WO2018233575A1 - 阻断型cd47纳米抗体及其用途 - Google Patents

阻断型cd47纳米抗体及其用途 Download PDF

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WO2018233575A1
WO2018233575A1 PCT/CN2018/091640 CN2018091640W WO2018233575A1 WO 2018233575 A1 WO2018233575 A1 WO 2018233575A1 CN 2018091640 W CN2018091640 W CN 2018091640W WO 2018233575 A1 WO2018233575 A1 WO 2018233575A1
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nanobody
seq
antibody
protein
vhh
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PCT/CN2018/091640
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English (en)
French (fr)
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安康
安文琪
马小伟
范蓓
张宝献
潘若文
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华兰生物工程股份有限公司
华兰基因工程有限公司
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    • 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
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • 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
    • C07K2317/567—Framework region [FR]
    • 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®

Definitions

  • the invention belongs to the field of biomedical or biopharmaceutical technology and relates to a blocking type Nanobody directed against 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
  • CD47 also known as integrin-associated protein (IAP)
  • IAP integrin-associated protein
  • the chemical property of CD47 is a 50 kD membrane glycoprotein comprising an extracellular Ig variable domain, five highly hydrophobic extended transmembrane segments, and a short selectively spliced carboxyl terminal cytoplasmic tail.
  • the multiple regulation of CD47 molecules in the collective is based on interactions with signal-regulated protein a, thrombospondin, and integrin, and is involved in transmembrane migration and phagocytosis of immune cells such as neutrophils, monocytes, and T cells. Adjustment of function.
  • 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.
  • CD47-SIRPa signal complex which has the function of mediating two-way signal regulation and regulating various immune processes.
  • malignant tumors such as leukemia, non-Hodgkin's 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.
  • blocking the interaction of CD47 and SIRPa by using an anti-CD47 antibody has the effect of targeted treatment.
  • Trillium's CD47 antibody project is a SIRPa-Fc fusion protein with similar CD47 affinity (nM level) to Hu5F9-G4.
  • 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 Nanobody.
  • the nano-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 penetration. Strong and so on. Due to its special structural properties, Nanobodies combine 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. Nanobodies with a molecular weight of only 1/10 of conventional antibodies are emerging as a new force in the diagnosis and treatment of next-generation antibodies. Therefore, the application of nano-antibody technology to develop CD47 therapeutic antibody drugs has broad prospects.
  • the present invention provides a Nanobody directed against the extracellular domain of CD47, which is capable of blocking the interaction of CD47 with its ligand SIRPa, and provides the coding sequence, preparation method, and Use in diagnostic treatment.
  • a VHH chain of an anti-CD47 Nanobody comprising a complementarity determining region CDR comprising the CDR1 set forth in SEQ ID NO.: 5, SEQ ID CDR2 shown by NO.: 6 and CDR3 shown by SEQ ID NO.: 7 (or consisting of CDR1, CDR2 and CDR3).
  • the CD47 is human CD47.
  • the amino acid sequence of any one of the above amino acid sequences further comprises at least one of adding, deleting, modifying and/or substituting, such as 1-3, preferably 1-2, more preferably 1) amino acid and retains a high affinity binding to CD47, blocking the derivative sequence of CD47 and SIRPa interaction.
  • the VHH chain further comprises a framework region FR, the CDR1, CDR2 and CDR3 being separated by framework regions FR1, FR2, FR3 and FR4 of the VHH chain.
  • the framework region FR is represented by FR1 shown in SEQ ID NO.: 1, FR2 shown in SEQ ID NO.: 2, FR3 shown in SEQ ID NO.: 3, and SEQ ID. NO.: FR4 composition shown in 4 (or containing the above-mentioned FR1, FR2, FR3 and FR4).
  • amino acid sequence of the VHH chain of the anti-CD47 Nanobody is as shown in SEQ ID NO.: 8.
  • a heavy chain variable region of an anti-human CD47 antibody comprising three complementarity determining regions CDR1, CDR2, and CDR3, and the three CDRs comprising SEQ ID NO. CDR1 shown in 5, CDR2 shown in SEQ ID NO.: 6, and CDR3 shown in SEQ ID NO.: 7.
  • an anti-CD47 Nanobody which is a Nanobody directed against a CD47 epitope and which has a VHH chain of the amino acid sequence set forth in SEQ ID NO.: 8.
