from the group consisting of Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces, Trichoderma, or a combination thereof.
  • the eukaryotic cells are selected from the group consisting of insect cells such as grass armyworm, plant cells such as tobacco, BHK cells, CHO cells, COS cells, myeloma cells, or a combination thereof.
  • the host cell is preferably a mammalian cell, more preferably a HEK293 cell, a CHO cell, a BHK cell, an NSO cell or a COS cell.
  • a method for producing an anti-CD47 single domain antibody or an Fc fusion protein thereof comprising the steps of:
  • the anti-CD47 single domain antibody has the amino acid sequence set forth in SEQ ID NO.: 8 or 14 or 16.
  • an immunoconjugate comprising:
  • a coupling moiety selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, or an enzyme.
  • the coupling moiety is a drug or a toxin.
  • the coupled moiety is a detectable label.
  • the conjugate is selected from the group consisting of: a fluorescent or luminescent label, a radioactive label, an MRI (magnetic resonance imaging) or CT (computer tomography) contrast agent, or is capable of producing a detectable agent
  • Product enzymes radionuclides, biotoxins, cytokines (such as IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles/nanorods, viral particles, liposomes, nanomagnetic particles, pre- A drug activating enzyme (eg, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), a chemotherapeutic agent (eg, cisplatin) or any form of nanoparticles, and the like.
  • DTD DT-diaphorase
  • BPHL biphenyl hydrolase-like protein
  • the immunoconjugate comprises: a multivalent (e.g., bivalent) VHH chain of an anti-CD47 single domain antibody of the second aspect of the invention, as described in the third aspect of the invention An anti-CD47 single domain antibody, or an anti-CD47 single domain antibody Fc fusion protein according to the fourth aspect of the invention.
  • a multivalent (e.g., bivalent) VHH chain of an anti-CD47 single domain antibody of the second aspect of the invention as described in the third aspect of the invention
  • An anti-CD47 single domain antibody, or an anti-CD47 single domain antibody Fc fusion protein according to the fourth aspect of the invention.
  • the multivalentity refers to a VHH chain comprising a plurality of repeating anti-CD47 single domain antibodies according to the second aspect of the present invention in the amino acid sequence of the immunoconjugate, the present invention
  • the anti-CD47 single domain antibody of the third aspect, or the anti-CD47 single domain antibody Fc fusion protein of the fourth aspect of the invention refers to a VHH chain comprising a plurality of repeating anti-CD47 single domain antibodies according to the second aspect of the present invention in the amino acid sequence of the immunoconjugate, the present invention
  • the anti-CD47 single domain antibody of the third aspect, or the anti-CD47 single domain antibody Fc fusion protein of the fourth aspect of the invention are preferred embodiment, the multivalentity to a VHH chain comprising a plurality of repeating anti-CD47 single domain antibodies according to the second aspect of the present invention in the amino acid sequence of the immunoconjugate, the present invention
  • the invention provides the use of the anti-CD47 single domain antibody of the third aspect of the invention and the anti-CD47 single domain antibody Fc fusion protein of the fourth aspect, for preparing (a) for detecting CD47 molecule Reagents; (b) drugs for the treatment of tumors.
  • the detection comprises flow detection, cellular immunofluorescence detection.
  • a pharmaceutical composition comprising:
  • the pharmaceutical composition is in the form of an injection.
  • the pharmaceutical composition is for the preparation of a medicament for treating a tumor
  • the tumor is selected from the group consisting of gastric cancer, liver cancer, leukemia, kidney tumor, lung cancer, small bowel cancer, bone cancer, prostate cancer. , colorectal cancer, breast cancer, colorectal cancer, prostate cancer, cervical cancer, lymphoma, adrenal tumor, or bladder tumor.
  • the use of one or more of the anti-CD47 single domain antibodies of the third aspect of the invention is provided:
  • the use is diagnostic and/or non-diagnostic, and/or
  • the use of one or more of the anti-CD47 single domain antibody Fc fusion proteins of the fourth aspect of the invention is provided:
  • the use is diagnostic and/or non-diagnostic, and/or
  • a recombinant protein comprising:
  • the tag sequence comprises a 6His tag, an HA tag, or an Fc tag.
  • the recombinant protein specifically binds to a CD47 protein.
  • a fifteenth aspect of the invention the VHH chain according to the second aspect of the invention, the single domain antibody of the third aspect of the invention, the anti-CD47 single domain antibody Fc fusion protein of the fourth aspect, or The use of the immunoconjugate of the ninth aspect of the invention, which is used for the preparation of a medicament, a reagent, a test plate or a kit;
  • the reagent, the detection plate or the kit is used for: detecting the CD47 protein in the sample;
  • the agent is for treating or preventing a tumor expressing a CD47 protein (ie, CD47-positive).
  • the tumor comprises: lymphoma, ovarian cancer, leukemia, melanoma, gastric cancer, lymphoma, liver cancer, leukemia, kidney tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, Breast, colorectal, prostate, or adrenal tumors.
  • a method for detecting a CD47 protein in a sample comprising the steps of:
  • the method is a non-diagnostic and non-therapeutic method.
  • a seventeenth aspect of the invention provides a method for treating a disease, which comprises administering a single domain antibody of the third aspect of the invention, or the anti-CD47 single domain antibody Fc of the fourth aspect, to a subject in need thereof A fusion protein or an immunoconjugate of the ninth aspect of the invention.
  • the subject comprises a mammal, such as a human.
  • a framework region FR of an anti-CD47 single domain antibody VHH chain is provided, wherein the framework region FR of the VHH chain is FR1, SEQ ID NO.: 2 represented by SEQ ID NO. FR2 is shown, FR3 shown in SEQ ID NO.: 3, and FR4 shown in SEQ ID NO.: 4.
  • Figure 1 is a SDS-PAGE diagram of antigenic protein and single domain antibody purification, in which A is a nucleic acid molecule standard and B is a purified hCD47 (ECD)-Fc protein expressed by HEK293F cells with a purity of over 90%.
  • A is a nucleic acid molecule standard
  • B is a purified hCD47 (ECD)-Fc protein expressed by HEK293F cells with a purity of over 90%.
  • FIG 2 is constructed capacity detecting FIG library, constructed by dilutions of the library were plated, there is shown taken 1/5 10 4 fold dilution series, 105 times, 106 times the number of clones, by calculating the number of monoclonal
  • the library size was determined and the library was calculated to have a library capacity of 2.5 x 109 CFU.
  • Figure 3 is a plot of the insertion rate of the constructed library showing the results of the insertion rate assay of the constructed single domain antibody library.
  • the DNA bands of the gel pores from left to right are: the first channel is a DNA molecular marker, and the remaining channels are PCR products for detecting the inserted fragment, and the PCR product band is about 500 bp; the insertion rate of the library is 100% after detection. .
  • Figure 4 shows the CD47 single domain antibody screening enrichment process.
  • the library did not show enrichment in the first round of panning.
  • the second round of panning showed 1.8 times enrichment
  • the third round of panning showed 5.6 times enrichment
  • the fourth round of panning showed 19 times enrichment.
  • FIG. 5 is a purification diagram of a HEK293F system expressing CD47 single domain antibody Fc fusion protein (No. Nb1-Fc), wherein the single domain antibody corresponds to the amino acid sequence of SEQ ID NO.: 8 and purified by Protein A affinity column affinity chromatography. After electrophoresis of SDS-PAGE of CD47 single domain antibody. The results showed that the CD47 single domain antibody Fc fusion protein Nb1-Fc had a purity of more than 90% after the purification process.
  • Figure 6 is a blockade effect of FACS detection of the CD47 single domain antibody Fc fusion protein Nb1-Fc, co-reacted with each group of antibodies and biotinylated hSIRPa (ECD)-Fc protein with HEK293T cells stably expressing human full length CD47 protein.
  • ECD biotinylated hSIRPa
  • the binding rate of hSIRPa(ECD)-Fc-biotin to stable cells decreased from 97.3% in the negative control group to 2.21%, indicating that the added antibody Nb1-Fc can significantly block the interaction between CD47 and SIRPa.
  • the results indicate that the Nb1-Fc specific for CD47 of the present invention has a good blocking effect on the binding of CD47 to SIRPa.
  • Figure 7 is a block diagram showing the blocking effect of humanized CD47 single domain antibody Nb1902-Fc by flow cytometry.
  • the stably transfected cells expressing CD47 protein were co-reacted with the humanized single domain antibody Nb1902-Fc and the biotinylated hSIRPa(ECD)-Fc protein, and hSIRP ⁇ (ECD)-Fc-biotin was stably transfected in the negative control group.
  • the binding rate of the strain cells was 97.2%, and the binding rate of hSIRPa(ECD)-Fc-biotin to the stably transfected cells was significantly decreased after the addition of the CD47 single domain antibody and the humanized single domain antibody; Humanized single-domain antibodies were able to significantly block the interaction of CD47 with hSIRPa.
  • FIG 8 is a flow cytometry single domain antibodies humanized Nb1902-Fc antibody and the positive control IC 50.
  • the results showed that humanized IC CD47 single domain antibody 50 of 1.51 ⁇ g / mL, whereas the control antibody B6H12 IC 50 of 12.34 ⁇ g / mL, the candidate humanized antibody is a single domain antibody blocking effect than the control.
  • Figure 9 is a graph showing the results of affinity detection of the humanized single domain antibody Nb1902-Fc.
  • the affinity of the humanized antibody Nb1902-Fc was determined using ForteBio's Octet System to be 1.65E-9M.
  • Figure 10 is a species-specific result of ELISA for detection of CD47 single domain antibodies. It can be seen that the humanized CD47 single domain antibody Nb1902-Fc interacts only with human CD47 and does not interact with CD47 in rats and mice. The candidate humanized single domain antibody has better species specificity. Sex.
  • Figure 11 is a graph showing the efficacy of a humanized CD47 single domain antibody Fc fusion protein in a human lymphoma model. The results showed that Nb1902-Fc had a significant tumor suppressive effect, and its tumor inhibition rate TGI was 80%.
  • Figure 12 is a graph showing the purity results of the humanized CD47 antibody Fc fusion protein (MY2238) expressed by the CHO-S system by SEC-HPLC. The results showed that the purity of the antibody MY2238, which was expressed by the system and purified by a step of Protein A affinity, was 93.11%.
  • Figure 13 is a graph showing the results of stability assay of a humanized CD47 antibody Fc fusion protein. The results showed that the antibody showed no obvious aggregation or degradation under the conditions of acceleration at 25 °C or strong destruction at 40 °C, showing good stability.
  • Figure 14 is a diagram showing the detection of phagocytosis of tumor cells by macrophages by a humanized CD47 antibody Fc fusion protein.
  • the results showed that MY2238 antibody can significantly promote the phagocytosis of Raji cells by macrophages, and the phagocytosis rate is about 50%.
  • the effect is similar to that of Hu5F9-G4 (FortySeven) in clinical stage I.
  • Figure 15 is a graph showing the efficacy of a humanized CD47 single domain antibody Fc fusion protein in a human ovarian cancer model.
  • the results showed that the candidate antibody MY2238 showed a good dose-dependent inhibition effect on the tumor in this pharmacodynamic model.
  • Figure 16 is a binding of a humanized CD47 single domain antibody Fc fusion protein to human erythrocytes.
  • Figure 17 is a graph showing the results of agglutination of human cells by a humanized CD47 single domain antibody Fc fusion protein. The results showed that the candidate antibody MY2238 did not cause agglutination of human red blood cells, whereas the control antibody Hu5F9-G4 significantly caused red blood cell agglutination.
  • Figure 18 is a graph showing the toxicological results of the humanized CD47 single domain antibody Fc fusion protein in cynomolgus monkeys. The results showed that different doses of the candidate antibody MY2238 did not cause obvious physiological toxicity in cynomolgus monkeys. Even under the high doses of 60mg/kg and 200mg/kg, the hemoglobin in cynomolgus monkeys returned to normal after reduction. Level, and no physiological abnormalities occurred during the study. Therefore, the candidate antibody MY2238 has excellent in vivo safety.
  • the inventors have successfully obtained a class of anti-CD47 single domain antibodies through extensive screening through extensive and intensive research.
  • the experimental results show that the CD47 single domain antibody obtained by the present invention can effectively bind to CD47.
  • the present invention immunizes a camel using a human CD47 antigen protein to obtain a high quality immune single domain antibody gene library.
