WO2022166802A1 - 抗人cd271的单克隆抗体及用途 - Google Patents

抗人cd271的单克隆抗体及用途 Download PDF

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WO2022166802A1
WO2022166802A1 PCT/CN2022/074522 CN2022074522W WO2022166802A1 WO 2022166802 A1 WO2022166802 A1 WO 2022166802A1 CN 2022074522 W CN2022074522 W CN 2022074522W WO 2022166802 A1 WO2022166802 A1 WO 2022166802A1
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seq
antibody
human
amino acid
cells
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路慧丽
杨慧
王紫嫣
朱建伟
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Shanghai Jiao Tong University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • 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
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies
    • 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
    • C07K2317/90—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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70578—NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30 CD40 or CD95

Definitions

  • the invention belongs to the field of biotechnology, and in particular relates to an anti-human CD271 monoclonal antibody and use thereof.
  • CD271 is a low-affinity nerve growth factor receptor (LNGFR), a member of the tumor necrosis factor receptor superfamily with a relative molecular mass of 75kD, also known as p75NTR, which contains 3 regions: cysteine-rich The extra-membrane domain, the transmembrane domain and the intracellular domain consisting of 155 amino acid residues.
  • LNGFR low-affinity nerve growth factor receptor
  • p75NTR a member of the tumor necrosis factor receptor superfamily with a relative molecular mass of 75kD, also known as p75NTR, which contains 3 regions: cysteine-rich The extra-membrane domain, the transmembrane domain and the intracellular domain consisting of 155 amino acid residues.
  • CD271 is not only expressed in the nervous system, but also closely related to the development, differentiation and survival of neural cells. It is also a suitable marker molecule for enriching mesenchymal stem cells. Compared with traditional sorting methods, CD271 has high specific
  • MSCs Mesenchymal stem cells
  • tissue damage repair such as repair of bone, cartilage, and joint damage
  • treatment of heart, liver, spinal cord injury, and nervous system diseases mainly include tissue damage repair, such as repair of bone, cartilage, and joint damage, and treatment of heart, liver, spinal cord injury, and nervous system diseases.
  • tissue damage repair such as repair of bone, cartilage, and joint damage
  • treatment of heart, liver, spinal cord injury, and nervous system diseases mainly include tissue damage repair, such as repair of bone, cartilage, and joint damage, and treatment of heart, liver, spinal cord injury, and nervous system diseases.
  • MSCs for targeted tissue repair has always been limited by the colonization and proliferation of stem cells in damaged sites. Therefore, in the treatment of tissue injury sites or neurological diseases, the removal or enrichment of specific cells in the graft has become the most critical issue in the current transplantation field.
  • CD271, CD105, CD133, etc. as surface markers of mesenchymal stem cells, can effectively isolate or enrich mesenchymal stem cells. Under specific induction conditions in vivo or in vitro, they can differentiate into cartilage, muscle, tendon, etc., to achieve tissue repair. the goal of.
  • CD271 is also one of the markers of various cancer stem cells (CSC).
  • CSC cancer stem cells
  • the purpose of the present invention is to provide an anti-human CD271 monoclonal antibody and use thereof.
  • the extracellular structure of native CD271 includes four cysteine-rich CRD structures (CRD1-4) and a linker region (stalk region).
  • CRD1-4 cysteine-rich CRD structures
  • Stalk region linker region
  • the CRD region is hydrolyzed by protease to be shed, so the binding of antibodies targeting the CRD region to CD271-positive cells is affected, and even the expression of CD271 cannot be detected at a certain stage of cell metabolism.
  • the stalk region near the cell membrane has no glycosylation, phosphorylation modification or advanced spatial structure, and the structure is stable.
  • the antibody that recognizes this region has good versatility in theory. Therefore, in order to obtain a monoclonal antibody that can stably recognize human CD271, we selected the stalk region as the target to develop a new type of anti-CD271 monoclonal antibody to provide new tools for research in this field.
  • the first aspect of the present invention provides a hybridoma cell line CD271T-2 which is anti-secreting anti-human CD271 monoclonal antibody, and the hybridoma cell line has a deposit number of CGMCC 21494. It is preserved in the China Ordinary Microbial Culture Collection and Management Center, and the preservation address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is January 25, 2021. The classification was named as the hybridoma cell line CD271T-2.
  • the second aspect of the present invention provides an anti-human CD271 monoclonal antibody, the anti-human CD271 monoclonal antibody is secreted by the CD271T-2 hybridoma cell line.
  • the anti-human CD271 monoclonal antibody has good binding affinity to human mesenchymal stem cells.
  • the antibody is a murine monoclonal antibody belonging to the IgM subtype.
  • a third aspect of the present invention provides an engineered antibody against human CD271, the engineered antibody against human CD271 includes a light chain and a heavy chain, the light chain includes LCDR1, LCDR2 and LCDR3, and the amino acid sequence of LCDR1 is as shown in SEQ ID As shown in NO.1, the amino acid sequence of LCDR2 is shown in SEQ ID NO.2, and the amino acid sequence of LCDR3 is shown in SEQ ID NO.3; the heavy chain includes HCDR1, HCDR2 and HCDR3, and the HCDR1
  • the amino acid sequence of HCDR2 is shown in SEQ ID NO.4, the amino acid sequence of HCDR2 is shown in SEQ ID NO.5, and the amino acid sequence of HCDR3 is shown in SEQ ID NO.6.
  • the light chain complementarity determining region LCDR1 has a length of 10aa, and the amino acid sequence is shown in SEQ ID NO.1, specifically: QSVDYDGDSY.
  • Light chain complementarity determining region LCDR2 length: 3aa, amino acid sequence as shown in SEQ ID NO.2, specifically: AAS.
  • Heavy chain complementarity determining region HCDR2 length: 7aa, amino acid sequence as shown in SEQ ID NO.5, specifically: IWWDDDK.
  • amino acid sequence of the light chain variable region of the engineered antibody against human CD271 is as shown in SEQ ID NO.7; or it is to perform certain substitutions, additions and/or deletions of one or more of SEQ ID NO.7 Amino acids obtain amino acid sequences with equivalent functions.
  • amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO.8, or an amino acid sequence with an equivalent function is obtained by performing certain substitutions, additions and/or deletions of one or several amino acids to SEQ ID NO.8.
  • sequence shown in SEQ ID NO.7 is specifically:
  • sequence shown in SEQ ID NO.8 is specifically:
  • the engineered antibody can be a monoclonal antibody.
  • the monoclonal antibody is of murine origin.
  • the monoclonal antibody subclass is IgM.
  • the antibody is produced by the CD271T-2 hybridoma cell line through reverse transcription-polymerase chain synthesis reaction.
  • the fourth aspect of the present invention provides a polynucleotide encoding the heavy chain variable region and/or light chain variable region or full-length amino acids of the aforementioned anti-human CD271 antibody.
  • the heavy chain variable region and/or light chain variable region encoding the aforementioned anti-human CD271 antibody are shown in SEQ ID NO.9-SEQ ID NO.14.
  • the light chain complementarity determining region LCDR1, DNA, length: 30 bp, and the nucleotide sequence is shown in SEQ ID NO. 9, specifically: CAAAGTGTTGATTATGATGGTGATAGTTAT.
  • Light chain complementarity determining region LCDR2 DNA, length: 9bp, nucleotide sequence as shown in SEQ ID NO.10, specifically: GCTGCATCC.
  • Light chain complementarity determining region LCDR3 DNA, length: 27bp, nucleotide sequence as shown in SEQ ID NO.11, specifically: CAGCAAAGTAATGAGGATCCATTCACG.
  • Heavy chain complementarity determining region HCDR1 DNA, length: 30bp, nucleotide sequence shown in SEQ ID NO. 12, specifically: GGGTTTTCACTGAGCACTTCTGGTATGGGT.
  • Heavy chain complementarity determining region HCDR2 DNA, length: 21bp, nucleotide sequence shown in SEQ ID NO.13, specifically: ATTTGGTGGGATGATGATGATAAG.
  • nucleotide sequence encoding the light chain variable region of the aforementioned anti-human CD271 antibody is shown in SEQ ID NO.15
  • nucleotide sequence encoding the heavy chain variable region of the aforementioned anti-human CD271 antibody is as shown in SEQ ID NO. NO.16 shows.
  • a fifth aspect of the present invention provides a polynucleotide encoding the recombination sequence of the light chain variable region and the heavy chain variable region of the Fab segment of the aforementioned anti-CD271 antibody, which can obtain a single chain with a smaller molecular weight Antibody.
  • a fifth aspect of the present invention provides a construct comprising the aforementioned polynucleotide.
  • the construct may be constructed by inserting the isolated polynucleotide into a multiple cloning site of an expression vector.
  • the expression vector in the present invention generally refers to various commercially available expression vectors well known in the art, such as bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenovirus, retrovirus or other vectors.
  • Specific expression vectors that can be used include but are not limited to: pET series expression vectors, pGEX series expression vectors, pcDNA series expression vectors, and the like.
  • the host cell of the present invention contains the aforementioned construct or the aforementioned polynucleotide of foreign origin integrated into the genome.
  • Any cell suitable for the expression of the expression vector can be used as a host cell, for example, the host cell can be a prokaryotic cell, such as a bacterial cell, etc.; or a lower eukaryotic cell, such as a yeast cell, etc.; or a higher eukaryotic cell , such as mammalian cells.
  • the host cell is a eukaryotic cell, and a mammalian host cell line that does not produce antibodies can be used.
  • Specific cell lines that can be used include but are not limited to: Chinese hamster ovary cells (CHO), baby hamster kidney cells (BHK, ATCC CCL 10), young mouse Sertoli cells (Sertoli cells), monkey kidney cells (COS cells), monkey kidney CVI cells transformed by SV40 (COS-7, ATCC CRL 1651), Human embryonic kidney cells (HEK-293), monkey kidney cells (CVI, ATCC CCL-70), African green monkey kidney cells (VERO-76, ATCC CRL-1587), human cervical cancer cells (HELA, ATCC CCL-2) and so on.
  • CHO Chinese hamster ovary cells
  • BHK baby hamster kidney cells
  • Sertoli cells young mouse Sertoli cells
  • COS cells monkey kidney cells
  • COS-7 monkey kidney CVI cells transformed by SV40
  • HEK-293 Human embryonic kidney cells
  • CVI ATCC CCL-70
  • African green monkey kidney cells VERO-76, ATCC CRL-1587
  • human cervical cancer cells HELA, AT
  • Appropriate conditions for expressing the antibody should be known to those skilled in the art, and those skilled in the art can select a suitable medium according to experience, and culture under conditions suitable for the growth of host cells. After the host cells have grown to an appropriate cell density, the promoter of choice is induced by a suitable method (eg, temperature switching or chemical induction), and the cells are cultured for an additional period of time.
  • a suitable method eg, temperature switching or chemical induction
  • the recombinant polypeptide in the above method can be expressed intracellularly, or on the cell membrane, or secreted outside the cell. If desired, recombinant proteins can be isolated and purified by various isolation methods utilizing their physical, chemical and other properties. These methods are well known to those skilled in the art.
  • Examples of these methods include, but are not limited to: conventional renaturation treatment, treatment with protein precipitants (salting-out method), centrifugation, osmotic disruption, ultratreatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • a sixth aspect of the present invention provides a host cell comprising the aforementioned construct or the aforementioned exogenous polynucleotide integrated into the genome.
  • the seventh aspect of the present invention provides the aforementioned cell line, or the aforementioned anti-human CD271 monoclonal antibody, or the aforementioned engineered antibody against human CD271, or the aforementioned polynucleotide, or the aforementioned construct, or the aforementioned host of the antibody Use of cells, selected from any of the following:
  • CD271 cells expressing CD271, including but not limited to mesenchymal stem cells, tumor stem cells, and nervous system cells, and their in vitro detection, identification, isolation, enrichment, and targeting;
  • the present invention has the following beneficial effects:
  • the monoclonal antibody secreted by the hybridoma cell line of the present invention has the advantages of high titer, good specificity and strong affinity with natural antigen.