  • an anti-CD47 antibody which is an antibody against a CD47 epitope and has a VH chain of the amino acid sequence shown in SEQ ID NO.: 8 is also provided.
  • the present invention provides a polynucleotide encoding a protein selected from the group consisting of the VHH chain of the anti-CD47 Nanobody of the first aspect of the invention, or the second aspect of the invention Said anti-CD47 Nanobody.
  • the polynucleotide has a nucleotide sequence as shown in SEQ ID NO.: 9.
  • the polynucleotide comprises DNA or RNA.
  • an expression vector comprising the polynucleotide of the third aspect of the invention is provided.
  • the expression vector is selected from the group consisting of DNA, RNA, viral vectors, plasmids, transposons, other gene transfer systems, or a combination thereof.
  • the expression vector comprises a viral vector, such as a lentivirus, an adenovirus, an AAV virus, a retrovirus, or a combination thereof.
  • a host cell comprising the expression vector of the fourth aspect of the invention, or a polynucleotide thereof according to the third aspect of the invention, is integrated into the host cell.
  • the host cell comprises a prokaryotic cell or a eukaryotic cell.
  • the host cell is selected from the group consisting of E. coli, yeast cells.
  • a method for producing an anti-CD47 Nanobody comprising the steps of:
  • the anti-CD47 Nanobody has the amino acid sequence set forth in SEQ ID NO.: 8.
  • an immunoconjugate comprising:
  • a coupled moiety selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, or a combination thereof.
  • 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 Nanobody of the first aspect of the invention, an anti-antibody according to the second aspect of the invention CD47 Nanobody.
  • a multivalent (e.g., bivalent) VHH chain of an anti-CD47 Nanobody of the first aspect of the invention an anti-antibody according to the second aspect of the invention CD47 Nanobody.
  • the polyvalent means a VHH chain comprising a plurality of repeating anti-CD47 Nanobodies according to the first aspect of the invention in the amino acid sequence of the immunoconjugate, the present invention
  • the anti-CD47 Nanobody of the second aspect of the invention for the preparation of (a) an agent for detecting a CD47 molecule; (b) a medicament for treating a tumor.
  • 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 used for preparing a medicament for treating a tumor, the tumor being 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 is non-diagnostic and non-therapeutic.
  • a recombinant protein comprising:
  • the tag sequence comprises a 6His tag and an HA tag.
  • the recombinant protein specifically binds to a CD47 protein.
  • a twelfth aspect of the invention provides the use of the VHH chain according to the first aspect of the invention, the Nanobody of the second aspect of the invention, or the immunoconjugate of the seventh aspect of the invention, Used to prepare medicaments, reagents, test plates or kits;
  • 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: melanoma, gastric cancer, lymphoma, liver cancer, leukemia, kidney tumor, lung cancer, small bowel cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, colon cancer, prostate cancer , adrenal tumors, or a combination thereof.
  • a method for detecting a CD47 protein in a sample comprising the steps of:
  • a method of treating a disease comprising administering a Nanobody of the second aspect of the invention or the immunoconjugate of the seventh aspect of the invention to a subject in need thereof.
  • the subject comprises a mammal, such as a human.
  • the framework region FR of the anti-CD47 Nanobody VHH chain is provided, and the framework region FR of the VHH chain is represented by FR1, SEQ ID NO.: 2 of SEQ ID NO.: FR2 shown, FR3 shown in SEQ ID NO.: 3, FR4 composition shown in SEQ ID NO.: 4 (or containing FR1, FR2, FR3 and FR4 as described).
  • a CAR-T cell comprising a chimeric antigen receptor CAR, the antigen binding domain of the CAR having a VHH chain according to the first aspect of the invention is provided Or the Nanobody of the second aspect of the invention.
  • Figure 1 shows the construction and screening results of a phage display CD47 Nanobody immunological library.
  • A is the PCR amplification result of the target fragment of VHH-CH1-CH2 during the construction of the library, and the fragment is about 700 bp in size;
  • B is the PCR amplification result of the VHH gene fragment, and the purification size is about 400 bp. fragments for subsequent library construction;
  • FIG capacity C is detected FIG construct libraries, library were plated after serial dilutions, showing taken 1/5 10 3-fold dilutions, 104-fold, 105-fold The number of clones was determined by calculating the number of clones. The library capacity was calculated to be 1.2 ⁇ 10 9 CFU.