  • the CD47 protein molecule is then coupled to the ELISA plate to display the correct spatial structure of the CD47 protein.
  • the antigen of this form is screened by the phage display technology to screen the immune single domain antibody gene bank (the camelid heavy chain antibody phage display gene library).
  • a single domain antibody gene specific for CD47 was further transferred into Escherichia coli to obtain a single domain antibody strain which was highly expressed in Escherichia coli and which was highly specific.
  • single domain antibody of the invention As used herein, the terms “single domain antibody of the invention”, “anti-CD47 single domain antibody of the invention”, “CD47 single domain antibody of the invention” are used interchangeably and both refer to the specific recognition and binding to CD47 (including human CD47). Single domain antibody. It is particularly preferred that the amino acid sequence of the VHH chain is a single domain antibody as set forth in SEQ ID NO.: 8 or 14.
  • antibody or "immunoglobulin” is an isotetrameric glycoprotein of about 150,000 daltons having the same structural features, consisting of two identical light chains (L) and two identical heavy chains. (H) Composition. Each light chain is linked to the heavy chain by a covalent disulfide bond, and the number of disulfide bonds between the heavy chains of different immunoglobulin isotypes is different. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end followed by a plurality of constant regions.
  • VH variable region
  • Each light chain has a variable region (VL) at one end and a constant region at the other end; the constant region of the light chain is opposite the first constant region of the heavy chain, and the variable region of the light chain is opposite to the variable region of the heavy chain .
  • Particular amino acid residues form an interface between the variable regions of the light and heavy chains.
  • single domain antibody VHH
  • single domain antibody sdAb, or nanobody
  • variable means that certain portions of the variable regions of an antibody differ in sequence, which form the binding and specificity of various specific antibodies for their particular antigen. However, the variability is not evenly distributed throughout the variable region of the antibody. It is concentrated in three segments in the variable region of the light and heavy chains called the complementarity determining region (CDR) or hypervariable region. The more conserved portion of the variable region is referred to as the framework region (FR).
  • the variable regions of the native heavy and light chains each comprise four FR regions which are substantially in a beta-sheet configuration and are joined by three CDRs forming a linker, in some cases forming a partial beta sheet structure.
  • the CDRs in each chain are closely joined together by the FR region and together with the CDRs of the other chain form the antigen binding site of the antibody (see Kabat et al, NIH Publ. No. 91-3242, Vol. I, pp. 647-669). (1991)).
  • the constant regions are not directly involved in the binding of the antibody to the antigen, but they exhibit different effector functions, such as antibody-dependent cytotoxicity of the participating antibodies.
  • immunoconjugates and fusion expression products include: drugs, toxins, cytokines, radionuclides, enzymes, and other diagnostic or therapeutic molecules that are combined with the antibodies or fragments thereof of the invention to form Conjugate.
  • the invention also encompasses cell surface markers or antigens that bind to the anti-CD47 protein antibody or fragment thereof.
  • variable region are used interchangeably with “complementarity determining region (CDR).
  • the heavy chain variable region of the antibody comprises three complementarity determining regions, CDR1, CDR2, and CDR3.
  • the heavy chain of the antibody comprises the heavy chain variable region and the heavy chain constant region described above.
  • the terms "antibody of the invention”, “protein of the invention”, or “polypeptide of the invention” are used interchangeably and refer to a polypeptide which specifically binds to a CD47 protein, such as a protein or polypeptide having a heavy chain variable region. . They may or may not contain an initial methionine.
  • the invention also provides other proteins or fusion expression products having the antibodies of the invention.
  • the invention encompasses any protein or protein conjugate having a heavy chain comprising a variable region and a fusion expression product (ie, an immunoconjugate and a fusion expression product), so long as the variable region is heavy with the antibody of the invention
  • the variable regions are identical or at least 90% homologous, preferably at least 95% homologous.
  • variable regions which are divided into four framework regions (FR), four FR amino acid sequences. Relatively conservative, not directly involved in the binding reaction.
  • CDRs form a cyclic structure in which the ⁇ -sheets formed by the FRs are spatially close to each other, and the CDRs on the heavy chain and the CDRs on the corresponding light chain constitute the antigen-binding site of the antibody.
  • the amino acid sequence of the same type of antibody can be compared to determine which amino acids constitute the FR or CDR regions.
  • variable regions of the heavy chains of the antibodies of the invention are of particular interest because at least some of them are involved in binding antigen.
  • the invention includes those molecules having an antibody heavy chain variable region with a CDR, as long as the CDRs thereof have 90% or more (preferably 95% or more, optimally 98% or more) homology to the CDRs identified herein. Sex.
  • the present invention encompasses not only intact antibodies, but also fragments of immunologically active antibodies or fusion proteins formed by antibodies with other sequences. Accordingly, the invention also includes fragments, derivatives and analogs of the antibodies.
  • fragment refers to a polypeptide that substantially retains the same biological function or activity of an antibody of the invention.
  • the polypeptide fragment, derivative or analog of the present invention may be (i) a polypeptide having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues It may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a mature polypeptide and another compound (such as a compound that extends the half-life of the polypeptide, for example Polyethylene glycol) a polypeptide formed by fusion, or (iv) a polypeptide formed by fused an additional amino acid sequence to the polypeptide sequence (such as a leader or secretion sequence or a sequence or proprotein sequence used to purify the polypeptide, or a fusion protein formed by the 6
  • the antibody of the present invention refers to a polypeptide comprising the above CDR regions having CD47 protein binding activity.
  • the term also encompasses variant forms of a polypeptide comprising the above-described CDR regions that have the same function as the antibodies of the invention. These variants include, but are not limited to, one or more (usually 1-50, preferably 1-30, more preferably 1-20, optimally 1-10) amino acid deletions , Insertion and/or Substitution, and the addition of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminus and/or N-terminus.
  • the function of the protein is generally not altered.
  • the addition of one or several amino acids at the C-terminus and/or N-terminus will generally not alter the function of the protein.
  • the term also encompasses active fragments and active derivatives of the antibodies of the invention.
  • Variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, DNA capable of hybridizing to the DNA encoding the antibody of the present invention under high or low stringency conditions.
  • the encoded protein, and the polypeptide or protein obtained using an antiserum against the antibody of the present invention.
  • the invention also provides other polypeptides, such as fusion proteins comprising a single domain antibody or a fragment thereof.
  • the invention also includes fragments of the single domain antibodies of the invention.
  • the fragment will have at least about 50 contiguous amino acids, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 100 contiguous amino acids of the antibody of the invention.
  • “conservative variant of the antibody of the present invention” means having up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3, compared to the amino acid sequence of the antibody of the present invention. Amino acids are replaced by amino acids of similar or similar nature to form a polypeptide.
  • the present invention also provides a polynucleotide molecule encoding the above antibody or a fragment thereof or a fusion protein thereof.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • DNA forms include cDNA, genomic DNA or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • the DNA can be a coding strand or a non-coding strand.
  • Polynucleotides encoding mature polypeptides of the invention include: coding sequences encoding only mature polypeptides; coding sequences for mature polypeptides and various additional coding sequences; coding sequences for mature polypeptides (and optionally additional coding sequences) and non-coding sequences .
  • polynucleotide encoding a polypeptide can be a polynucleotide comprising the polypeptide, or a polynucleotide further comprising additional coding and/or non-coding sequences.
  • the invention also relates to polynucleotides 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 particularly relates to polynucleotides that hybridize to the polynucleotides of the invention under stringent conditions.
  • stringent conditions means: (1) hybridization and elution at a lower ionic strength and higher temperature, such as 0.2 x SSC, 0.1% SDS, 60 ° C; or (2) hybridization a denaturing agent such as 50% (v/v) formamide, 0.1% calf serum / 0.1% Ficoll, 42 ° C, etc.; or (3) at least 90% identity between the two sequences, more It is good that hybridization occurs more than 95%.
  • the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.
  • the full-length nucleotide sequence of the antibody of the present invention or a fragment thereof can be usually obtained by a PCR amplification method, a recombinant method or a synthetic method.
  • One possible method is to synthesize related sequences by artificial synthesis, especially when the fragment length is short.
  • a long sequence of fragments can be obtained by first synthesizing a plurality of small fragments and then performing the ligation.
  • the coding sequence of the heavy chain and the expression tag (such as 6His) can be fused together to form a fusion protein.
  • the recombinant sequence can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it to a cell, and then isolating the relevant sequence from the proliferated host cell by conventional methods.
  • the biomolecule (nucleic acid, protein, etc.) to which the present invention relates includes biomolecules existing in an isolated form.
  • DNA sequence encoding the protein of the present invention (or a fragment thereof, or a derivative thereof) completely by chemical synthesis.
  • the DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art.
  • mutations can also be introduced into the protein sequences of the invention by chemical synthesis.
  • the invention also relates to vectors comprising the appropriate DNA sequences described above, as well as appropriate promoters or control sequences. These vectors can be used to transform appropriate host cells to enable them to express proteins.
  • the host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.
  • a prokaryotic cell such as a bacterial cell
  • a lower eukaryotic cell such as a yeast cell
  • a higher eukaryotic cell such as a mammalian cell.
  • Representative examples are: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; animal cells of CHO, COS7, 293 cells, and the like.
  • Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art.
  • the host is a prokaryote such as E. coli
  • competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated by the CaCl2 method, and the procedures used are well known in the art.
  • Another method is to use MgCl2. Conversion can also be carried out by electroporation if desired.
  • the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, and the like.
  • 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 cultivation is carried out under conditions suitable for the growth of the host cells.
  • the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction) and the cells are cultured for a further period of time.
  • the recombinant polypeptide in the above method can be expressed intracellularly, or on the cell membrane, or secreted outside the cell.
  • 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 (salting method), centrifugation, osmotic sterilizing, super treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • the antibodies of the invention may be used alone or in combination or in combination with a detectable label (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modifying moiety, or a combination of any of these.
  • Detectable labels for diagnostic purposes include, but are not limited to, fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (computer tomography) contrast agents, or capable of producing detectable products. Enzyme.
  • Therapeutic agents that can be bound or conjugated to the antibodies of the invention include, but are not limited to: 1. radionuclides; 2. biotoxicity; 3. cytokines such as IL-2, etc.; 4. gold nanoparticles/nanorods; Particles; 6. liposomes; 7. nanomagnetic particles; 8. drug activating enzymes (eg, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)); 9. therapeutic agents (eg , cisplatin) or any form of nanoparticles, etc.
  • DTD DT-diaphorase
  • BPHL biphenyl hydrolase-like protein
  • Integrin-associated protein is a 50 kD membrane glycoprotein belonging to the immunoglobulin superfamily. It is called integrin-associated protein (IAP) because it is first isolated from leukocytes and placenta in the form of membrane surface proteins together with integrin ⁇ V ⁇ 3, and its function is mostly related to integrin. CD47 is widely expressed on the surface of hematopoietic cells (erythrocytes, lymphocytes, platelets, monocytes, and neutrophils) as well as on placenta, liver, and brain tissue.
  • IAP integrin-associated protein
  • CD47 and the inhibitory receptor signal-modulating protein a are receptors and ligands, which form a CD47-SIRPa signal complex, which has the function of mediating two-way signal regulation and regulating various immune processes. As a self-marking of red blood cells, it inhibits the clearance of red blood cells and participates in the pathogenesis of hemolytic anemia.
  • CD47 On normal hematopoietic stem cells (HSCs), the significance of CD47 expression is to maintain its relative stability in the body; in leukemia, non-ho In malignant tumors such as Qijin lymphoma, bladder cancer and breast cancer, CD47 is highly expressed on the surface of tumor cells, suggesting a poor clinical prognosis. Tumor cells evade tumor immunity by taking the "Don't Eat Me” signal.
  • the invention also provides a composition.
  • the composition is a pharmaceutical composition comprising the above antibody or active fragment thereof or a fusion protein thereof, and a pharmaceutically acceptable carrier.
  • these materials can be formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium wherein the pH is usually from about 5 to about 8, preferably from about 6 to about 8, although the pH may be The nature of the formulation and the condition to be treated vary.
  • the formulated pharmaceutical compositions can be administered by conventional routes including, but not limited to, intratumoral, intraperitoneal, intravenous, or topical administration.
  • the pharmaceutical composition of the present invention can be directly used for binding to a CD47 protein molecule, and thus can be used for treating tumors.
  • other therapeutic agents can be used simultaneously.