  • the engineered antibody against human CD271 is produced by CD271T-2 hybridoma through reverse transcription-polymerase chain synthesis reaction.
  • the antibody can specifically bind to human CD271 molecule and has a certain affinity for human mesenchymal stem cells in vitro.
  • CD271 is expressed on the surface of mesenchymal stem cells, tumor stem cells and cells of nervous system-related diseases, and is a potential target for mediating mesenchymal stem cells or targeting nervous system-related cells and tumors. Therefore, the anti-human CD271 monoclonal antibody is a medical antibody with great potential.
  • the anti-human CD271 antibody of the present invention has high specificity and affinity, and can be coupled with magnetic nanomaterials to prepare immunomagnetic beads for sorting mesenchymal stem cells, and can also be combined with other targets to design bispecificity Antibodies, or used in combination with other chemical molecules, radioactive molecules, polypeptides or proteins, nanomaterials, etc., are used for the treatment of certain tissue repair diseases and nervous system or tumor-related diseases.
  • the preservation information of strains of the present invention is as follows:
  • Cell line name hybridoma cell line CD271T-2;
  • the deposit number is: CGMCC 21494;
  • preservation unit China General Microorganism Culture Preservation and Management Center;
  • CGMCC depositary unit
  • the address of the preservation unit No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.
  • Figure 1 shows the detection of serum titer of mice after anti-human CD271 immunization by ELISA: the coating antigen is full-length CD271 protein antigen, the antigen concentration is 4 ⁇ g/mL, and the loading serum is diluted from 1:2000 to 1:256000 dilution, 1
  • the serum titers of No. 2, No. 3, No. 4 and No. 5 mice were all above 1:128000, and the 5 mice had high reactivity to the antigen;
  • Figure 2 is the SDS-PAGE detection chart of mouse CD271T-2 ascites purified antibody: in the non-reducing condition, the ascites antibody band is relatively single, and in the reducing condition, the light chain and heavy chain of the antibody are at 26kDa and 53kDa, respectively;
  • Figure 3 shows the ELISA identification of purified antibody from mouse ascites: the coated antigen is full-length CD271 protein antigen, the antigen concentration is 2 ⁇ g/mL, each well is 100 ⁇ L, and the concentration gradient of loading antibody is 0.625 ⁇ g/mL, 1.25 ⁇ g/mL, 2.5 ⁇ g /mL, 5 ⁇ g/mL, 10 ⁇ g/mL, 20 ⁇ g/mL, 40 ⁇ g/mL, 100 ⁇ L per well, the results show that CD271T-2 antibody can recognize and bind human CD271 antigen;
  • Figure 4 is the antibody affinity map after verification and purification by ForteBio
  • Figure 5 is the PCR result of the heavy and light chain variable region gene cloning of the anti-human CD271 antibody of the present invention:
  • Figure 5A is the gene cloning PCR result of the heavy and light chain variable region of the antibody of the CD271T-2 hybridoma
  • Figure 5B is the CD271T- 2 PCR results of hybridoma antibody heavy and light chain variable region chimeric ScFv gene cloning;
  • Figure 6 is the anti-human CD271 monoclonal antibody to detect the expression of CD271 on the surface of mesenchymal stem cells:
  • Figure 6A is the positive control antibody of APC Goat anti human CD271 from Biolegend, the immunogen comes from melanoma cells, which highly express CD271 on the cell surface molecule, this antibody has a slight migration compared with the negative control;
  • Figure 6B shows the negative control antibody of Rabbit anti human CD271 derived from Abcam, the immunogen comes from the 350-450 amino acid region in the human CD271 cell membrane, and compared with the positive control, there is no bias
  • Figure 6C shows the purified antibody of CD271T-2 ascites. Compared with the positive and negative controls, the degree of deviation is higher, indicating that the prepared anti-human CD271 monoclonal antibody has very high affinity and specificity, and the proportion of positive cells superior to the commercially available APC mouse anti human CD271 control antibody.
  • the term "antibody” refers to an immunoglobulin molecule generally composed of two pairs of polypeptide chains, each pair having one "light” (L) chain and one "heavy” (H) chain .
  • a heavy chain can be understood as a polypeptide chain with a larger molecular weight in an antibody
  • a light chain refers to a polypeptide chain with a smaller molecular weight in an antibody.
  • Light chains can be classified as kappa and lambda light chains.
  • Heavy chains can generally be classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.
  • variable and constant regions are linked by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids.
  • Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH).
  • the heavy chain constant region consists of 3 domains (CH1, CH2 and CH3).
  • Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL).
  • the light chain constant region consists of one domain, CL.
  • the constant regions of the antibodies mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.
  • VH and VL regions can also be subdivided into regions of high variability called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • Each VH and VL consists of 3 CDRs and 4 FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from amino terminus to carboxy terminus.
  • the assignment of amino acids to regions or domains follows the Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol.
  • the heavy chain may also contain more than 3 CDRs, eg, 6, 9, or 12.
  • the heavy chain may be a ScFv in which the C-terminus of the heavy chain of an IgG antibody is linked to another antibody, in which case the heavy chain contains 9 CDRs.
  • the term "antibody” is not limited by any particular method of producing an antibody. For example, it includes, in particular, recombinant antibodies, monoclonal antibodies and polyclonal antibodies.
  • Antibodies can be of different isotypes, eg, IgG (eg, IgGl, IgG2, IgG3, or IgG4 subtype), IgAl, IgA2, IgD, IgE, or IgM antibodies.
  • IgG eg, IgGl, IgG2, IgG3, or IgG4 subtype
  • IgAl IgA2, IgD, IgE, or IgM antibodies.
  • the experimental methods, detection methods and preparation methods disclosed in the present invention all adopt the conventional molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology and related fields in the technical field. conventional technology.
  • CD271-T2 used in the present invention refers to a cell line when used together with a hybridoma cell line; when used alone or together with a monoclonal antibody, antibody, etc., it refers to the monoclonal antibody or engineered antibody of the present invention.