  • D is the insertion rate detection map of the library.
  • the DNA bands from left to right are: The channel is a DNA molecular marker, and the remaining channels are PCR products for detecting the inserted fragment.
  • the PCR product band is about 500 bp, and the insertion rate of the library reaches 100%.
  • Figure 2 is the result of flow cytometry to detect the blocking function of CD47 Nanobody.
  • the results indicate that a CD47 Nanobody (amino acid sequence as shown in SEQ ID NO.: 8) is capable of blocking the interaction of CD47 with SIRPa.
  • Figure 3 is a flow cytometry assay for IC50 of CD47 Nanobody and positive control antibody. The results showed that the IC50 of the CD47 Nanobody was 1.316 nM, while the IC50 of the positive control antibody (CELGENE) was 4.391 nM. The blocking effect of the candidate CD47 Nanobody was better than that of the control antibody.
  • Figure 4 shows the manner in which the test solution is added in one embodiment of the present invention.
  • Figure 5 is the result of affinity detection of CD47 Nanobody.
  • the affinity of the CD47 Nanobody was determined to be 4.92E-10M using a ForteBio's Octet System.
  • Figure 6 is a species-specific result of ELISA for detection of CD47 Nanobody.
  • the CD47 Nanobody interacts only with human SIRP ⁇ and does not interact with CD47 in rats and mice.
  • the candidate CD47 Nanobody has better species specificity.
  • Figure 7 is the result of agglutination of erythrocytes by CD47 Nanobody. The results show that the Nanobody does not cause agglutination of red blood cells.
  • the inventors have successfully obtained an anti-CD47 Nanobody through extensive screening through extensive and in-depth research.
  • the experimental results show that the CD47 Nanobody obtained by the present invention can effectively bind to CD47 and block CD47 and its ligand.
  • the interaction of SIRPa The interaction of SIRPa.
  • the present invention utilizes a human CD47 antigen protein to immunize a camel to obtain a high quality immune nanobody gene library. Then, the CD47 protein molecule was coupled to the ELISA plate to display the correct spatial structure of the CD47 protein. The antigen of this form was screened by the phage display technology to screen the immuno Nanobody gene library (the camelid heavy chain antibody phage display gene library), thereby obtaining CD47-specific Nanobody gene. This gene was further transferred to Escherichia coli to obtain a highly specific Nanobody strain which was highly expressed in Escherichia coli.
  • the terms “nanobody of the invention”, “anti-CD47 Nanobody of the invention”, “CD47 Nanobody of the invention” are used interchangeably and refer to a nanoparticle that specifically recognizes and binds to CD47 (including human CD47).
  • antibody particularly preferred is the amino acid sequence of the VHH chain as shown in SEQ ID NO.: 8.
  • 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 a 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
  • nanobody single domain antibody
  • VHH single domain antibody
  • CH1 light chain and heavy chain constant region 1
  • 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 complementarity determining region CDR comprises CDR1 set forth in SEQ ID NO.: 5, CDR2 set forth in SEQ ID NO.: 6, and CDR3 set forth in SEQ ID NO.: 7 (or Consists of CDR1, CDR2 and CDR3).
  • AAAPPSVPCRLVVARYNY (SEQ ID NO.: 7)
  • the VHH chain further comprises a framework region FR, the CDR1, CDR2 and CDR3 being separated by framework regions FR1, FR2, FR3 and FR4 of the VHH chain.
  • the framework region FR is represented by FR1 shown in SEQ ID NO.: 1, FR2 shown in SEQ ID NO.: 2, FR3 shown in SEQ ID NO.: 3, and SEQ ID. NO.: FR4 composition shown in 4 (or containing the above-mentioned FR1, FR2, FR3 and FR4).
  • FYTDSVKGRFTISRDNAKNTLFLQMNSLKPEDTATYYC (SEQ ID NO.: 3)
  • 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 linked to the heavy chain of an 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 (typically 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 Nanobodies or fragments thereof.
  • the invention also includes fragments of the Nanobodies 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 polynucleotide has a nucleotide sequence as shown in SEQ ID NO.: 9.
  • 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 CaCl 2 method, and the procedures used are well known in the art.
  • Another method is to use MgCl 2 .
  • 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 cell.
  • 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. biotoxic; 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
  • 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 Nanobody of the present invention (or a conjugate thereof) and pharmaceutically An acceptable carrier or excipient.