  • the pharmaceutical composition of the present invention contains a safe and effective amount (e.g., 0.001 to 99% by weight, preferably 0.01 to 90% by weight, more preferably 0.1 to 80% by weight) of the above-described single domain antibody (or a conjugate thereof) of the present invention and pharmacy An acceptable carrier or excipient.
  • Such carriers include, but are not limited to, saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the pharmaceutical preparation should be matched to the mode of administration.
  • the pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants.
  • compositions such as injections and solutions are preferably prepared under sterile conditions.
  • the amount of active ingredient administered is a therapeutically effective amount, for example from about 10 micrograms per kilogram body weight to about 50 milligrams per kilogram body weight per day.
  • the polypeptides of the invention may also be used with other therapeutic agents.
  • a safe and effective amount of the immunoconjugate is administered to the mammal, wherein the safe and effective amount is typically at least about 10 micrograms per kilogram of body weight, and in most cases no more than about 50 milligrams per kilogram of body weight, Preferably, the dosage is from about 10 micrograms per kilogram of body weight to about 10 milligrams per kilogram of body weight.
  • specific doses should also consider factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled physician.
  • the single domain antibody carries a detectable label. More preferably, the label is selected from the group consisting of an isotope, a colloidal gold label, a colored label or a fluorescent label.
  • the colloidal gold label can be carried out by methods known to those skilled in the art.
  • the anti-CD47 single domain antibody is labeled with colloidal gold to provide a colloidal gold labeled single domain antibody.
  • the anti-CD47 single domain antibody of the present invention has good specificity and high potency.
  • the invention also relates to methods of detecting CD47 protein.
  • the method steps are substantially as follows: obtaining a cell and/or tissue sample; dissolving the sample in a medium; detecting the level of CD47 protein in the dissolved sample.
  • the sample to be used is not particularly limited, and a representative example is a cell-containing sample present in the cell preservation solution.
  • the present invention also provides a kit comprising the antibody (or a fragment thereof) or a test plate of the present invention.
  • the kit further comprises a container, instructions for use, a buffer, and the like.
  • the invention also provides a detection kit for detecting the level of CD47, which comprises an antibody for recognizing CD47 protein, a lysis medium for dissolving the sample, detecting a common reagent and a buffer required, such as various buffers, detection Mark, detect substrates, etc.
  • the test kit can be an in vitro diagnostic device.
  • the single domain antibody of the present invention has broad biological application value and clinical application value, and its application relates to various fields such as diagnosis and treatment of diseases associated with CD47, basic medical research, and biological research.
  • a preferred application is for clinical diagnosis and targeted therapy against CD47.
  • the single domain antibody of the present invention is highly specific for human CD47 protein having the correct spatial structure.
  • the single domain antibody of the present invention has good binding activity, blocking activity and affinity, and the humanized antibody has excellent species specificity.
  • the single domain antibody of the present invention can effectively enhance the phagocytosis of tumor cells by macrophages, and exhibits significant antitumor activity in both human lymphoma models and human ovarian cancer models.
  • the single domain antibody of the present invention does not cause human red blood cell agglutination not only in vitro, but also exhibits excellent safety in cynomolgus monkeys.
  • the single domain antibody of the present invention is simple to produce and has excellent stability.
  • Antibody Screening Identification Briefly, (1) 10 ⁇ g of hCD47(ECD)-Fc antigen (10 ⁇ g Fc in NaHCO3 as a control) dissolved in 100 mM NaHCO 3 , pH 8.2 was coupled to NUNC plate and placed at 4 ° C overnight.
  • Example 3 Expression and purification of CD47 single domain antibody in eukaryotic cell HEK293 and flow cytometry to detect blocking function of single domain antibody
  • the eukaryotic cell HEK293F expresses the CD47 Nb1-Fc fusion protein: (1) The CD47 Nb1 sequence with the correct sequencing result was cloned into the pFUSE-IgG4 vector (purchased from Invivogen), and the pFUSE-IgG4-Nb1 plasmid was extracted with the Omega plasmid extract kit; (2) HEK293F cells were cultured to an OD of 2.0 ⁇ 10 6 cells/mL; (3) The plasmid and the transfection reagent PEI were uniformly mixed 1:3, and then allowed to stand for 20 min, and then added to HEK293F cells at 37 ° C, 6%.
  • hSIRPa ECD
  • Fc-Biotin protein biotin method reference biotin reagent instructions
  • 5 ⁇ 10 per sample Five CD47-stabilized cells were added to 0.5% BSA-PBS buffer, and 5 ⁇ g of the above purified CD47 single-domain antibody was added, and a negative control (hIgG1) and a blank group (PBS) were set, and 5 ⁇ g of hSIRPa (ECD) was simultaneously added to all samples.
  • Fc-Biotin was incubated at 4 °C for 20 min; (3) cells were washed twice with PBS, added with eBioscience SA-PE, incubated at 4 °C for 20 min, washed twice with PBS, and detected by flow cytometry (BD FACS Calibur).
  • Example 5 Detection of blocking effect of humanized CD47 single domain antibody Fc fusion protein by flow cytometry
  • the humanized CD47 single domain antibody Nb1902 sequence (SEQ ID NO.: 15) was synthesized into the pFUSE-IgG4PE vector, and the pFUSE-IgG4PE-Nb1902 plasmid was extracted with the Omega Plasmid Large Kit; then the HEK293F system was used.
  • the humanized single domain antibody Nb1902-Fc was expressed in the same manner as in Example 3.
  • the two antibodies and the control antibody (B6H12) before and after humanization were diluted to 1 ⁇ g/uL, 1/4 ⁇ g/uL, and 1/8 ⁇ g/uL, respectively.
  • the humanized single domain antibody Nb1902-Fc was serially diluted from 100 nM with PBST: 100 nM, 66.7 nM, 44.4 nM, 29.6 nM, 19.8 nM, 13.2 nM.
  • the antigenic proteins hCD47 (ECD)-Fc and Fc were each diluted to 30 ⁇ g/mL.
  • the antibody was captured using a probe coated with ProteinA (Fortebio Part No: 18-5010) for a capture time of 180 s; combined with a gradient diluted antigen, binding time of 180 s; dissociation time of 300 s; 10 mM glycine (pH 1.7) was regenerated 3 times. 5s each time. (3) On-board inspection with ForteBio's Octet System.
  • Example 8 Species-specific detection of humanized CD47 single domain antibody
  • the humanized CD47 single domain antibody gene was cloned into the E. coli expression vector pMECS, and the expression and purification process was the same as in Example 4; (2) coated antigen protein CD47 (human), CD47 (rat), CD47 ( Mouse): 0.5 ⁇ g per well (5 ⁇ g/mL, 100 ⁇ L), coated with IgG4 as control, overnight at 4 °C; (3) washed 3 times with PBST on the next day, and blocked with 200 ⁇ L of 1% BSA for 2 h at room temperature; 4) Wash PBST three times, add 100 uL of humanized single-domain antibody at a concentration of 10 ⁇ g/mL, and react at room temperature for 1 h; (5) wash away unbound antibody with PBST and add mouse anti-HA antibody (COVENCE).
  • the specificity of the single domain antibody was determined based on the absorbance value, and the results of the assay are shown in Figure 10: the humanized single domain antibody was able to interact with human CD47 without interacting with murine CD47.
  • Example 9 Efficacy of humanized CD47 single domain antibody Fc fusion protein in human lymphoma model
  • the Raji cells were inoculated into NOG mice, and the tumors were divided into two groups for administration (negative group: IgG4; experimental group: Nb1902-Fc).
  • Dosing frequency once a day for 28 days.
  • Dosage 20 mg/kg.
  • the experimental results are shown in Fig. 11: Nb1902-Fc has a remarkable tumor suppressing effect, and its tumor suppressing rate TGI is 80%.
  • Example 10 Expression of humanized CD47 single domain antibody in CHO-S system
  • the Nb1902-Fc amino acid sequence is converted into a CHO codon-optimized base sequence (as shown in SEQ ID NO. 17), which is then synthesized into a pCHO1.0 vector, and a specific signal peptide is selected for subsequent production of the antibody.
  • Expression, the antibody expressed by this system is numbered MY2238.
  • the synthesized plasmid was extracted with OMEGA plasmid extract kit in large amount, and then mixed with PEI and transfected into CHO-S cells.
  • the specific transfection protocol was the same as that of Example 1;
  • (2) Protein purification The same procedure as in Example 1; (3)
  • the one-step purified sample was subjected to SEC-HPLC analysis, and as shown in FIG. 12, the antibody was purified by one-step affinity, and its purity reached 93.11%, which was used for subsequent analysis.
  • Example 11 Stability of humanized CD47 single domain antibody Fc fusion protein
  • the antibody showed no significant aggregation or degradation under the conditions of acceleration at 25 ° C or strong destruction at 40 ° C, and showed good stability.
  • Example 12 Humanized CD47 single domain antibody Fc fusion protein mediates phagocytosis of tumor cells by macrophages
  • MY2238 antibody can significantly promote the phagocytosis of Raji cells by macrophages, and the phagocytosis rate is about 50%.
  • the effect is similar to that of Hu5F9-G4 (FortySeven) in clinical stage I.
  • Example 13 Efficacy of humanized CD47 single domain antibody Fc fusion protein in human ovarian cancer model
  • mice were inoculated with SK-OV-3 cells and divided into five groups after tumor formation (negative group: PBS, positive control group: 5 mg/kg Hu5F9-G4, experimental group: 5 mg/kg MY2238, 1 mg/) Kg MY2238, 0.2mg/kg MY2238).
  • Dosing frequency once a day, 12 days.
  • the experimental results are shown in Figure 15.
  • the candidate antibody MY2238 showed a good dose-dependent inhibition effect on the tumor in this pharmacodynamic model.
  • Example 14 Binding of humanized CD47 single domain antibody Fc fusion protein to human erythrocytes
  • the control antibody Hu5F9-G4 was serially diluted (5 ⁇ g/ml, 2.5 ⁇ g/ml, 1.25 ⁇ g/ml, 0.625 ⁇ g/ml, 0.3125 ⁇ g/ml, 0.15625 ⁇ g/ml, 0.078125 ⁇ g/ml, 0.039 ⁇ g/ Ml, 0.0195 ⁇ g/ml, 0.00975 ⁇ g/ml, 0.0049 ⁇ g/ml, 0.0024 ⁇ g/ml) and MY2238 were serially diluted (40 ⁇ g/ml, 20 ⁇ g/ml, 10 ⁇ g/ml, 5 ⁇ g/ml, 2.5 ⁇ g/ml, 1.25 Gg/ml, 0.625 ⁇ g/ml, 0.3125 ⁇ g/ml, 0.15625 ⁇ g/ml, 0.078125 ⁇ g/ml, 0.039 ⁇ g/ml, 0.0195 ⁇ g/ml); (2) The above 100 ⁇ L diluted
  • Example 15 Agglutination of humanized red blood cells by humanized CD47 single domain antibody Fc fusion protein
  • the surface of the red blood cells has high expression of CD47, it is easier to preferentially bind to the drug of the CD47 antibody, and the drug is concentrated on the surface to function as a "reservoir". Therefore, this situation is prone to anemia. Only when the drug enters the body, it is necessary to break through the "absorption pool" effect of the platelet on the CD47 antibody in order to effectively reach the action site.
  • the humanized CD47 single domain antibody Fc fusion protein, positive control antibody (B6H12, sequence derived from patent WO 2011143624 A2), positive control antibody (Hu5F9-G4), negative control (IgG4) were serially diluted to 8000 nM, 2000 nM, 500 nM, respectively. , 125 nM, 31.25 nM, 7.81 nM, 1.95 nM, 0 nM. 50 ⁇ L of the gradient-diluted antibody was separately added to 50 ⁇ L of human red blood cell suspension (2%), and the reaction was observed after overnight reaction at 37 °C.
  • the results are shown in Figure 18.
  • the candidate antibody MY2238 was reduced from 140 g/L to a minimum of 108 g/L at a single dose of 10 mg/kg, a decrease of 22.85%; at a single dose of 30 mg/kg, the hemoglobin was 134 g/L. Dropped to a minimum of 105g/L, a decrease of 21.64%; and according to Jie Liu, Lijuan Wang et al., Plos One. 2015 Sept 21; 10(9): e0137345, the reported literature indicates that Hu5F9-G4 is at a dose of 30 mg/kg. Hemoglobin was reduced from 125 g/L to a minimum of 70 g/L with a decrease of approximately 44%.