  • amino acid sequence of the immunization antigen is shown in SEQ ID NO.17, specifically:
  • Antigen preparation for secondary immunity and tertiary immunity take a 1 mL sterile EP tube, dilute 100 ⁇ g of immunogen with PBS to 100 ⁇ L and put it in the EP tube; shake the incomplete Freund’s adjuvant well to mix the precipitated bifidobacteria evenly ; Take 100 ⁇ L of incomplete Freund’s adjuvant and add it to a 5 mL sterile EP tube, then add the diluted immunogen to make the volume ratio of antigen and adjuvant 1:1, and place the centrifuge tube in an ice box; prepare Good sonicator, put the centrifuge tube in the ice box to sonicate.
  • Ultrasonic conditions were total power 200W, total time 10min, working time 5s, and interval time 6s. Observe the emulsification of the solution in real time; inhale the emulsified antigen with a 1 ml disposable syringe, and place it in an ice box for later use.
  • the immunizing antigen was inhaled with a 1 ml syringe, divided into 2-3 points, each point of about 0.1 mL, and subcutaneously injected into the back (or abdomen) of the mice; blood was collected on the third day after the three immunizations for antibody titer ELISA detection, the specific results are shown in the figure 1. Immunization can be boosted when the titer reaches 1:100000.
  • Feeder cells can promote the growth and reproduction of single or a few scattered cells.
  • Cells Sp2/0-Ag14
  • medium DMEM+10% FBS
  • passaged 48 hours before fusion and the density should be about 80% of the bottom of the bottle covered after 48 hours.
  • mice Balb/c mice were killed by tail clipping and neck pulling, and immersed in 70% alcohol for 10 min.
  • each nude mouse was intraperitoneally injected with 0.5 mL of autoclaved pristane (paraffin oil).
  • mice Female Balb/c mice were injected intraperitoneally for 8 weeks.
  • ascites begins to appear. Ascites can be drawn when the animal is the size of a pregnant female. With a 5mL syringe, ascites was drawn from the periphery of the abdominal cavity, about 2-3mL per animal.
  • the extraction can be repeated at intervals of 1-3 days according to the situation of ascites.
  • mice ascites was centrifuged immediately after collection, and the supernatant was frozen at -80°C. Before purification, thaw at room temperature or 4 degrees, centrifuge to take the supernatant, add 1/10 volume of 1M Tris-HCl, 0.5M NaCl, pH 8.0 to adjust the pH value, filter at 0.22 ⁇ m to be the sample to be loaded on the column, and sample S;
  • the purified ascites antibody CD271T-2 was analyzed by SDS-PAGE protein electrophoresis, and the specific results are shown in Figure 2.
  • the protein in all collection tubes was collected, and the protein concentration was determined after ultrafiltration and centrifugation.
  • the CD271T-1 and CD271T-2 were identified by ELISA.
  • the substrate was CD271 full-length protein.
  • the primary antibody was mouse ascites purified antibody, and the secondary antibody was Goat Anti Mouse. IgG(H+L)-HRP, the specific results are shown in Figure 3.
  • the full-length antigen CD271 was immobilized with proA sensor, and 30, 50, 70, 90, 110, 130nM CD271T-1, CD271T-2 antibody affinity was detected. Cure for 5 minutes (cure height is about 1 nm), combine for 5 minutes, dissociate for 5 minutes (dissociation fitting for the first 1 minute). Regenerate for 5s, neutralize for 15s, and cycle three times. All antigen-antibody dilutions were PBS, regeneration buffer was pH 1.5 10 mM glycine buffer, equilibration eluent was 0.02% Tween PBS, and neutralization was 0.05% Tween PBS. The concentration of the solidified substance (ie, antigen) was 10 ⁇ g/mL. The curing height is around 1nm. The specific parameters are shown in Table 1, and the graph is shown in Figure 4.
  • RNA extraction using Trizol one-step method, take about 10 6 hybridoma cells CD271T-2, add Trizol, extract RNA according to Kangwei Century Ultrapure RNA Kit CW0581S kit, and identify RNA integrity by nucleic acid electrophoresis; hybridoma cells CD271T- 2 has a deposit number of CGMCC 21494. It is preserved in the China General Microorganism Culture Collection and Management Center, and the preservation address is No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is January 25, 2021. The classification is named as hybridoma cell line CD271T-2;
  • Reverse transcription into cDNA (20 ⁇ L): take 1 ⁇ L of Oligo dT Primer (50 ⁇ M), 1 ⁇ L of dNTP Mixture (10 nM), 5 ⁇ g of template RNA, add water to 10 ⁇ L, incubate at 65°C for 5 min, and quickly cool on ice; add the above denaturation reaction solution 10 ⁇ L, 5x primerscript II Buffer 4 ⁇ L, RNase Inhibitor (40U/ ⁇ L) 0.5 ⁇ L, PrimeScirpt II RTase (200U/ ⁇ L) 1 ⁇ L, add water to 20 ⁇ L, react at 42°C for 60min, and inactivate at 70°C for 15min;
  • PCR amplification of heavy and light chain variable region genes of anti-CD271 antibody PCR amplification of light and heavy chain variable region genes was performed in Suzhou Hongxun Biotechnology Co., Ltd. The PCR results are shown in Figure 5A&5B;
  • PMD19-T vector was purchased from takara, the heavy and light chain variable region gene PCR products were recovered, and after ligation with T vector, DH5 ⁇ was transformed, and positive clones were screened at 100 ⁇ g/mL ampicillin concentration, and sent for sequencing. Alignment with several conserved framework amino acids possessed by protein databases.
  • the obtained monoclonal antibody comprises a light chain and a heavy chain
  • the light chain comprises LCDR1, LCDR2 and LCDR3, the amino acid sequence of the LCDR1 is as shown in SEQ ID NO.1, and the amino acid sequence of the LCDR2 is as shown in SEQ ID NO.2,
  • the amino acid sequence of the LCDR3 is shown in SEQ ID NO.3;
  • the heavy chain includes HCDR1, HCDR2 and HCDR3, the amino acid sequence of the HCDR1 is shown in SEQ ID NO.4, and the amino acid sequence of the HCDR2 is shown in SEQ ID As shown in NO.5, the amino acid sequence of the HCDR3 is shown in SEQ ID NO.6.