  • 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
  • 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 Nanobody is provided with 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 Nanobody is labeled with colloidal gold to provide a colloidal gold-labeled Nanobody.
  • the anti-CD47 Nanobody 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 Nanobody of the present invention has a wide range of biological application value and clinical application value, and its application relates to various fields such as diagnosis and treatment of diseases related to CD47, basic medical research, and biological research.
  • a preferred application is for clinical diagnosis and targeted therapy against CD47.
  • Nanobody of the present invention is highly specific for human CD47 protein having the correct spatial structure.
  • the nanobody of the present invention has a strong affinity.
  • Example 1 Construction and screening of phage display CD47 Nanobody library
  • pFUSE-hCD47 (ECD) plasmid (3) HEK293F cells were cultured to an OD of 2.0 ⁇ 10 6 cells/mL; (4) The plasmid was mixed with the transfection reagent PEI 1:3 and allowed to stand for 20 min, then added to HEK293F cells were cultured for 5 days at 37 ° C in a 6% CO 2 shaker incubator; (5) Cell supernatants were collected and combined with Protein A column for 1 h at room temperature; (6) phosphate buffered with pH 7.0 After washing the column with liquid, the protein was eluted with 0.1 M pH 3.0 Glycine; (7) The eluted protein was ultrafiltered into PBS to obtain hCD47(ECD)-Fc antigen.
  • a fragment of about 400 bp in size was purified for subsequent library construction); (4) 20 ⁇ g of pMECS phage was digested with restriction endonucleases Pst I and Not I.
  • the display vector supplied by Biovector
  • 10 ⁇ g of VHH were ligated into two fragments; (5) the ligation product was transformed into electroporation competent cell TG1, and a CD47 nanobody library was constructed and the storage capacity was determined.
  • the storage capacity was 1.2 ⁇ 10 9 CFU (results such as Figure 1C)).
  • 24 clones were randomly picked for colony PCR detection.
  • Figure 1D shows the results of colony PCR. The results showed that the insertion rate of the constructed library was 100%.
  • Antibody Screening Identification Briefly, (1) 10 ⁇ g of hCD47(ECD)-Fc antigen (10 ⁇ g Fc in NaHCO 3 as a control) dissolved in 100 mM NaHCO 3 , pH 8.2 was coupled to a NUNC plate, placed at 4 ° C.
  • Example 2 Expression of CD47 Nanobody by HEK293F System and Identification of Its Blocking Function
  • the eukaryotic cell HEK293F expresses the CD47Nb-Fc fusion protein: (1) The CD47Nb sequence with the correct sequencing result was cloned into the pFUSE-IgG4 vector (purchased from Invivogen), and the pFUSE-IgG4-Nb plasmid was extracted with the Omega plasmid extract kit; The 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% CO 2 Incubate for 5-6 days in a shaker incubator; (4) Collect the cell supernatant and bind it with Protein A beads for 1 h at room temperature; (5) Wash the beads with phosphate buffer pH 7.0, then use 0.1 M pH 3.0 Glycine The protein was eluted; (6) The eluted
  • hSIRPa ECD
  • Fc-Biotin protein biotin method reference biotin reagent instructions
  • 5 ⁇ 10 5 per sample CD47 stably transfected cells were added to 0.5% BSA-PBS buffer, 5 ⁇ g of the above purified CD47 Nanobody was added, and a negative control (hIgG1) and a blank group (PBS) were set, and 5 ⁇ g of hSIRP ⁇ (ECD)-Fc- was simultaneously added to all samples.
  • 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).
  • the test results are shown in Figure 2.
  • the results indicate that the CD47 Nanobody (amino acid sequence as shown in SEQ ID NO.: 8) is capable of blocking the interaction of CD47 with SIRPa.
  • CD47 Nanobody (Nb-Fc) was subjected to 2-fold gradient dilution from 200 nM with PBST: 200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.125 nM.
  • the antigenic proteins hCD47 (ECD)-Fc and Fc were each diluted to 20 ug/mL.
  • the CD47 Nanobody is expressed by an E. coli system, and the expressed Nanobody contains His-tag and HA-tag.
  • the method see Zhu min et al., 2014, Nanoscale Research Letters, 9: 528; (2) coated antigenic protein.