Abstract

一种CD47单域抗体及其用途，具体为一种针对整合素相关蛋白(CD47)的阻断型单域抗体及其衍生蛋白。具体而言，公开了一种整合素相关蛋白(CD47)结合分子及其用途，特别是在治疗和/或预防、或诊断CD47相关疾病例如肿瘤中的用途。涉及的CD47单域抗体能够有效阻断CD47与其配体SIRPa的相互作用，具有良好的结合活性、阻断活性、亲和力及稳定性，能有效增强巨噬细胞对肿瘤细胞的吞噬，在人淋巴瘤模型及人卵巢癌模型中均表现出非常显著的抗肿瘤活性。此外，该抗体不仅在体外不会引起人红细胞凝集，而且在食蟹猴体内也表现出极好的安全性。

Description

CD47单域抗体及其用途 技术领域
本发明属于生物医学或生物制药技术领域，涉及一种针对整合素相关蛋白(CD47)分子胞外段的阻断型单域抗体及其衍生蛋白。还公开了其编码序列、相关制备方法及其用途，特别是在治疗和/或预防、或诊断CD47相关疾病例如肿瘤中的用途。
背景技术
近年来，抗体药物以其独有的优势成为21世纪全球生物医药领域的研究热点，越来越多的抗体药物进入了临床实践。通过使用抗CD47抗体阻断CD47和SIRPa的相互作用具有靶向性治疗的效果。目前处于Phase I的三个药物分别为Forty Seven的Hu5F9-G4、Celgene的CC-90002以及Trillium的TTI-621。Trillium的CD47抗体项目为SIRPa-Fc融合蛋白，与Hu5F9-G4具有相类似的CD47亲和力(nM级别)。而SIRPa-Fc分子量更小约80kDa，相对于抗体分子的150kDa具有更好的穿透性和组织分布性；SIRPa-Fc对于红细胞的亲和力要远远低于Hu5F9-G4，表明其可能具有更好的安全性。
然而，传统单克隆抗体分子量较大、难以渗透进入组织间，并且单抗的生产周期长、人源化较为困难，因此，寻找具有更小分子量的抗体尤为重要。除抗原结合片段(Fab)、单链抗体(scFv)等基于传统单抗进行改造的小分子抗体外，在自然界骆驼科与鲨鱼科的体内存在一种天然的目前发现的最小抗原结合片段。这种抗体在1989年由布鲁塞尔自由大学Muyldermans等发现，他们在分离检测骆驼血清中的抗体时首次发现了一种重链抗体，该抗体缺失了两条轻链CL和恒定区CH1，仅由N端可变区(VHH)，铰链区和两个恒定区(CH1、CH2)构成，其可变区(VHH)即称为单域抗体(nanobody)。单域抗体分子量仅有约15kDa，其纳米级的分子大小和独特的结构赋予它优于传统抗体的多种特性，如稳定性高、水溶性好、人源化简单、靶向性高、穿透性强等。单域抗体因其特殊的结构性质，兼具了传统抗体和小分子药物的优势，几乎完美地克服了传统抗体的开发周期长，稳定性低，保存条件苛刻等缺陷。这种分子量仅为常规抗体1/10的单域抗体逐渐成为新一代抗体诊断及治疗中的新兴力量。因此应用单域抗体技术研发CD47治疗性抗体药物具有广阔的前景。
发明内容
本发明所要解决的技术问题是提供一种针对CD47胞外段的单域抗体，该单域抗体能够有效阻断CD47与其配体SIRPa的相互作用，具有良好的结合活性、阻断活性、亲和力及稳定性，能有效增强巨噬细胞对肿瘤细胞的吞噬，在人淋巴瘤模型及人卵巢癌模型中均表现出非常显著的抗肿瘤活性。此外，该抗体不仅在体外不会引起人红细胞凝集，而且在食蟹猴体内也表现出极好的安全性。本发明同时提供该单域抗体及其衍生物的编码序列、制备方法、及在诊断治疗中的用途。
为实现上述目的，本发明的第一方面，提供了一种抗CD47单域抗体VHH链的互补决定区CDR区，所述VHH链的互补决定区CDR由SEQ ID NO.:5所示的CDR1,SEQ ID NO.:6所示的CDR2，SEQ ID NO.:7所示的CDR3组成。
在另一优选例中，所述的CDR1、CDR2和CDR3由VHH链的框架区FR1、FR2、FR3和FR4所隔开。
本发明第二方面，提供了一种抗CD47单域抗体的VHH链，所述VHH链包括框架区FR和本发明第一方面所述的互补决定区CDR，所述的框架区FR由
(a)SEQ ID NO.:1所示的FR1，SEQ ID NO.:2所示的FR2，SEQ ID NO.:3所示的FR3，SEQ ID NO.:4所示的FR4组成；或
(b)SEQ ID NO.:10所示的FR1，SEQ ID NO.:11所示的FR2，SEQ ID NO.:12所示的FR3，SEQ ID NO.:13所示的FR4组成。
在另一优选例中，所述的抗CD47单域抗体的VHH链如SEQ ID NO.:8或14所示。
本发明第三方面，提供了一种抗CD47单域抗体，它是针对CD47表位的单域抗体，并且具有如SEQ ID NO.:8或SEQ ID NO.:14中所示的氨基酸序列的VHH链。
本发明第四方面，提供了一种抗CD47单域抗体Fc融合蛋白，所述融合蛋白从N端到C端的结构如式Ia或Ib所示：
A-L-B (Ia)；
B-L-A (Ib)
其中，
A为权利要求3所述的抗CD47单域抗体；
B为IgG的Fc片段；和
L为无或柔性接头。
在另一优选例中，所述的柔性接头为肽接头。
在另一优选例中，所述的肽接头具有1-50个氨基酸，较佳地1-20个氨基酸。
在另一优选例中，所述的肽接头具有(GGGGS)n的结构，其中n为1-5的正整数。
在另一优选例中，所述IgG的Fc片段包括人的IgG的Fc片段。
在另一优选例中，所述IgG的Fc片段选自下组：IgG1、IgG2、IgG3、IgG4的Fc片段、或其组合。
在另一优选例中，所述IgG的Fc片段为hIgG4PE。
在另一优选例中，所述Fc片段的氨基酸序列为SEQ ID NO.:18。
在另一优选例中，所述融合蛋白的氨基酸序列如SEQ ID NO.:16所示。
在另一优选例中，所述融合蛋白为针对CD47表位的单域抗体Fc融合蛋白。
本发明第五方面，提供了一种多核苷酸，所述多核苷酸编码选自下组的蛋白质：本发明第一方面所述的抗CD47单域抗体VHH链的CDR区、本发明第二方面所述的抗CD47单域抗体的VHH链、本发明第三方面所述的抗CD47单域抗体、或本发明第四方面所述的抗CD47单域抗体Fc融合蛋白。
在另一优选例中，所述多核苷酸具有如SEQ ID NO.:9、15或17所示的核苷酸序列。
在另一优选例中，所述的多核苷酸包括DNA或RNA。
本发明第六方面，提供了一种表达载体，所述表达载体含有本发明第五方面所述的多核苷酸。
本发明第七方面，提供了一种宿主细胞，所述宿主细胞含有本发明第六方面所述的表达载体，或其基因组中整合有本发明第五方面所述的多核苷酸。
在另一优选例中，所述的宿主细胞包括原核细胞或真核细胞。
在另一优选例中，所述的宿主细胞选自下组：哺乳动物细胞、大肠杆菌、酵母细胞、噬菌体、或其组合。
在另一优选例中，所述原核细胞选自下组：大肠杆菌、枯草杆菌、乳酸菌、链霉菌、奇异变形菌、或其组合。
在另一优选例中，所述真核细胞选自下组：巴斯德毕赤酵母、酿酒酵母、裂殖酵母、木霉、或其组合。
在另一优选例中，所示真核细胞选自下组：草地粘虫等昆虫细胞、烟草等植物细胞、BHK细胞、CHO细胞、COS细胞、骨髓瘤细胞、或其组合。
在另一优选例中，所述宿主细胞优选为哺乳动物细胞，更优选HEK293细胞、CHO细胞、BHK细胞、NSO细胞或COS细胞。
本发明第八方面，提供了一种产生抗CD47单域抗体或其Fc融合蛋白的方法，包括步骤：
(a)在适合产生单域抗体及其Fc融合蛋白的条件下，培养本发明第七方面所述的宿主细胞，从而获得含所述抗CD47单域抗体或其Fc融合蛋白的培养物；以及
(b)从所述培养物中分离或回收所述的抗CD47单域抗体或其Fc融合蛋白。
在另一优选例中，所述的抗CD47单域抗体具有如SEQ ID NO.:8或14或16所示的氨基酸序列。
本发明第九方面，提供了一种免疫偶联物，该免疫偶联物含有：
(a)如本发明第二方面所述的抗CD47单域抗体的VHH链、或如本发明第三方面所述的抗CD47单域抗体、或如本发明第四方面所述的抗CD47单域抗体Fc融合蛋白；和
(b)选自下组的偶联部分：可检测标记物、药物、毒素、细胞因子、放射性核素、或酶。
在另一优选例中，所述偶联部分为药物或毒素。
在另一优选例中，所述偶联部分为可检测标记物。
在另一优选例中，所述偶联物选自：荧光或发光标记物、放射性标记物、MRI(磁共振成像)或CT(电子计算机X射线断层扫描技术)造影剂、或能够产生可检测产物的酶、放射性核素、生物毒素、细胞因子(如IL-2等)、抗体、抗体Fc片段、抗体scFv片段、金纳米颗粒/纳米棒、病毒颗粒、脂质体、纳米磁粒、前药激活酶(例如，DT-心肌黄酶(DTD)或联苯基水解酶-样蛋白质(BPHL))、化疗剂(例如，顺铂)或任何形式的纳米颗粒等。
在另一优选例中，所述免疫偶联物含有：多价(如二价)的如本发明第二方面所述的抗CD47单域抗体的VHH链、如本发明第三方面所述的抗CD47单域抗体、或如本发明第四方面所述的抗CD47单域抗体Fc融合蛋白。
在另一优选例中，所述多价是指，在所述免疫偶联物的氨基酸序列中包含多个重复的如本发明第二方面所述的抗CD47单域抗体的VHH链、本发明第三方面所述的抗CD47单域抗体、或如本发明第四方面所述的抗CD47单域抗体Fc融合蛋白。
本发明第十方面，提供了本发明第三方面所述的抗CD47单域抗体及第四方 面所述的抗CD47单域抗体Fc融合蛋白的用途，用于制备(a)用于检测CD47分子的试剂；(b)用于治疗肿瘤的药物。
在另一优选例中，所述的检测包括流式检测、细胞免疫荧光检测。
本发明第十一方面，提供了一种药物组合物，含有：
(i)本发明第一方面抗CD47单域抗体VHH链的互补决定区CDR、本发明第二方面所述的抗CD47单域抗体的VHH链、本发明第三方面所述的抗CD47单域抗体、本发明第四方面所述的抗CD47单域抗体Fc融合蛋白或本发明第九方面所述的免疫偶联物；以及
(ii)药学上可接受的载体。
在另一优选例中，所述的药物组合物为注射剂型。
在另一优选例中，所述的药物组合物为用于制备治疗肿瘤的药物，所述的肿瘤选自下组：胃癌、肝癌、白血病、肾脏肿瘤、肺癌、小肠癌、骨癌、前列腺癌、结直肠癌、乳腺癌、大肠癌、前列腺癌、宫颈癌、淋巴癌、肾上腺肿瘤、或膀胱肿瘤。
本发明第十二方面，提供了本发明第三方面所述抗CD47单域抗体的一种或多种的用途：
(i)用于检测人CD47分子；
(ii)用于流式检测；
(iii)用于细胞免疫荧光检测；
(iv)用于治疗肿瘤；
(v)用于肿瘤诊断。
在另一优选例中，所述用途为诊断性和/或非诊断性的，和/或
治疗性和/或非治疗性的。
本发明第十三方面，提供了本发明第四方面所述抗CD47单域抗体Fc融合蛋白的一种或多种的用途：
(i)用于检测人CD47分子；
(ii)用于流式检测；
(iii)用于细胞免疫荧光检测；
(iv)用于治疗肿瘤；
(v)用于肿瘤诊断。
在另一优选例中，所述用途为诊断性和/或非诊断性的，和/或
治疗性和/或非治疗性的。
本发明第十四方面，提供了一种重组蛋白，所述的重组蛋白具有：
(i)如本发明第二方面所述的重链可变区VHH的序列、或如本发明第三方面所述的单域抗体的序列、或如第四方面所述抗CD47单域抗体Fc融合蛋白；以及
(ii)任选的协助表达和/或纯化的标签序列。
在另一优选例中，所述的标签序列包括6His标签、HA标签、或Fc标签。
在另一优选例中，所述的重组蛋白特异性结合于CD47蛋白。
本发明第十五方面，提供了如本发明第二方面所述的VHH链、如本发明第三方面所述的单域抗体、如第四方面所述抗CD47单域抗体Fc融合蛋白、或本发明第九方面所述的免疫偶联物的用途，它们被用于制备药剂、试剂、检测板或试剂盒；