  • the amino acid sequence of the light chain variable region is shown in SEQ ID NO.7.
  • the heavy chain variable region amino acid sequence is shown in SEQ ID NO.8.
  • the nucleotide sequence encoding the light chain complementarity determining region LCDR1 is shown in SEQ ID NO.9; the nucleotide sequence encoding the light chain complementary determining region LCDR2 is shown in SEQ ID NO.10; the nucleotide sequence encoding the light chain complementarity determining region LCDR3 is shown in SEQ ID NO.10.
  • nucleotide sequence encoding the heavy chain complementarity determining region HCDR3 is shown in SEQ ID NO.14.
  • the nucleotide sequence of the light chain variable region is shown in SEQ ID NO.15, and the nucleotide sequence of the heavy chain variable region is shown in SEQ ID NO.16.
  • Hu-MSC cells were collected by trypsin digestion and centrifugation, resuspended and washed with FACS (PBS+2% FBS), centrifuged at 1000 rpm for 3 min, and the supernatant was discarded; primary antibody was added according to Table 3, and incubated at room temperature for 30 min in the dark.
  • this antibody has a slight migration ( Figure 6A); derived from Abcam Rabbit anti human CD271 positive control antibody, the immunogen is derived from the 350-450 amino acid region in the human CD271 cell membrane, and there is no offset compared with the negative control (Fig. 6B); the ascites purified antibody of CD271T-2 was compared with the positive and negative pairs. Compared with the control, the degree of offset is higher, indicating that the prepared anti-human CD271 monoclonal antibody has very high affinity and specificity (Fig. 6C).

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Abstract

提供了一种分泌抗人CD271单克隆抗体的杂交瘤细胞株CD271T-2以及由该杂交瘤细胞株分泌的抗人CD271单克隆抗体，该杂交瘤细胞株的保藏号为CGMCC 21494。还提供了具有分别如SEQ ID NOs.1-6所示的LCDR1-3和HCDR1-3的抗人CD271工程抗体。还提供了该细胞株或抗体的用途。

Description

抗人CD271的单克隆抗体及用途 技术领域
本发明属于生物技术领域，具体涉及一种抗人CD271的单克隆抗体及用途。
背景技术
CD271是一种低亲和力的神经生长因子受体(LNGFR)，属于肿瘤坏死因子受体超家族成员，相对分子质量为75kD，又称为p75NTR，它包含3个区域：富含半胱氨酸的膜外区、跨膜区和155个氨基酸残基组成的胞内区。CD271不仅表达在神经系统，与神经细胞的发育、分化和存活密切相关，也是富集间充质干细胞比较合适的标志分子，与传统分选方法相比，具有特异性高、纯度高、集落形成能力强等优点。
间充质干细胞(mesenchymal stem cells,MSC)是一类具有自我更新和多向分化潜能的成体干细胞，在再生医学研究中具有广泛的应用前景。间充质干细胞进行的临床试验主要包括组织损伤修复，如骨、软骨、关节损伤的修复，心脏、肝脏、脊髓损伤和神经系统疾病的治疗。但MSC用于靶向组织修复一直受到干细胞在受损部位定植、增殖的限制。因此，在组织损伤部位或者神经系统疾病相关的治疗中，清除或富集移植物中特定的细胞成为当前移植领域中最为关键的问题。CD271、CD105、CD133等作为间充质干细胞的表面标记物，可以有效分离或者富集间充质干细胞，在体内或体外特定的诱导条件下，可分化为软骨、肌肉、肌腱等，达到组织修复的目的。另一方面，CD271也是多种肿瘤干细胞(CSC)的标志物之一。CD271作为MSC和CSC的表面标记分子，相对于CD44、CD105等分子具有对干细胞更好的特异性，其单克隆抗体不但可用于MSC的筛选和鉴定，也能用于抗肿瘤研究，具有重要的研究和应用价值。但目前仍缺乏对间充质干细胞及肿瘤干细胞等具有广泛的识别能力的抗CD271单克隆抗体。