  • CD47 human
  • CD47 mouse
  • CD47 monkey
  • CD47 monkey
  • CD47 monkey
  • CD47 monkey
  • the results are shown in Figure 6.
  • the CD47 Nanobody is capable of interacting with human and monkey-derived CD47, but not with murine CD47, which has better species specificity.
  • CD47 Nanobody, positive control antibody (CELGENE), negative control (IgG4) were serially diluted (4000 nM, 1000 nM, 250 nM, 62.5 nM, 15.625 nM, 3.906 nM, 0.976 nM, 0 nM). The gradient-diluted antibodies were separately added to the monkey's red blood cell suspension, and the reaction was observed at 37 ° C for 30 min.

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Abstract

一种阻断型CD47纳米抗体。该抗体不仅能够特异结合人CD47，还能够有效阻断CD47与其配体SIRPa的相互作用。该抗体在治疗和/或预防、或诊断CD47相关疾病例如肿瘤中的用途。

Description

阻断型CD47纳米抗体及其用途 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纳米抗体。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
CD47也被称为整合素相关蛋白(integrin-associatedprotein，IAP)，是免疫球蛋白超家族中的重要成员，在不同组织细胞中均广泛表达。CD47的化学特性是一个50kD的膜糖蛋白，包含1个胞外类Ig可变结构域、5个高度疏水延伸的跨膜片段以及1个短选择性拼接的羧基端胞质尾区。CD47分子在集体中多重调控作用的发挥主要基于与信号调节蛋白a、血小板反应蛋白、及整合素间的相互作用，参与中性粒细胞、单核细胞和T细胞等免疫细胞跨膜迁移以及吞噬作用的调节。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。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。然而，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。
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)即称为纳米抗体。纳米抗体分子量仅有约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。纳米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的纳米抗体逐渐成为新一代抗体诊断及治疗中的新兴力量。因此应用纳米抗体技术研发CD47治疗性抗体药物具有广阔的前景。
发明内容
发明目的：本发明提供一种针对CD47胞外段的纳米抗体，该纳米抗体能够阻断CD47与其配体SIRPa的相互作用，同时提供该纳米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。
本发明的第一方面，提供了一种抗CD47纳米抗体的VHH链，所述VHH链包括互补决定区CDR，所述的互补决定区CDR包括SEQ ID NO.:5所示的CDR1，SEQ ID NO.:6所示的CDR2，和SEQ ID NO.:7所示的CDR3(或由CDR1、CDR2和CDR3构成)。
在另一优选例中，所述CD47为人CD47。
在另一优选例中，上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代至少一个(如1-3个，较佳地1-2个，更佳地1个)氨基酸并能保留与CD47高亲和力结合、阻断CD47与SIRPa相互作用的衍生序列。
在另一优选例中，所述VHH链还包括框架区FR，所述的CDR1、CDR2和CDR3由VHH链的框架区FR1、FR2、FR3和FR4所隔开。
在另一优选例中，所述的框架区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，和SEQ ID NO.:4所示的FR4组成(或含有所述的FR1、FR2、FR3和FR4)。
在另一优选例中，所述的抗CD47纳米抗体的VHH链的氨基酸序列如SEQ ID NO.:8所示。
此外，还提供一种抗人CD47抗体的重链可变区，所述的重链可变区包括三个互补决定区CDR1、CDR2、和CDR3，并且所述3个CDR包括SEQ ID NO.:5所示的CDR1，SEQ ID NO.:6所示的CDR2，和SEQ ID NO.:7所示的CDR3。
本发明第二方面，提供了一种抗CD47纳米抗体，它是针对CD47表位的纳米抗体，并且具有如SEQ ID NO.:8中所示的氨基酸序列的VHH链。
此外，还提供了一种抗CD47抗体，所述抗CD47抗体是针对CD47表位的抗体，并且具有如SEQ ID NO.:8中所示的氨基酸序列的VH链。
本发明第三方面，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纳米抗体的VHH链、或本发明第二方面所述的抗CD47纳米抗体。
在另一优选例中，所述多核苷酸具有如SEQ ID NO.:9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。
本发明第四方面，提供了一种表达载体，所述表达载体含有本发明第三方面所述的多核苷酸。
在另一优选例中，所述的表达载体选自下组：DNA、RNA、病毒载体、质粒、转座子、其他基因转移系统、或其组合。优选地，所述表达载体包括病毒载体，如慢病毒、腺病毒、AAV病毒、逆转录病毒、或其组合。