其中，所述试剂、检测板或试剂盒用于：检测样品中CD47蛋白；
其中，所述药剂用于治疗或预防表达CD47蛋白(即CD47阳性)的肿瘤。
在另一优选例中，所述肿瘤包括：淋巴癌、卵巢癌、白血病、黑色素瘤、胃癌、淋巴瘤、肝癌、白血病、肾脏肿瘤、肺癌、小肠癌、骨癌、前列腺癌、结直肠癌、乳腺癌、大肠癌、前列腺癌、或肾上腺肿瘤。
本发明第十六方面，提供了一种检测样品中CD47蛋白的方法，所述方法包括步骤：
(1)将样品与本发明第三方面所述的单域抗体或是第四方面所述抗CD47单域抗体Fc融合蛋白接触；
(2)检测是否形成抗原-抗体复合物，其中形成复合物就表示样品中存在CD47蛋白。
在另一优选例中，所述方法为非诊断和非治疗性的方法。
本发明第十七方面，提供了一种治疗疾病的方法，所述方法包括，给需要的对象施用本发明第三方面所述的单域抗体、或第四方面所述抗CD47单域抗体Fc融合蛋白或本发明第九方面所述的免疫偶联物。
在另一优选例中，所述的对象包括哺乳动物，如人。
本发明第十八方面，提供了一种抗CD47单域抗体VHH链的框架区FR，所述的VHH链的框架区FR由SEQ ID NO.:1所示的FR1，SEQ ID NO.:2所示的FR2，SEQ ID NO.:3所示的FR3，SEQ ID NO.:4所示的FR4组成。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1是抗原蛋白及单域抗体纯化SDS-PAGE图，图中A为核酸分子标准，B为纯化的hCD47(ECD)-Fc蛋白，该蛋白由HEK293F细胞表达，纯度达到90％以上。
图2是构建文库的库容检测图，构建好的文库被梯度稀释后涂板，图中显示取1/5梯度稀释10 4倍、10 5倍、10 6倍的克隆数目，通过计算单克隆数确定文库大小，计算该文库的库容为2.5x10 9CFU。
图3是构建文库的插入率检测图，展示的是构建的单域抗体文库的插入率检测结果。从左到右凝胶孔的DNA条带分别是：第一道为DNA分子标记，其余孔道为检测插入片段的PCR产物，PCR产物带约为500bp；经检测，该文库的插入率达到100％。
图4展示的是CD47单域抗体筛选富集过程。文库经第一轮淘选未出现富集，第二轮淘选出现1.8倍富集，第三轮淘选出现5.6倍富集，第四轮淘选出现19倍富集。
图5是1株HEK293F系统表达CD47单域抗体Fc融合蛋白(编号Nb1-Fc)的纯化图，其中单域抗体对应SEQ ID NO.:8氨基酸序列，经Protein A亲和柱亲和层析纯化后，CD47单域抗体的SDS-PAGE的电泳图。结果显示，CD47单域抗体Fc融合蛋白Nb1-Fc经过该纯化过程，其纯度可达到90％以上。
图6是FACS检测CD47单域抗体Fc融合蛋白Nb1-Fc的阻断效果，用稳定表达人全长CD47蛋白的HEK293T细胞与各组抗体和生物素化的hSIRPa(ECD)-Fc蛋白共反应。由图可知，hSIRPa(ECD)-Fc-生物素与稳转细胞的结合率由阴性对照组的97.3％降至2.21％，这表明加入的抗体Nb1-Fc能够明显阻断CD47与SIRPa的相互作用。结果表明本发明特异性针对CD47的Nb1-Fc对CD47与SIRPa的结合具有很好的阻断效果。
图7是流式细胞术检测人源化CD47单域抗体Nb1902-Fc的阻断效果。用表达CD47蛋白的稳转株细胞与人源化单域抗体Nb1902-Fc和生物素化的hSIRPa(ECD)-Fc蛋白共反应，阴性对照组中hSIRPa(ECD)-Fc-生物素与稳转株细胞的结合率为97.2％，而加入CD47单域抗体和人源化单域抗体后， hSIRPa(ECD)-Fc-生物素与稳转细胞的结合率显著降低；这表明人源化前及人源化后的单域抗体均能够明显阻断CD47与hSIRPa的相互作用。
图8是流式细胞术检测人源化单域抗体Nb1902-Fc与阳性对照抗体的IC 50。结果表明，人源化CD47单域抗体的IC 50为1.51μg/mL，而对照抗体B6H12的IC 50为12.34μg/mL，候选人源化单域抗体的阻断效果优于对照抗体。
图9是人源化单域抗体Nb1902-Fc的亲和力检测结果。利用ForteBio's Octet System测定人源化抗体Nb1902-Fc的亲和力为1.65E-9M。
图10是ELISA检测CD47单域抗体的种属特异性结果。可见人源化后的CD47单域抗体Nb1902-Fc只与人源的CD47相互作用，不与大鼠及小鼠的CD47发生相互作用，候选的人源化单域抗体具有较好的种属特异性。
图11是人源化CD47单域抗体Fc融合蛋白在人淋巴瘤模型中的药效结果。结果表明，Nb1902-Fc有显著的肿瘤抑制效果，其肿瘤抑制率TGI为80％。
图12是SEC-HPLC检测CHO-S系统表达的人源化CD47抗体Fc融合蛋白(MY2238)的纯度结果。结果表明，利用该系统表达及一步Protein A亲和纯化的抗体MY2238的纯度为93.11％。
图13是人源化CD47抗体Fc融合蛋白的稳定性检测结果。结果表明，该抗体不论是在25℃加速条件下还是在40℃强力破坏条件下，均无明显的聚集或是降解发生，表现出良好的稳定性。
图14是人源化CD47抗体Fc融合蛋白介导巨噬细胞对肿瘤细胞的吞噬作用检测。结果表明，MY2238抗体能够明显促进巨噬细胞对Raji细胞的吞噬，吞噬率约为50％，其效果与处于临床I期的Hu5F9-G4产品(FortySeven公司)效果相似。
图15是人源化CD47单域抗体Fc融合蛋白在人卵巢癌模型中的药效结果。结果表明，候选抗体MY2238在该药效模型中对肿瘤的抑制效果表现出良好的剂量依赖性，同等剂量下候选抗体MY2238的药效(TGI＝62％)明显优于对照抗体Hu5F9-G4药效，且MY2238抗体1mg/kg的药效(TGI＝40％)相当于对照抗体Hu5F9-G4 5mg/kg的药效(TGI＝42％)。
图16是人源化CD47单域抗体Fc融合蛋白与人红细胞的结合。结果表明候选抗体MY2238与红细胞表面CD47的结合活性(Donor 1 EC50＝0.6625μg/mL，Donor 2 EC 50＝0.6443μg/mL)远远低于Hu5F9-G4与红细胞的结合活性(Donor 1 EC 50＝0.2208μg/mL，Donor 2 EC 50＝0.1583μg/mL)。
图17是人源化CD47单域抗体Fc融合蛋白对红细胞的凝集作用结果。结果表 明候选抗体MY2238不会引起人红细胞的凝集反应，而对照抗体Hu5F9-G4会明显引起红细胞凝集。
图18是人源化CD47单域抗体Fc融合蛋白在食蟹猴体内的毒理结果。结果表明，不同剂量的候选抗体MY2238在食蟹猴体内均未引起明显的生理毒性，即使在高剂量60mg/kg及200mg/kg的注射条件下，食蟹猴体内的血红蛋白降低后也恢复到正常水平，并且在研究过程中未出现任何生理异常。因此候选抗体MY2238具有极好的体内安全性。
具体实施方式
本发明人通过广泛而深入的研究，经过大量的筛选，成功获得一类抗CD47单域抗体，实验结果表明，本发明获得的CD47单域抗体能有效与CD47结合。
具体地，本发明利用人源的CD47抗原蛋白免疫骆驼，获得高质量的免疫单域抗体基因文库。然后将CD47蛋白分子偶联在酶标板上，展示CD47蛋白的正确空间结构，以此形式的抗原利用噬菌体展示技术筛选免疫单域抗体基因库(骆驼重链抗体噬菌体展示基因库)，从而获得了CD47特异性的单域抗体基因。再将此基因转至大肠杆菌中，从而获得了能在大肠杆菌中高效表达的、且特异性高的单域抗体株。
如本文所用，术语“本发明单域抗体”、“本发明的抗CD47单域抗体”、“本发明CD47单域抗体”可互换使用，均指特异性识别和结合于CD47(包括人CD47)的单域抗体。特别优选的是VHH链的氨基酸序列如SEQ ID NO.:8或14所示的单域抗体。
如本文所用，术语“抗体”或“免疫球蛋白”是有相同结构特征的约150000道尔顿的异四聚糖蛋白，其由两个相同的轻链(L)和两个相同的重链(H)组成。每条轻链通过一个共价二硫键与重链相连，而不同免疫球蛋白同种型的重链间的二硫键数目不同。每条重链和轻链也有规则间隔的链内二硫键。每条重链的一端有可变区(VH)，其后是多个恒定区。每条轻链的一端有可变区(VL)，另一端有恒定区；轻链的恒定区与重链的第一个恒定区相对，轻链的可变区与重链的可变区相对。特殊的氨基酸残基在轻链和重链的可变区之间形成界面。
如本文所用，术语“单域抗体(VHH)”、“单域抗体”(single domain antibody,sdAb，或纳米抗体nanobody)具有相同的含义，指克隆抗体重链的可 变区，构建仅由一个重链可变区组成的单域抗体(VHH)，它是具有完整功能的最小的抗原结合片段。通常先获得天然缺失轻链和重链恒定区1(CH1)的抗体后，再克隆抗体重链的可变区，构建仅由一个重链可变区组成的单域抗体(VHH)。
如本文所用，术语“可变”表示抗体中可变区的某些部分在序列上有所不同，它形成了各种特定抗体对其特定抗原的结合和特异性。然而，可变性并不均匀地分布在整个抗体可变区中。它集中于轻链和重链可变区中称为互补决定区(CDR)或超变区中的三个片段中。可变区中较保守的部分称为构架区(FR)。天然重链和轻链的可变区中各自包含四个FR区，它们大致上呈β-折叠构型，由形成连接环的三个CDR相连，在某些情况下可形成部分β折叠结构。每条链中的CDR通过FR区紧密地靠在一起并与另一链的CDR一起形成了抗体的抗原结合部位(参见Kabat等,NIH Publ.No.91-3242,卷I，647-669页(1991))。恒定区不直接参与抗体与抗原的结合，但是它们表现出不同的效应功能，例如参与抗体的依赖于抗体的细胞毒性。
如本领域技术人员所知，免疫偶联物及融合表达产物包括：药物、毒素、细胞因子(cytokine)、放射性核素、酶和其他诊断或治疗分子与本发明的抗体或其片段结合而形成的偶联物。本发明还包括与所述的抗CD47蛋白抗体或其片段结合的细胞表面标记物或抗原。
如本文所用，术语“重链可变区”与“VH”可互换使用。
如本文所用，术语“可变区”与“互补决定区(complementarity determining region,CDR)”可互换使用。
在本发明的一个优选的实施方式中，所述抗体的重链可变区包括三个互补决定区CDR1、CDR2、和CDR3。
在本发明的一个优选的实施方式中，所述抗体的重链包括上述重链可变区和重链恒定区。
在本发明中，术语“本发明抗体”、“本发明蛋白”、或“本发明多肽”可互换使用，都指特异性结合CD47蛋白的多肽，例如具有重链可变区的蛋白或多肽。它们可含有或不含起始甲硫氨酸。
本发明还提供了具有本发明抗体的其他蛋白质或融合表达产物。具体地， 本发明包括具有含可变区的重链的任何蛋白质或蛋白质偶联物及融合表达产物(即免疫偶联物及融合表达产物)，只要该可变区与本发明抗体的重链可变区相同或至少90％同源性，较佳地至少95％同源性。
一般，抗体的抗原结合特性可由位于重链可变区的3个特定区域来描述，称为可变区域(CDR)，将该段间隔成4个框架区域(FR)，4个FR的氨基酸序列相对比较保守，不直接参与结合反应。这些CDR形成环状结构，通过其间的FR形成的β折叠在空间结构上相互靠近，重链上的CDR和相应轻链上的CDR构成了抗体的抗原结合位点。可以通过比较同类型的抗体的氨基酸序列来确定是哪些氨基酸构成了FR或CDR区域。
本发明抗体的重链的可变区特别令人感兴趣，因为它们中至少部分涉及结合抗原。因此，本发明包括那些具有带CDR的抗体重链可变区的分子，只要其CDR与此处鉴定的CDR具有90％以上(较佳地95％以上，最佳地98％以上)的同源性。