发明内容
鉴于以上所述现有技术的缺点，本发明的目的在于提供一种抗人CD271的单克隆抗体及用途。
天然CD271的胞外结构包括4个富含半胱氨酸的CRD结构(CRD1-4)以及一个连接区域(stalk区)。在CD271表达后，CRD区域会被蛋白酶水解从而脱落，因此靶向CRD区的抗体与CD271阳性细胞的结合会受到影响，甚至在细胞代谢的某个阶段无法 检测到CD271的表达。而靠近细胞膜的stalk区无糖基化、磷酸化修饰或高级空间结构，结构稳定，识别此区域的抗体理论上具有较好的通用性。因此，为了获得能够稳定识别人CD271的单克隆抗体，我们选取stalk区作为靶点开发新型的抗CD271单克隆抗体，为该领域研究提供新的工具。
为了实现上述目的以及其他相关目的，本发明采用如下技术方案：
本发明第一方面提供一种抗分泌抗人CD271单克隆抗体的杂交瘤细胞株CD271T-2，所述杂交瘤细胞株的保藏号为CGMCC 21494。保藏于中国普通微生物菌种保藏管理中心，保藏地址为北京市朝阳区北辰西路1号院3号。保藏日期为2021年1月25日。分类命名为杂交瘤细胞株CD271T-2。
本发明第二方面提供一种抗人CD271单克隆抗体，抗人CD271单克隆抗体由CD271T-2杂交瘤细胞株分泌。
所述的抗人CD271单克隆抗体与人间充质干细胞有很好的结合亲和力。
可选的，所述述抗体是鼠源性单克隆抗体，属于IgM亚型。
本发明第三方面提供一种抗人CD271的工程抗体，所述抗人CD271的工程抗体包括轻链和重链，所述轻链包括LCDR1、LCDR2和LCDR3，所述LCDR1的氨基酸序列如SEQ ID NO.1所示，所述LCDR2的氨基酸序列如SEQ ID NO.2所示，所述LCDR3的氨基酸序列如SEQ ID NO.3所示；所述重链包括HCDR1、HCDR2和HCDR3，所述HCDR1的氨基酸序列如SEQ ID NO.4所示，所述HCDR2的氨基酸序列如SEQ ID NO.5所示，所述HCDR3的氨基酸序列如SEQ ID NO.6所示。
具体的，轻链互补决定区LCDR1，长度：10aa，氨基酸序列如SEQ ID NO.1所示，具体为：QSVDYDGDSY。
轻链互补决定区LCDR2，长度：3aa，氨基酸序列如SEQ ID NO.2所示，具体为：AAS。
轻链互补决定区LCDR3，长度：9aa，氨基酸序列如SEQ ID NO.3所示，具体为：QQSNEDPFT。
重链互补决定区HCDR1，长度：10aa，氨基酸序列如SEQ ID NO.4所示，具体为：GFSLSTSGMG。
重链互补决定区HCDR2，长度：7aa，氨基酸序列如SEQ ID NO.5所示，具体为：IWWDDDK。
重链互补决定区HCDR3，长度：13aa，氨基酸序列如SEQ ID NO.6所示，具体为：ARRDYGNYYAMDY。
进一步的，所述抗人CD271的工程抗体的轻链可变区氨基酸序列如SEQ ID NO.7所示；或为对SEQ ID NO.7进行一定的取代、添加和/或缺失一个或几个氨基酸获得具有同等功能的氨基酸序列。重链可变区氨基酸序列如SEQ ID NO.8所示，或为对SEQ ID NO.8进行一定的取代、添加和/或缺失一个或几个氨基酸获得具有同等功能的氨基酸序列。
具体的，SEQ ID NO.7所示序列，具体为：
Figure PCTCN2022074522-appb-000001
SEQ ID NO.8所示序列，具体为：
Figure PCTCN2022074522-appb-000002
可选的，所述工程抗体可以为单克隆抗体。
可选的，所述单克隆抗体是鼠源性的。
一种实施方式中，所述单克隆抗体亚类为IgM。
可选的，所述抗体由CD271T-2杂交瘤细胞株经反转录-聚合酶链式合成反应制备产生。
本发明第四方面提供一种多核苷酸，编码前述的抗人CD271抗体的重链可变区和/或轻链可变区或全长氨基酸。
编码前述的抗人CD271抗体的重链可变区和/或轻链可变区如SEQ ID NO.9-SEQ ID NO.14所示。
具体的，轻链互补决定区LCDR1，DNA，长度：30bp，核苷酸序列如SEQ ID NO.9所示，具体为：CAAAGTGTTGATTATGATGGTGATAGTTAT。
轻链互补决定区LCDR2，DNA，长度：9bp，核苷酸序列如SEQ ID NO.10所示，具体为：GCTGCATCC。
轻链互补决定区LCDR3，DNA，长度：27bp，核苷酸序列如SEQ ID NO.11所示，具体为：CAGCAAAGTAATGAGGATCCATTCACG。
重链互补决定区HCDR1，DNA，长度：30bp，核苷酸序列如SEQ ID NO.12所示，具体为：GGGTTTTCACTGAGCACTTCTGGTATGGGT。
重链互补决定区HCDR2，DNA，长度：21bp，核苷酸序列如SEQ ID NO.13所示，具体为：ATTTGGTGGGATGATGATAAG。
重链互补决定区HCDR3，DNA，长度：39bp，核苷酸序列如SEQ ID NO.14所示，具体为：GCTCGAAGGGACTATGGTAACTACTATGCTATGGACTAC。
进一步的，编码前述抗人CD271抗体的轻链可变区的核苷酸序列如SEQ ID NO.15所示，编码前述的抗人CD271抗体的重链可变区的核苷酸序列如SEQ ID NO.16所示。
具体的，SEQ ID NO.15所示序列：
Figure PCTCN2022074522-appb-000003
SEQ ID NO.16所示序列：
Figure PCTCN2022074522-appb-000004
本发明第五方面提供一种多核苷酸，所述多核苷酸编码前述的抗CD271抗体的Fab段的轻链可变区和重链可变区的重组序列，能够获得分子量更小的单链抗体。
本发明第五方面提供一种构建体，含有前述的多核苷酸。
所述构建体可以由所述分离的多核苷酸插入到表达载体的多克隆位点构建而成。
本发明中的表达载体通常指本领域熟知的各种市售表达载体，例如可以是细菌质粒、噬菌体、酵母质粒、植物细胞病毒、哺乳动物细胞病毒如腺病毒、逆转录病毒或其他载体。具体可采用的表达载体包括但不限于：pET系列表达载体、pGEX系列表达载体、pcDNA系列表达载体等。