本发明第五方面，提供了一种宿主细胞，所述宿主细胞含有本发明第四方面所述的表达载体，或其基因组中整合有本发明第三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：大肠杆菌、酵母细胞。
本发明第六方面，提供了一种产生抗CD47纳米抗体的方法，包括步骤：
(a)在适合产生纳米抗体的条件下，培养本发明第五方面所述的宿主细胞，从而获得含所述抗CD47纳米抗体的培养物；以及
(b)从所述培养物中分离或回收所述的抗CD47纳米抗体。
在另一优选例中，所述的抗CD47纳米抗体具有如SEQ ID NO.:8所示的氨基酸序列。
本发明第七方面，提供了一种免疫偶联物，该免疫偶联物含有：
(a)如本发明第一方面所述的抗CD47纳米抗体的VHH链、或如本发明第二方面所述的抗CD47纳米抗体；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、酶、或其组合。
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)的如本发明第一方面所述的抗CD47纳米抗体的VHH链、如本发明第二方面所述的抗CD47纳米抗体。
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一方面所述的抗CD47纳米抗体的VHH链、本发明第二方面所述的抗CD47纳米抗体。
本发明第八方面，提供了本发明第二方面所述的抗CD47纳米抗体的用途，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。
本发明第九方面，提供了一种药物组合物，含有：
(i)本发明第一方面所述的抗CD47纳米抗体的VHH链、或如本发明第二方面所述的抗CD47纳米抗体、或本发明第七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。
本发明第十方面，提供了本发明第二方面所述抗CD47纳米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，所述用途为非诊断的和非治疗的。
本发明第十一方面，提供了一种重组蛋白，所述的重组蛋白具有：
(i)如本发明第一方面所述的重链可变区VHH的序列或如本发明第二方面所述的纳米抗体的序列；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标签和HA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。
本发明第十二方面，提供了如本发明第一方面所述的VHH链、如本发明第二方面所述的纳米抗体、或本发明第七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：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、肾上腺肿瘤、或其组合。
本发明第十三方面，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)将样品与本发明第二方面所述的纳米抗体接触；
(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。
本发明第十四方面，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二方面所述的纳米抗体或本发明第七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。
本发明第十五方面，提供了一种抗CD47纳米抗体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(或含有所述的FR1、FR2、FR3和FR4)。
本发明第十六方面，提供了一种CAR-T细胞，所述CAR-T细胞表达嵌合抗原受体CAR，所述CAR的抗原结合结构域具有如本发明第一方面所述的VHH链、或本发明第二方面所述的纳米抗体。
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是噬菌体展示CD47纳米抗体免疫文库的构建及筛选结果。图中A为文库构建过程中VHH-CH1-CH2的目的片段的PCR扩增结果，割胶回收大小约为700bp的片段；图中B为VHH基因片段的PCR扩增结果，纯化大小约为400bp的片段用于后续文库构建；图中C为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 3倍、10 4倍、10 5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1.2x10 9CFU；图中D为文库的插入率检测图，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，该文库的插入率达到100％。
图2是流式细胞术检测CD47纳米抗体阻断功能的结果。结果表明，一株CD47纳米抗体(氨基酸序列如SEQ ID NO.:8所示)能够阻断CD47与SIRPa的相互作用。
图3是流式细胞术检测CD47纳米抗体与阳性对照抗体的IC50。结果表明，CD47纳米抗体的IC50为1.316nM，而阳性对照抗体(CELGENE)的IC50为4.391nM，候选CD47纳米抗体的阻断效果优于对照抗体。
图4显示了本发明一个实施例中加入检测液的方式。
图5是CD47纳米抗体的亲和力检测结果。利用ForteBio’s Octet System测定该CD47纳米抗体的亲和力为4.92E-10M。
图6是ELISA检测CD47纳米抗体的种属特异性结果。该CD47纳米抗体只与人源的SIRPa相互作用，不与大鼠及小鼠的CD47发生相互作用，候选的CD47纳米抗体具有较好的种属特异性。
图7是CD47纳米抗体对红细胞的凝集作用结果。结果表明，该纳米抗体不会引起红细胞的凝集反应。
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，成功获得一株抗CD47纳米抗体，实验结果表明，本发明获得的CD47纳米抗体既能有效与CD47结合，还能够阻断CD47与其配体SIRPa的相互作用。