本发明不仅包括完整的抗体，还包括具有免疫活性的抗体的片段或抗体与其他序列形成的融合蛋白。因此，本发明还包括所述抗体的片段、衍生物和类似物。
如本文所用，术语“片段”、“衍生物”和“类似物”是指基本上保持本发明抗体相同的生物学功能或活性的多肽。本发明的多肽片段、衍生物或类似物可以是(i)有一个或多个保守或非保守性氨基酸残基(优选保守性氨基酸残基)被取代的多肽，而这样的取代的氨基酸残基可以是也可以不是由遗传密码编码的，或(ii)在一个或多个氨基酸残基中具有取代基团的多肽，或(iii)成熟多肽与另一个化合物(比如延长多肽半衰期的化合物，例如聚乙二醇)融合所形成的多肽，或(iv)附加的氨基酸序列融合到此多肽序列而形成的多肽(如前导序列或分泌序列或用来纯化此多肽的序列或蛋白原序列，或与6His标签形成的融合蛋白)。根据本文的教导，这些片段、衍生物和类似物属于本领域熟练技术人员公知的范围。
本发明抗体指具有CD47蛋白结合活性的、包括上述CDR区的多肽。该术语还包括具有与本发明抗体相同功能的、包含上述CDR区的多肽的变异形式。这些变异形式包括(但并不限于)：一个或多个(通常为1-50个，较佳地1-30个，更佳地1-20个，最佳地1-10个)氨基酸的缺失、插入和/或取代，以及在C末端 和/或N末端添加一个或数个(通常为20个以内，较佳地为10个以内，更佳地为5个以内)氨基酸。例如，在本领域中，用性能相近或相似的氨基酸进行取代时，通常不会改变蛋白质的功能。又比如，在C末端和/或N末端添加一个或数个氨基酸通常也不会改变蛋白质的功能。该术语还包括本发明抗体的活性片段和活性衍生物。
该多肽的变异形式包括：同源序列、保守性变异体、等位变异体、天然突变体、诱导突变体、在高或低的严紧度条件下能与本发明抗体的编码DNA杂交的DNA所编码的蛋白、以及利用抗本发明抗体的抗血清获得的多肽或蛋白。
本发明还提供了其他多肽，如包含单域抗体或其片段的融合蛋白。除了几乎全长的多肽外，本发明还包括了本发明单域抗体的片段。通常，该片段具有本发明抗体的至少约50个连续氨基酸，较佳地至少约50个连续氨基酸，更佳地至少约80个连续氨基酸，最佳地至少约100个连续氨基酸。
在本发明中，“本发明抗体的保守性变异体”指与本发明抗体的氨基酸序列相比，有至多10个，较佳地至多8个，更佳地至多5个，最佳地至多3个氨基酸被性质相似或相近的氨基酸所替换而形成多肽。
本发明还提供了编码上述抗体或其片段或其融合蛋白的多核苷酸分子。本发明的多核苷酸可以是DNA形式或RNA形式。DNA形式包括cDNA、基因组DNA或人工合成的DNA。DNA可以是单链的或是双链的。DNA可以是编码链或非编码链。
编码本发明的成熟多肽的多核苷酸包括：只编码成熟多肽的编码序列；成熟多肽的编码序列和各种附加编码序列；成熟多肽的编码序列(和任选的附加编码序列)以及非编码序列。
术语“编码多肽的多核苷酸”可以是包括编码此多肽的多核苷酸，也可以是还包括附加编码和/或非编码序列的多核苷酸。
本发明还涉及与上述的序列杂交且两个序列之间具有至少50％，较佳地至少70％，更佳地至少80％相同性的多核苷酸。本发明特别涉及在严格条件下与本发明所述多核苷酸可杂交的多核苷酸。在本发明中，“严格条件”是指：(1)在较低离子强度和较高温度下的杂交和洗脱，如0.2×SSC,0.1％SDS,60℃；或(2)杂交时加有变性剂,如50％(v/v)甲酰胺，0.1％小牛血清/0.1％Ficoll，42℃等；或(3)仅在两条序列之间的相同性至少在90％以上，更好是95％以上时 才发生杂交。并且,可杂交的多核苷酸编码的多肽与成熟多肽有相同的生物学功能和活性。
本发明的抗体的核苷酸全长序列或其片段通常可以用PCR扩增法、重组法或人工合成的方法获得。一种可行的方法是用人工合成的方法来合成有关序列，尤其是片段长度较短时。通常，通过先合成多个小片段，然后再进行连接可获得序列很长的片段。此外，还可将重链的编码序列和表达标签(如6His)融合在一起，形成融合蛋白。
一旦获得了有关的序列，就可以用重组法来大批量地获得有关序列。这通常是将其克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。本发明所涉及的生物分子(核酸、蛋白等)包括以分离的形式存在的生物分子。
目前，已经可以完全通过化学合成来得到编码本发明蛋白(或其片段，或其衍生物)的DNA序列。然后可将该DNA序列引入本领域中已知的各种现有的DNA分子(或如载体)和细胞中。此外，还可通过化学合成将突变引入本发明蛋白序列中。
本发明还涉及包含上述的适当DNA序列以及适当启动子或者控制序列的载体。这些载体可以用于转化适当的宿主细胞，以使其能够表达蛋白质。
宿主细胞可以是原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如哺乳动物细胞。代表性例子有：大肠杆菌，链霉菌属；鼠伤寒沙门氏菌的细菌细胞；真菌细胞如酵母；果蝇S2或Sf9的昆虫细胞；CHO、COS7、293细胞的动物细胞等。
用重组DNA转化宿主细胞可用本领域技术人员熟知的常规技术进行。当宿主为原核生物如大肠杆菌时，能吸收DNA的感受态细胞可在指数生长期后收获，用CaCl2法处理，所用的步骤在本领域众所周知。另一种方法是使用MgCl2。如果需要，转化也可用电穿孔的方法进行。当宿主是真核生物，可选用如下的DNA转染方法：磷酸钙共沉淀法，常规机械方法如显微注射、电穿孔,脂质体包装等。
获得的转化子可以用常规方法培养，表达本发明的基因所编码的多肽。根据所用的宿主细胞，培养中所用的培养基可选自各种常规培养基。在适于宿主 细胞生长的条件下进行培养。当宿主细胞生长到适当的细胞密度后，用合适的方法(如温度转换或化学诱导)诱导选择的启动子，将细胞再培养一段时间。
在上面的方法中的重组多肽可在细胞内、或在细胞膜上表达、或分泌到细胞外。如果需要，可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化重组的蛋白。这些方法是本领域技术人员所熟知的。这些方法的例子包括但并不限于：常规的复性处理、用蛋白沉淀剂处理(盐析方法)、离心、渗透破菌、超处理、超离心、分子筛层析(凝胶过滤)、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结合。
本发明的抗体可以单独使用，也可与可检测标记物(为诊断目的)、治疗剂、PK(蛋白激酶)修饰部分或任何以上这些物质的组合结合或偶联。
用于诊断目的可检测标记物包括但不限于：荧光或发光标记物、放射性标记物、MRI(磁共振成像)或CT(电子计算机X射线断层扫描技术)造影剂、或能够产生可检测产物的酶。
可与本发明抗体结合或偶联的治疗剂包括但不限于：1.放射性核素；2.生物毒；3.细胞因子如IL-2等；4.金纳米颗粒/纳米棒；5.病毒颗粒；6.脂质体；7.纳米磁粒；8.药激活酶(例如，DT-心肌黄酶(DTD)或联苯基水解酶-样蛋白质(BPHL))；9.疗剂(例如，顺铂)或任何形式的纳米颗粒等。
整合素相关蛋白CD47
整合素相关蛋白(CD47)是一种50kD的膜糖蛋白，属于免疫球蛋白超家族成员。因其最先以膜表面蛋白的形式与整合素αVβ3一起从白细胞和胎盘中分离出来，且其功能多与整合素有关，因此称之为整合素相关蛋白(integrin-associatedprotein，IAP)。CD47广泛表达于造血细胞(红细胞、淋巴细胞、血小板、单核细胞和中性粒细胞)表面以及胎盘、肝和脑组织上。通过与其配体间的相互作用，参与生物体的多种生理活动，如血小板的激活、清除，巨噬细胞的趋化、吞噬作用，基质细胞支持的造血生成以及中性粒细胞的迁移及激活过程。CD47与抑制性受体信号调节蛋白a互为受体和配体，可形成CD47-SIRPa信号复合体，该信号复合物具有介导双向信号调节并调控多种免疫反应进程的作用。作为红细胞的自身标志抑制红细胞的清除，参与溶血性贫血 的发病机制；在正常造血干细胞(hematopoietic stem cells,HSCs)上，CD47表达的意义在于保持其在机体内的相对稳定；在白血病、非霍奇金淋巴瘤、膀胱癌和乳腺癌等恶性肿瘤中，CD47高表达于肿瘤细胞表面，提示临床预后不良。肿瘤细胞通过借此“别吃我”信号，逃避了肿瘤免疫。
药物组合物
本发明还提供了一种组合物。优选地，所述的组合物是药物组合物，它含有上述的抗体或其活性片段或其融合蛋白，以及药学上可接受的载体。通常，可将这些物质配制于无毒的、惰性的和药学上可接受的水性载体介质中，其中pH通常约为5-8，较佳地pH约为6-8，尽管pH值可随被配制物质的性质以及待治疗的病症而有所变化。配制好的药物组合物可以通过常规途径进行给药，其中包括(但并不限于)：瘤内、腹膜内、静脉内、或局部给药。
本发明的药物组合物可直接用于结合CD47蛋白分子，因而可用于治疗肿瘤。此外，还可同时使用其他治疗剂。
本发明的药物组合物含有安全有效量(如0.001-99wt％,较佳地0.01-90wt％，更佳地0.1-80wt％)的本发明上述的单域抗体(或其偶联物)以及药学上可接受的载体或赋形剂。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、及其组合。药物制剂应与给药方式相匹配。本发明的药物组合物可以被制成针剂形式，例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。药物组合物如针剂、溶液宜在无菌条件下制造。活性成分的给药量是治疗有效量，例如每天约10微克/千克体重-约50毫克/千克体重。此外，本发明的多肽还可与其他治疗剂一起使用。
使用药物组合物时，是将安全有效量的免疫偶联物施用于哺乳动物，其中该安全有效量通常至少约10微克/千克体重，而且在大多数情况下不超过约50毫克/千克体重，较佳地该剂量是约10微克/千克体重-约10毫克/千克体重。当然，具体剂量还应考虑给药途径、病人健康状况等因素，这些都是熟练医师技能范围之内的。
标记的单域抗体
在本发明的一个优选例中，所述单域抗体带有可检测标记物。更佳地，所述的标记物选自下组：同位素、胶体金标记物、有色标记物或荧光标记物。
胶体金标记可采用本领域技术人员已知的方法进行。在本发明的一个优选的方案中，抗CD47的单域抗体用胶体金标记，得到胶体金标记的单域抗体。
本发明的抗CD47单域抗体具有很好的特异性，很高的效价。
检测方法
本发明还涉及检测CD47蛋白的方法。该方法步骤大致如下：获得细胞和/或组织样本；将样本溶解在介质中；检测在所述溶解的样本中CD47蛋白的水平。
在本发明的检测方法中，所使用的样本没有特别限制，代表性的例子是存在于细胞保存液中的含细胞的样本。
试剂盒
本发明还提供了一种含有本发明的抗体(或其片段)或检测板的试剂盒，在本发明的一个优选例中，所述的试剂盒还包括容器、使用说明书、缓冲剂等。
本发明还提供了用于检测CD47水平的检测试剂盒，该试剂盒包括识别CD47蛋白的抗体，用于溶解样本的裂解介质，检测所需的通用试剂和缓冲液，如各种缓冲液、检测标记、检测底物等。该检测试剂盒可以是体外诊断装置。
应用
如上所述，本发明的单域抗体有广泛生物应用价值和临床应用价值，其应用涉及到与CD47相关的疾病的诊断和治疗、基础医学研究、生物学研究等多个领域。一个优选的应用是用于针对CD47的临床诊断和靶向治疗。
本发明的主要优点包括：
(a)本发明单域抗体高特异性针对人的具有正确空间结构的CD47蛋白。
(b)本发明单域抗体具有良好的结合活性、阻断活性及亲和力，且人源化抗体具有优异的种属特异性。