本发明的宿主细胞，所述宿主细胞含有前述的构建体或基因组中整合有外源的前述的 多核苷酸。任何适用于表达载体进行表达的细胞都可以作为宿主细胞，例如，所述宿主细胞可以是原核细胞，如细菌细胞等；或是低等真核细胞，如酵母细胞等；或是高等真核细胞，如哺乳动物细胞等。具体的，可以为例如，酵母、昆虫、植物等的细胞。优选的，所述宿主细胞为真核细胞，可采用不会产生抗体的哺乳动物宿主细胞系，具体可采用的细胞系包括但不限于：中国仓鼠的卵巢细胞(CHO)、幼仓鼠的肾脏细胞(BHK,ATCC CCL 10)、幼鼠的塞尔托利细胞(Sertoli cells)、猴的肾脏细胞(COS细胞)、通过SV40(COS-7,ATCC CRL 165 1)转化的猴的肾脏CVI细胞、人的胚肾细胞(HEK-293)、猴肾脏细胞(CVI，ATCC CCL-70)、非洲绿猴的肾脏细胞(VERO-76,ATCC CRL-1587)、人的子宫颈癌细胞(HELA,ATCC CCL-2)等。
合适的表达所述抗体的条件对于本领域技术人员来说应该是已知的，本领域技术人员可根据经验选择适用的培养基，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。
本发明第六方面提供一种宿主细胞，所述宿主细胞含有前述的构建体或基因组中整合有外源的前述的多核苷酸。
本发明第七方面提供前述的细胞株，或前述的抗人CD271单克隆抗体，或前述的抗人CD271的工程抗体，或前述的多核苷酸，或前述的构建体，或前述的抗体的宿主细胞的用途，选自以下任一种：
a)表达CD271的细胞，所述细胞包括但不限于间充质干细胞、肿瘤干细胞、神经系统细胞，其体外的检测、鉴定、分离、富集、靶向；
b)介导间充质干细胞靶向修复组织；
c)干细胞、肿瘤干细胞、神经系统细胞的检测及示踪；
d)制备神经系统靶向药物；
e)制备肿瘤治疗药物；
f)与其他化学小分子、生物大分子、放射性分子、纳米材料等偶联，用于上述a)-e) 的各类用途。
与现有技术相比，本发明具有如下有益效果：
本发明的由杂交瘤细胞株分泌的单克隆抗体具有效价高、特异性好、与天然抗原亲和力强的优势。抗人CD271的工程抗体由CD271T-2杂交瘤经反转录-聚合酶链式合成反应制备产生，该抗体能特异性结合人CD271分子，在体外对于人间充质干细胞有一定的亲和力。CD271在间充质干细胞、肿瘤干细胞和神经系统相关疾病细胞表面均有表达，是介导间充质干细胞或者靶向神经系统相关细胞及肿瘤的潜在靶点。因此抗人CD271单克隆抗体是一种具有巨大潜力的医疗用抗体。
本发明的抗人CD271抗体具有高特异性和亲和力，可以将其与磁性纳米材料偶联，制备免疫磁珠来分选间充质干细胞，也可将其与其他靶点联用设计双特异性抗体，或与其他化学分子、放射性分子、多肽或蛋白、纳米材料等联合使用，用于某些组织修复疾病及神经系统或者肿瘤相关疾病的治疗。
本发明菌株保藏信息如下：
细胞株名称：杂交瘤细胞株CD271T-2；
保藏号为：CGMCC 21494；
保藏日期：2021年1月25日；
保藏单位名称：中国普通微生物菌种保藏管理中心；
保藏单位简称：CGMCC；
保藏单位地址：北京市朝阳区北辰西路1号院3号。
附图说明
图1是ELISA检测抗人CD271免疫后小鼠血清效价检测图：包被抗原为全长CD271蛋白抗原，抗原浓度为4μg/mL，上样血清从1:2000稀释到1:256000稀释，1号、2号、3号、4号、5号小鼠血清效价都在1：128000以上，5只小鼠对抗原的反应性都较高；
图2是小鼠CD271T-2腹水纯化抗体SDS-PAGE检测图：在非还原情况下，腹水抗体条带较为单一，还原情况下，在26kDa和53kDa处分别是抗体的轻链和重链；
图3是小鼠腹水纯化抗体ELISA鉴定：包被抗原为全长CD271蛋白抗原，抗原浓度为2μg/mL，每孔100μL,上样抗体浓度梯度为0.625μg/mL、1.25μg/mL、2.5μg/mL、5μg/mL、10μg/mL、20μg/mL、40μg/mL，每孔100μL，结果表明CD271T-2抗体能够识别结合人CD271抗原；
图4是ForteBio验证纯化后抗体亲和力图谱；
图5是本发明所述抗人CD271抗体的重轻链可变区基因克隆PCR结果：图5A为CD271T-2杂交瘤的抗体重轻链可变区的基因克隆PCR结果，图5B为CD271T-2杂交瘤的抗体重轻链可变区嵌合ScFv基因克隆PCR结果；
图6是抗人CD271单克隆抗体检测间充质干细胞表面CD271的表达情况：图6A为来源于Biolegend的APC Goat anti human CD271的阳性对照抗体，免疫原来自黑色素瘤细胞，其细胞表面高表达CD271分子，此抗体与阴性对照相比有略微迁移；图6B为来源于Abcam的Rabbit anti human CD271阴性对照抗体，免疫原来自人CD271细胞膜内350-450氨基酸区域，与阳性对照相比，无任何偏移；图6C为CD271T-2腹水纯化抗体，与阳性和阴性对照相比，其偏移程度较高，说明制备的抗人CD271单克隆抗体具有非常高的亲和力和特异性，且从阳性细胞比例上优于市售的APC mouse anti human CD271对照抗体。
具体实施方式
如本文中所使用的，术语“抗体”是指，是指通常由两对多肽链(每对具有一条“轻”(L)链和一条“重”(H)链)组成的免疫球蛋白分子。从一般意义上，重链可以理解为抗体中分子量较大的多肽链，轻链是指抗体中分子量较小的多肽链。轻链可分类为κ和λ轻链。重链通常可分类为μ、δ、γ、α或ε，并且分别将抗体的同种型定义为IgM、IgD、IgG、IgA和IgE。在轻链和重链内，可变区和恒定区通过大约12或更多个氨基酸的“J”区连接，重链还包含大约3个或更多个氨基酸的“D”区。各重链由重链可变区(VH)和重链恒定区(CH)组成。重链恒定区由3个结构域(CH1、CH2和CH3)组成。各轻链由轻链可变区(VL)和轻链恒定区(CL)组成。轻链恒定区由一个结构域CL组成。抗体的恒定区可介导免疫球蛋白与宿 主组织或因子，包括免疫系统的各种细胞(例如，效应细胞)和经典补体系统的第一组分(C1q)的结合。