具体地，本发明利用人源的CD47抗原蛋白免疫骆驼，获得高质量的免疫纳米抗体基因文库。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纳米抗体基因库(骆驼重链抗体噬菌体展示基因库)，从而获得了CD47特异性的纳米抗体基因。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、且特异性高的纳米抗体株。
如本文所用，术语“本发明纳米抗体”、“本发明的抗CD47纳米抗体”、“本发明CD47纳米抗体”可互换使用，均指特异性识别和结合于CD47(包括人CD47)的纳米抗体。特别优选的是VHH链的氨基酸序列如SEQ ID NO.:8所示的纳米抗体。
Figure PCTCN2018091640-appb-000001
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)”、“纳米抗体”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“V H”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。
在另一优选例中，所述的互补决定区CDR包括SEQ ID NO.:5所示的CDR1，SEQ ID NO.:6所示的CDR2，和SEQ ID NO.:7所示的CDR3(或由CDR1、CDR2和CDR3构成)。
GYTYSSYCMG(SEQ ID NO.:5)
IYPDAGIT(SEQ ID NO.:6)
AAAPPSVPCRLVVARYNY(SEQ ID NO.:7)
在另一优选例中，所述VHH链还包括框架区FR，所述的CDR1、CDR2和CDR3由VHH链的框架区FR1、FR2、FR3和FR4所隔开。
在另一优选例中，所述的框架区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，和SEQ ID NO.:4所示的FR4组成(或含有所述的FR1、FR2、FR3和FR4)。
QVQLQESGGGSVQAGGSLRLSCAAS(SEQ ID NO.:1)
WFRQAPGKEREGVAA(SEQ ID NO.:2)
FYTDSVKGRFTISRDNAKNTLFLQMNSLKPEDTATYYC(SEQ ID NO.:3)
WGQGTQVTVSS(SEQ ID NO.:4)
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，如包含纳米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纳米抗体的片段。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。
在另一优选例中，所述多核苷酸具有如SEQ ID NO.:9所示的核苷酸序列。
Figure PCTCN2018091640-appb-000002
Figure PCTCN2018091640-appb-000003
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CaCl 2法处理，所用的步骤在本领域众所周知。另一种方法是使用MgCl 2。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。
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％)的本发明上述的纳米抗体(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。
标记的纳米抗体
在本发明的一个优选例中，所述纳米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的纳米抗体用胶体金标记，得到胶体金标记的纳米抗体。
本发明的抗CD47纳米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，本发明的纳米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)本发明纳米抗体高特异性针对人的具有正确空间结构的CD47蛋白。
(b)本发明纳米抗体的亲和力强。
(c)本发明纳米抗体的生产简便。
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：噬菌体展示CD47纳米抗体文库的构建及筛选
抗原制备：(1)将hCD47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载体上；(2)用Omega质粒大提试剂盒提取构建的pFUSE-hCD47(ECD)质粒；(3)将HEK293F细胞培养至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天；(5)收集细胞上清，与Protein A层析柱在室温下结合1h；(6)用pH 7.0的磷酸盐缓冲液洗涤层析柱后，再用0.1M pH 3.0Glycine洗脱蛋白；(7)将洗脱的蛋白超滤至PBS中，即得hCD47(ECD)-Fc抗原。
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3次，刺激B细胞表达抗原特异性的纳米抗体；(2)3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(第一次PCR结果如图1A，割胶回收大小约为700bp的片段，第二次PCR结果如图1B，纯化大小约为400bp的片段用于后续文库构建)；(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纳米抗体文库并测定库容，库容大小为1.2×10 9CFU(结果如图1C所示)。与此同时，随机挑取24颗克隆进行菌落PCR检测，图1D显示菌落PCR结果，结果表明所建文库的插入率为100％。
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 NaHCO 3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免疫骆驼纳米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3轮使阳性的克隆被富集。(6)从富集后含有噬菌体的细胞培养皿中，挑选96个单菌落并接种于含有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2：HEK293F系统表达CD47纳米抗体及其阻断功能鉴定