(c)本发明单域抗体能有效增强巨噬细胞对肿瘤细胞的吞噬，在人淋巴瘤模型及人卵巢癌模型中均表现出显著的抗肿瘤活性。
(d)本发明单域抗体不仅在体外不会引起人红细胞凝集，而且在食蟹猴体内也表现出极好的安全性。
(e)本发明单域抗体的生产简便，且具有很好的稳定性。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
实施例1：人CD47蛋白的表达纯化
(1)将人CD47的核苷酸序列合成在pCDNA3.1(-)载体上，然后将其胞外段序列亚克隆至pFUSE-IgG1载体上；(2)用Omega质粒大提试剂盒提取构建的pFUSE-IgG1-hCD47(ECD)质粒；(3)培养HEK293F细胞至OD为2.0×10 6个/mL；(4)将质粒与转染试剂PEI 1:3混合均匀后静置20min，然后加入到HEK293F细胞中，37℃，6％CO 2摇床培养箱中培养5-6天；(5)收集细胞上清，与Protein A珠子在室温下结合1h；(6)用磷酸盐缓冲液pH 7.0洗涤珠子后，再用0.1M pH 3.0 Glycine洗脱蛋白；(7)将洗脱的蛋白超滤至PBS中，测定产量后取样进行SDS-PAGE检测(检测结果如图1所示)，其余蛋白保存于-80℃冰箱；
实施例2：CD47单域抗体文库的构建及筛选
文库构建：简要地，(1)将1mg hCD47(ECD)-Fc抗原与弗氏佐剂等体积混合，免疫一只新疆双峰驼，每周一次，共免疫7次，刺激B细胞表达抗原特异性的单域抗体；(2)7次免疫结束后，提取100mL骆驼外周血淋巴细胞并提取总RNA；(3)合成cDNA并利用套式PCR扩增VHH；(4)利用限制性内切酶Pst I及Not I酶切20μg pMECS噬菌体展示载体(Biovector供应)及10μg VHH并连接两个片段；(5)将连接产物转化至电转感受态细胞TG1中，构建CD47单域抗体文库并测定库容，库容大小为2.5×10 9CFU(结果如图2所示)。与此同时，随机挑取24颗克隆进行菌落PCR检测，结果表明所建文库的插入率为100％，图3显示菌落PCR结果。
抗体筛选鉴定：简要地，(1)将溶解在100mM NaHCO 3、pH 8.2中的10μg hCD47(ECD)-Fc抗原(10μg Fc in NaHCO3作为对照)偶联在NUNC酶标板上，4℃放置过夜；(2)第二天加入100μL 0.1％BSA，室温封闭2h；(3)2h后，加入100μL噬菌体(2×10 11CFU免疫骆驼单域抗体噬菌展示基因库)，室温作用1h；(4)用0.05％PBS+Tween-20洗5遍，以洗掉非特异的噬菌体；(5)用100mM三乙醇胺将与CD47特异性结合的噬菌体解离下，并感染处于对数期生长的大肠杆菌TG1细胞，37℃培养1h，产生并纯化噬菌体用于下一轮的筛选，相同筛选过程重复4轮使阳性的克隆被富集(图4)。(6)从富集后含有噬菌体的细胞培养皿中，挑选200个单个菌落并接种于含有100μg/mL的氨苄青霉素的TB培养基(1L TB培养基中含有2.3g KH 2PO 4，12.52g K 2HPO 4，12g蛋白胨，24g酵母提取物，4mL甘油中，生长至对数期后，加终浓度1mM的IPTG，28℃培养过夜。(7)利用渗透法获得粗提抗体，并将抗体转移到经抗原包被的ELISA板中，在室温下放置1h。(8)用PBST洗去未结合的抗体，加入鼠抗HA抗体(COVENCE)，在室温下放置1h。(9)用PBST洗去未结合的抗体，加入山羊抗小鼠碱性磷酸酶标记抗体，在室温下放置1h。(10)用PBST洗去未结合的抗体，加入碱性磷酸酶显色液，于ELISA仪上，在405nm波长，读取吸收值。(11)当样品孔OD值大于对照孔OD值3倍以上时(Ratio+/-＞3)，判为阳性克隆孔。(12)将阳性克隆孔的菌转摇在含有100μg/mL Amp的LB液体中以便提取质粒并进行测序。
实施例3：CD47单域抗体在真核细胞HEK293中的表达纯化及流式细胞术检测单域抗体的阻断功能
真核细胞HEK293F表达CD47 Nb1-Fc融合蛋白：(1)将测序结果正确的CD47 Nb1序列克隆至pFUSE-IgG4载体(购自Invivogen)，用Omega质粒大提试剂盒提取pFUSE-IgG4-Nb1质粒；(2)培养HEK293F细胞至OD为2.0×10 6个/mL；(3)将质粒与转染试剂PEI按照1:3混合均匀后静置20min，然后加入到HEK293F细胞中，37℃，6％CO 2摇床培养箱中培养5-6天；(4)收集细胞上清，与Protein A珠子在室温下结合1h；(5)用磷酸盐缓冲液pH 7.0洗涤珠子后，再用0.1M pH 3.0 Glycine洗脱蛋白；(6)将洗脱的蛋白超滤至PBS中，测定产量后取样进行SDS-PAGE检测(检测结果图5所示)，其余蛋白保存于-80℃冰箱。
流式细胞术鉴定单域抗体的阻断功能：简要地，(1)制备hSIRPa(ECD)-Fc-Biotin，蛋白生物素的方法参照生物素试剂说明书；(2)每个样品取5×10 5个CD47稳转细胞于0.5％BSA-PBS buffer中，加入上述纯化的CD47单域抗体5μg，同时设置阴性对照(hIgG1)和空白组(PBS)，所有样本中同时加入5μg hSIRPa(ECD)-Fc-Biotin，4℃孵育20min；(3)PBS洗涤2次细胞，加入eBioscience的SA-PE，4℃孵育20min，PBS洗涤2次细胞后用流式细胞仪(BD FACS Calibur)检测。
检测结果如图6所示，hSIRPa(ECD)-Fc-生物素与稳转细胞的结合率由阴性对照组的97.3％降至2.21％，这表明加入的单域抗体Nb1-Fc能够明显阻断CD47与SIRPa的相互作用。结果表明本发明特异性针对CD47的单域抗体对CD47与SIRPa的结合具有很好的阻断效果。
实施例4：CD47单域抗体的人源化改造
首先，以SEQ ID NO.:8所示的CD47单域抗体序列为模板在结构数据库中同源结构的搜索，取其中E value＝0.0并且序列等同性≥70％的结构；其次，对这些结构进行结构比对，并依据晶体结构分辨率大小和构建的进化树，最终选取包括3dwt在内的蛋白，进行基于SEQ ID NO.:8所示的CD47单域抗体序列的多模板同源模建，再依据打分函数的高低排序，选取molpdf最低的结构，继续下面的工作；然后对模建的最优结构，利用ProtSA服务器计算残基的溶剂可接触性，即残基的折叠态相对于去折叠态的溶剂可接触面积的比值为判据，取大于40％的残基为暴露于溶剂外的残基；最后，对模建的最优结构和DP-47进行序列比对，替换相应的暴露于溶剂的残基。最终确定出一种人源化CD47单域抗体(编号为Nb1902)，由SEQ ID NO.14所示的氨基酸序列编码。人源化前后抗体序列对应如下表1：
Figure PCTCN2018116006-appb-000001
Figure PCTCN2018116006-appb-000002
实施例5：流式细胞术检测人源化CD47单域抗体Fc融合蛋白的阻断效果
首先，将人源化后的CD47单域抗体Nb1902序列(SEQ ID NO.:15)合成至pFUSE-IgG4PE载体上，用Omega质粒大提试剂盒提取pFUSE-IgG4PE-Nb1902质粒；然后利用HEK293F系统将人源化的单域抗体Nb1902-Fc表达出来，表达方法同实施例3。随后，将人源化前后的两株抗体及对照抗体(B6H12)分别稀释至1μg/uL、1/4μg/uL、1/8μg/uL。分别于配体5μg hSIRPa(ECD)-Fc-Biotin混合后与稳定表达hCD47的细胞混合，实验流程及检测方法同实施例3。检测结果如图7所示。
由图7可见，阴性对照组中hSIRPa(ECD)-Fc-生物素与稳转株细胞的结合率为97.2％，而加入CD47单域抗体和人源化单域抗体后，hSIRPa(ECD)-Fc-生物素与稳转细胞的结合率显著降低；这表明人源化前及人源化后的单域抗体均能够明显阻断CD47与hSIRPa的相互作用。
实施例6：流式细胞术检测人源化CD47单域抗体Fc融合蛋白的IC 50
(1)每个样品取5×10 5个CD47稳转细胞于0.5％BSA-PBS buffer中，加入梯度稀释的CD47人源化单域抗体(Nb1902-Fc)及对照抗体B6H12(抗体稀释梯度为30μg/mL、25μg/mL、20μg/mL、15μg/mL、10μg/mL、5μg/mL、3.333μg/mL、2.5μg/mL、1.667μg/mL、1.25μg/mL、1μg/mL、0.833μg/mL、0.625μg/mL、0.5μg/mL、0.417μg/mL、0.313μg/mL)，每个样品加入100uL，同时设置阴性对照(hIgG4)，所有样本中同时加入5μg hSIRPa(ECD)-Fc-Biotin，4℃孵育20min；(2)PBS洗涤2次细胞，加入eBioscience的SA-PE，4℃孵育20min，PBS洗涤2次细胞后用流式细胞仪(BD FACS Calibur)检测，使用graphpad prism 6软件进行数据处理。
结果如图8所示，人源化的CD47单域抗体的IC 50为1.51μg/mL，而对照抗体B6H12的IC 50为12.34μg/mL。可见候选人源化单域抗体的阻断效果显著优于对 照抗体。
实施例7：人源化单域抗体Fc融合蛋白的亲和力检测
(1)用PBST将人源化的单域抗体Nb1902-Fc从100nM进行梯度稀释，分别为：100nM、66.7nM、44.4nM、29.6nM、19.8nM、13.2nM。将抗原蛋白hCD47(ECD)-Fc和Fc分别稀释至30μg/mL。(2)设置仪器运行条件：温度30℃，Shake speed 1000rpm。使用已包被ProteinA的探针(Fortebio Part No:18-5010)捕获抗体，捕获时间180s；结合梯度稀释的抗原，结合时间180s；解离时间300s；10mM甘氨酸(PH1.7)再生3次，每次5s。(3)用ForteBio's Octet System进行上机检测。
检测结果如图9所示：人源化Nb1902-Fc的亲和力为1.65E-9M，与已报导的对照抗体(B6H12)的亲和力相似，参考文献Zeng D,Sun Q等人，Oncotarget.2016 Dec 13；7(50):83040-83050。
实施例8：人源化CD47单域抗体的种属特异性检测
(1)将人源化的CD47单域抗体基因克隆至大肠杆菌表达载体pMECS上，表达纯化过程同实施例4；(2)包被抗原蛋白CD47(人)、CD47(大鼠)、CD47(小鼠)：每孔0.5μg(5μg/mL，100μL)，包被IgG4为对照，4℃过夜；(3)第二天用PBST洗涤3次，加入200μL的1％BSA室温下封闭2h；(4)PBST洗涤三次，分别加入100uL浓度为10μg/mL人源化后的单域抗体，室温下反应1h；(5)用PBST洗去未结合的抗体，加入鼠抗HA抗体(COVENCE)，在室温下放置1h；(6)用PBST洗去未结合的抗体，加入山羊抗小鼠碱性磷酸酶标记抗体，在室温下放置1h；(7)用PBST洗去未结合的抗体，加入碱性磷酸酶显色液，于ELISA仪上，在405nm波长，读取吸收值。
根据吸收值判断单域抗体的特异性，检测结果如图10所示：人源化后的单域抗体能够与人源的CD47相互作用，而不与鼠源的CD47相互作用。
实施例9：人源化CD47单域抗体Fc融合蛋白在人淋巴瘤模型中的药效