真核细胞HEK293F表达CD47Nb-Fc融合蛋白：(1)将测序结果正确的CD47Nb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-Nb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 3.0Glycine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，其余蛋白保存于-80℃冰箱。
流式细胞术鉴定纳米抗体的阻断功能：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纳米抗体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2所示。结果表明，CD47纳米抗体(氨基酸序列如SEQ ID NO.:8所示)能够阻断CD47与SIRPa的相互作用。
实施例3：流式细胞术检测CD47纳米抗体的IC50
(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纳米抗体及对照抗体(抗体稀释梯度为25nM、20nM、15nM、10nM、5nM、3.33nM、2.5nM、1.67nM、1.25nM、1.0nM、0.83nM、0.625nM)，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3所示，该CD47纳米抗体的IC50为1.316nM，而对照抗体(Celgene)的IC50为4.39nM。
实施例4：CD47纳米抗体的亲和力检测
(1)用PBST将CD47纳米抗体(Nb-Fc)从200nM进行2倍梯度稀释，分别为：200nM、100nM、50nM、25nM、12.5nM、6.25nM、3.125nM。将抗原蛋白hCD47(ECD)-Fc和Fc分别稀释至20ug/mL。(2)按照图4指示加入各种检测液。(3)用ForteBio’s Octet System进行上机检测。
检测结果如图5所示：该CD47纳米抗体的亲和力为4.92×10 -10M。
实施例5：CD47纳米抗体的种属特异性检测
(1)利用大肠杆菌系统表达该CD47纳米抗体，表达的纳米抗体含有His-tag及HA-tag，方法参见Zhu min等人，2014，Nanoscale Research Letters，9：528；(2)包被抗原蛋白CD47(人)、CD47(鼠)、CD47(猴)：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，加入100uL浓度为10μg/mL CD47纳米抗体，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。根据吸收值判断纳米抗体的特异性。
检测结果如图6所示：该CD47纳米抗体能够与人源和猴源的CD47相互作用，而不与鼠源的CD47相互作用，该抗体具有较好的种属特异性。
实施例6：CD47纳米抗体对红细胞的凝集反应
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CD47纳米抗体、阳性对照抗体(CELGENE)、阴性对照(IgG4)分别梯度稀释(4000nM、1000nM、250nM、62.5nM、15.625nM、3.906nM、0.976nM、0nM)。将梯度稀释好的抗体分别加入到猴子的红细胞悬液中，置于37℃下反应30min后观察结果。
结果表明(图7)，该CD47纳米抗体和对照抗体均不会引起红细胞的凝集反应。其中对照抗体(Celgene)的实验结果与Penka S.Petrova等人，2016，Clin.Cancer.Res.23(4)报道的结果一致。
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 (10)

  1. 一种抗CD47纳米抗体的VHH链，其特征在于，所述VHH链包括互补决定区CDR，所述的互补决定区CDR包括SEQ ID NO：5所示的CDR1，SEQ ID NO：6所示的CDR2，和SEQ ID NO：7所示的CDR3。
  2. 如权利要求1所述的VHH链，其特征在于，所述VHH链还包括框架区FR，所述的框架区FR包括SEQ ID NO：1所示的FR1，SEQ ID NO：2所示的FR2，SEQ ID NO：3所示的FR3，和SEQ ID NO：4所示的FR4。
  3. 一种抗CD47纳米抗体，其特征在于，它是针对CD47表位的纳米抗体，并且具有如SEQ ID NO.:8中所示的氨基酸序列的VHH链。
  4. 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所述的抗CD47纳米抗体的VHH链、或权利要求3所述的抗CD47纳米抗体。
  5. 如权利要求4所述的多核苷酸，其特征在于，具有如SEQ ID NO.:9所示的核苷酸序列。
  6. 一种表达载体，其特征在于，所述表达载体含有权利要求4所述的多核苷酸。
  7. 一种宿主细胞，其特征在于，所述宿主细胞含有权利要求6所述的表达载体，或其基因组中整合有权利要求4所述的多核苷酸。
  8. 一种产生抗CD47纳米抗体的方法，其特征在于，包括步骤：(a)在适合产生纳米抗体的条件下，培养权利要求7所述的宿主细胞，从而获得含所述抗CD47纳米抗体的培养物；以及(b)从所述培养物中分离或回收所述的抗CD47纳米抗体。
  9. 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1所述的抗CD47纳米抗体的VHH链、或如权利要求3所述的抗CD47纳米抗体；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、酶、或其组合。
  10. 权利要求3所述的抗CD47纳米抗体的用途，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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