对NOG小鼠接种Raji细胞，成瘤后分为两组进行给药(阴性组：IgG4；实验组：Nb1902-Fc)。给药频率：每天1次，连续给药28天。给药剂量：20mg/kg。 实验结果如图11所示：Nb1902-Fc有显著的肿瘤抑制效果，其肿瘤抑制率TGI为80％。
实施例10：人源化CD47单域抗体在CHO-S系统中的表达
将Nb1902-Fc氨基酸序列转化成CHO密码子优化的碱基序列(如SEQ ID NO.17所示)，随后将该序列合成至pCHO1.0载体，选择特定信号肽，用于后续该抗体的生产表达，将该系统表达的抗体编号为MY2238。(1)将合成好的质粒用OMEGA质粒大提试剂盒大量抽提，然后与PEI混合后转染CHO-S细胞，具体转染方案与实施例1的转染方案相同；(2)蛋白纯化方案也同实施例1；(3)将一步纯化的样品进行SEC-HPLC分析，结果如图12所示，该抗体经一步亲和纯化，其纯度达到93.11％，可用于后续分析研究。
实施例11：人源化CD47单域抗体Fc融合蛋白的稳定性研究
(1)将MY2238样品浓缩或稀释至10mg/ml；(2)用0.22um的针式滤器将样品过滤至新的离心管内；(3)用0.22um的针式滤器将稀释用的样品保存液(1xPBS,pH7.0)过滤至新的离心管内；(4)将10mg/mL过滤后的样品，每管100uL分装并按照下表对应的名称标记；
Figure PCTCN2018116006-appb-000003
(5)将样品配置成终浓度为5mg/mL,充分混匀，每管100uL,分装并按照下表5mg/mL对应的名称标记；
Figure PCTCN2018116006-appb-000004
(6)将样品配置成终浓度为1mg/mL,充分混匀，每管100uL,分装并按照下表对应 的名称标记；
Figure PCTCN2018116006-appb-000005
(7)将样品放置相应的培养箱内；(8)在相应的检测时间点取样检测；(9)用流动相将样品稀释至1mg/ml,作为供试品；(10)将供试品10000rpm离心3min,用移液枪将样品上清移至进样瓶中，放入HPLC自动进样器中，进样检测。
结果如13A-F所示，该抗体不论是在25℃加速条件下还是在40℃强力破坏条件下，均无明显的聚集或是降解发生，表现出良好的稳定性。
实施例12：人源化CD47单域抗体Fc融合蛋白介导巨噬细胞对肿瘤细胞的吞噬作用
(1)提前5天将M1巨噬细胞按照1E5个/孔加入24孔板中；(2)重悬Raji细胞，转移至50mL离心管，计数后取3E6个细胞；(3)1000rpm，离心5min，用PBS洗一次，再用2mL PBS重悬，加入终浓度1.5μM的CFSE，室温染色10min；
(4)加入6ml完全培养基，置于冰上5min，1000rpm，离心5min；(5)将细胞用1.2ml完全培养基重悬，分至4个96孔中，加入抗体孵育1h；(6)将M1巨噬细胞培养基吸出，加入染色的Raji细胞，孵育3h；(7)荧光显微镜下观察M1巨噬细胞对肿瘤细胞的吞噬情况。
如图14表明：MY2238抗体能够明显促进巨噬细胞对Raji细胞的吞噬，吞噬率约为50％，其效果与处于临床I期的Hu5F9-G4产品(FortySeven公司)效果相似。
实施例13：人源化CD47单域抗体Fc融合蛋白在人卵巢癌模型中的药效
对BABL/C小鼠接种SK-OV-3细胞，成瘤后分为五组给药(阴性组：PBS、阳性对照组：5mg/kg Hu5F9-G4、实验组：5mg/kg MY2238、1mg/kg MY2238、0.2mg/kg MY2238)。给药频率：每天1次，12天。实验结果如图15所示：候选抗体MY2238在该药效模型中对肿瘤的抑制效果表现出良好的剂量依赖性，同等剂量下候选 抗体MY2238的药效(TGI＝62％)明显优于对照抗体Hu5F9-G4药效，且MY2238抗体1mg/kg的药效(TGI＝40％)相当于对照抗体Hu5F9-G4 5mg/kg的药效(TGI＝42％)。
实施例14：人源化CD47单域抗体Fc融合蛋白与人红细胞的结合
(1)将对照抗体Hu5F9-G4进行梯度稀释(5μg/ml，2.5μg/ml，1.25μg/ml，0.625μg/ml，0.3125μg/ml，0.15625μg/ml，0.078125μg/ml，0.039μg/ml，0.0195μg/ml，0.00975μg/ml，0.0049μg/ml，0.0024μg/ml)以及MY2238进行梯度稀释(40μg/ml，20μg/ml，10μg/ml，5μg/ml，2.5μg/ml，1.25μg/ml，0.625μg/ml，0.3125μg/ml，0.15625μg/ml，0.078125μg/ml，0.039μg/ml，0.0195μg/ml)；(2)将以上100μL稀释好的抗体分别与3x10 5个人红细胞进行4℃孵育20min(本实验同时采取2个人的外周血并分离其红细胞)；(3)3000rpm，4℃离心4min，去除上清，加200uL PBS/well，重悬；(4)3000rpm，4℃离心4min，在离心的过程中将anti-human Fc-FITC按照1：200稀释，配好后避光置于冰上；离心结束，去除上清，再加入100uL/well，重悬，4℃孵育20min；(5)加入200uL PBS/well洗涤细胞，3000rpm，4℃离心4min，去除上清，加入200uL PBS/孔，重悬，转移至流式管中，上机检测每个样本FITC信号。
结果如图16所示：候选抗体MY2238与红细胞表面CD47的结合活性(Donor 1 EC 50＝0.6625μg/mL，Donor 2 EC 50＝0.6443μg/mL)远远低于Hu5F9-G4与红细胞的结合活性(Donor 1 EC 50＝0.2208μg/mL，Donor 2 EC 50＝0.1583μg/mL)。
实施例15：人源化CD47单域抗体Fc融合蛋白对人红细胞的凝集反应
由于红细胞表面有CD47高表达，因此会更容易优先和CD47抗体药物结合，把药物富集在表面起到“储水池”的作用。因此，该种情况容易出现贫血。只有药物进入体内后首先需要突破血小板对CD47抗体的“吸收池”作用，才能有效到达作用位置发挥作用。
将人源化CD47单域抗体Fc融合蛋白、阳性对照抗体(B6H12，序列源自专利WO 2011143624 A2)、阳性对照抗体(Hu5F9-G4)、阴性对照(IgG4)分别梯度稀释至8000nM、2000nM、500nM、125nM、31.25nM、7.81nM、1.95nM、0nM。将50μL梯度稀释好的抗体分别加入到50μL人的红细胞悬液(2％)中，置于37℃下反 应过夜后观察结果。
结果如图17所示表明，人源化CD47单域抗体Fc融合蛋白不会引起人红细胞的凝集反应。其中对照抗体(B6H12及Hu5F9-G4)的实验结果与Penka S.Petrova等人，2016，Clin.Cancer.Res.23(4)报道的结果一致。
实施例16：人源化CD47单域抗体Fc融合蛋白在食蟹猴体内的毒理研究
由于CD47抗体能够与红细胞表面的CD47结合，可能存在引起贫血等副反应的风险，因此需要探究候选抗体在动物体内的毒副作用。对4只食蟹猴分别进行四种条件的抗体注射：(1)单次注射10mg/kg；(2)单次注射30mg/kg；(3)第一周注射3mg/kg，第二周注射60mg/kg；(4)第一周注射3mg/kg，第二周注射200mg/kg。随后定期观察并检测食蟹猴的各项生理指标。
结果如图18所示：候选抗体MY2238在10mg/kg的单剂量下，血红蛋白由140g/L降至最低108g/L，降幅为22.85％；在30mg/kg的单剂量下，血红蛋白由134g/L降至最低105g/L，降幅为21.64％；而根据Jie Liu，Lijuan Wang等人，Plos One.2015 Sept 21；10(9):e0137345已报到的文献表明Hu5F9-G4在30mg/kg剂量下，血红蛋白由125g/L降至最低70g/L，降幅约为44％。由此表明MY2238具有更好的安全性。此外，在高剂量60mg/kg及200mg/kg的条件下，食蟹猴体内的血红蛋白降低后也恢复到正常水平，并且在研究过程中未出现任何生理异常。因此表明：候选抗体MY2238具有极好的体内安全性。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (11)

  1. 一种抗CD47单域抗体VHH链的互补决定区CDR区，其特征在于，所述VHH链的互补决定区CDR由SEQ ID NO.:5所示的CDR1、SEQ ID NO.:6所示的CDR2，SEQ ID NO.:7所示的CDR3组成。
  2. 一种抗CD47单域抗体的VHH链，其特征在于，所述VHH链包括框架区FR和权利要求1所述的互补决定区CDR，所述的框架区FR由(a)SEQ ID NO.:1所示的FR1，SEQ ID NO.:2所示的FR2，SEQ ID NO.:3所示的FR3，SEQ ID NO.:4所示的FR4组成；或(b)SEQ ID NO.:10所示的FR1，SEQ ID NO.:11所示的FR2，SEQ ID NO.:12所示的FR3，SEQ ID NO.:13所示的FR4组成。
  3. 一种抗CD47单域抗体，其特征在于，它是针对CD47表位的单域抗体，并且具有如SEQ ID NO.:8或SEQ ID NO.:14中所示的氨基酸序列的VHH链。
  4. 一种抗CD47单域抗体Fc融合蛋白，其特征在于，所述融合蛋白从N端到C端的结构如式Ia或Ib所示：
    A-L-B(Ia)；
    B-L-A(Ib)
    其中，
    A为权利要求3所述的抗CD47单域抗体；
    B为IgG的Fc片段；和
    L为无或柔性接头。
  5. 一种多核苷酸，其特征在于，所述多核苷酸编码选自下组的蛋白质：权利要求1所述的CDR区、权利要求2所述的抗CD47单域抗体的VHH链、权利要求3所述的抗CD47单域抗体、或权利要求4所述的抗CD47单域抗体Fc融合蛋白。
  6. 如权利要求5所述的多核苷酸，其特征在于，具有如SEQ ID NO.:9、SEQ ID NO.:15、或SEQ ID NO.:17所示的核苷酸序列。
  7. 一种表达载体，其特征在于，所述表达载体含有权利要5所述的多核苷酸。
  8. 一种宿主细胞，其特征在于，所述宿主细胞含有权利要求7所述的表达载体，或其基因组中整合有权利要求5所述的多核苷酸。
  9. 一种产生抗CD47单域抗体或其Fc融合蛋白的方法，其特征在于，包括步骤：
    (a)在适合产生单域抗体或其Fc融合蛋白的条件下，培养权利要求8所述的宿主细胞，从而获得含所述抗CD47单域抗体或其Fc融合蛋白的培养物；以及(b)从所述培养物中分离或回收所述的抗CD47单域抗体或其Fc融合蛋白。
  10. 一种免疫偶联物，其特征在于，该免疫偶联物含有：
    (a)如权利要求2所述的抗CD47单域抗体的VHH链、如权利要求3所述的抗CD47单域抗体、或如权利要求4所述的抗CD47单域抗体Fc融合蛋白；和(b)选自下组的偶联部分：可检测标记物、药物、毒素、细胞因子、放射性核素、或酶。
  11. 权利要求2所述的抗CD47单域抗体的VHH链、权利要求3所述的抗CD47单域抗体及权利要求4所述的抗CD47单域抗体Fc融合蛋白的用途，其特征在于，用于制备(a)用于检测CD47分子的试剂；(b)用于治疗肿瘤的药物。
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