VH和VL区还可被细分为具有高变性的区域(称为互补决定区(CDR))，其间散布有较保守的称为构架区(FR)的区域。各VH和VL由按下列顺序：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4从氨基末端至羧基末端排列的3个CDR和4个FR组成。各重链/轻链对的可变区(VH和VL)分别形成抗体结合部位。氨基酸至各区域或结构域的分配遵循Kabat Sequences of Proteins of ImmunologicalInterest(National Institutes of Health,Bethesda,Md.(1987and 1991))，或Chothia&Lesk(1987)J.Mol.Biol.196:901-917；Chothia等人(1989)Nature 342:878-883的定义。特别地，重链还可以包含3个以上CDR，例如6、9、或12个。例如在本发明的双功能抗体中，重链可以是IgG抗体的重链的C端连接另一个抗体的ScFv，这种情况下重链含有9个CDR。术语“抗体”不受任何特定的产生抗体的方法限制。例如，其包括，特别地，重组抗体、单克隆抗体和多克隆抗体。抗体可以是不同同种型的抗体，例如，IgG(例如，IgG1，IgG2，IgG3或IgG4亚型)，IgA1，IgA2，IgD，IgE或IgM抗体。
为了使本发明的发明目的、技术方案和有益技术效果更加清晰，以下结合实施例对本发明进一步详细说明，熟悉此技术的人士可由本说明书所揭露的内容容易地了解本申请发明的其他优点及功效。
在进一步描述本发明具体实施方式之前，应理解，本发明的保护范围不局限于下述特定的具体实施方案；还应当理解，本发明实施例中使用的术语是为了描述特定的具体实施方案，而不是为了限制本发明的保护范围。下列实施例中未注明具体条件的试验方法，通常按照常规条件，或者按照各制造商所建议的条件。
当实施例给出数值范围时，应理解，除非本发明另有说明，每个数值范围的两个端点以及两个端点之间任何一个数值均可选用。除非另外定义，本发明中使用的所有技术和科学术语与本技术领域技术人员通常理解的意义相同。除实施例中使用的具体方法、设备、材料外，根据本技术领域的技术人员对现有技术的掌握及本发明的记载，还可以使用与本发明实施例中所述的方法、设备、材料相似或等同的现有技术的任何方法、设备和材料来实现本发明。
除非另外说明，本发明中所公开的实验方法、检测方法、制备方法均采用本技术领域常规的分子生物学、生物化学、染色质结构和分析、分析化学、细胞培养、重组DNA技术及相关领域的常规技术。
本发明所用的“CD271-T2”当跟杂交瘤细胞株一起使用时时表示细胞株；当单独使用或者跟单克隆抗体、抗体等一起时表示为本发明所述的单克隆抗体或工程抗体。
【实施例1】抗人CD271杂交瘤细胞株及单克隆抗体的制备
1.小鼠免疫
免疫抗原的氨基酸序列如SEQ ID NO.17所示，具体为：
Figure PCTCN2022074522-appb-000005
1)初免抗原制备：取1mL无菌EP管，将免疫原100μg用PBS稀释至100μL放于EP管中；充分摇匀弗氏完全佐剂，使沉淀的双歧杆菌均匀混合；取100μL弗氏完全佐剂加入5mL的无菌EP管，再加入稀释好的免疫原，使抗原的体积与佐剂的体积比为1：1，将离心管置于冰盒中；准备好超声破碎仪，将离心管置于冰盒中超声。超声条件是总功率200W，总时间10min，工作时间5秒，间隔时间6秒。实时观察溶液乳化的情况；用1毫升一次性注射器吸入乳化好的抗原，置于冰盒中备用。
2)二免、三免抗原制备：取1mL无菌EP管，将免疫原100μg用PBS稀释至100μL放于EP管中；充分摇匀弗氏不完全佐剂，使沉淀的双歧杆菌均匀混合；取100μL弗氏不完全佐剂加入5mL的无菌EP管，再加入稀释好的免疫原，使抗原的体积与佐剂的体积比为1：1，将离心管置于冰盒中；准备好超声破碎仪，将离心管置于冰盒中超声。超声条件是总功率200W，总时间10min，工作时间5s，间隔时间6s。实时观察溶液乳化的情况；用1毫升一次性注射器吸入乳化好的抗原，置于冰盒中备用。
3)加强免疫抗原制备：取1mL无菌EP管，将免疫原200μg用PBS稀释至400μL，用1毫升一次性注射器吸入，置于冰盒备用。
4)小鼠免疫方法
用1毫升注射器吸入免疫抗原，分2-3点，每点0.1mL左右，给小鼠进行背部(或腹部)皮下注射；三免后第三天采血进行抗体滴度ELISA检测，具体结果见图1，滴度达到1:100000时即可加强免疫。
2.饲养细胞的准备
饲养细胞可促进单个或少数分散细胞的生长繁殖。
1)准备好灭菌的手术器具和玻璃平皿，96孔板，5mL一次性注射器，300mL 70％的酒精，Balb/c小鼠若干，无血清DMEM培养液，灭菌的PBS。
2)拉颈处死Balb/c小鼠，浸入70％酒精10min。
3)拿出小鼠沥去多余的酒精，置于灭菌的玻璃平皿上。
4)剪开并分离小鼠腹部的皮肤(勿剪破肌肉层)。用5mL注射器吸取5mL DMEM无 血清培养液，注入小鼠腹部，轻轻按摩并摇动小鼠身体，使巨噬细胞充分混入培养液。用注射器小心吸回培养液，放入15mL离心管。
5)按上述方法重复1-2次，以尽量多的获得巨噬细胞。
6)1500rpm，离心10min，弃上清。用培养液悬浮备，1只成年Balb/c小鼠可取约10 6个腹腔细胞，可铺3-4块96孔板。
3.PEG介导大鼠脾脏B细胞和小鼠骨髓瘤细胞融合
1)细胞：Sp2/0-Ag14，培养基：DMEM+10％FBS，融合前48小时传代，密度视48小时后覆盖瓶底80％左右为宜。
2)小鼠剪尾拉颈处死Balb/c小鼠，浸入70％酒精10min。
3)剪开左侧上腹部直至小鼠左背部，分离出脾脏，除去脾脏上的结缔组织，放入干净平皿中，加少量DMEM基础培养基(无血清)。
4)将小鼠脾脏剪成4-5小段，用5mL注射器内塞顶部研磨，边磨边加入少量DMEM，将磨出的脾脏细胞冲入平皿。
5)将平皿中的细胞全部移入15mL离心管，加入适量的DMEM基础培养基，混匀离心，200g，5min。
6)用PBS洗涤二遍，离心，200g，5min。
7)收集所有的Sp2/0-Ag14细胞，并用PBS洗涤一遍待用并计数。
8)将10 8个脾脏细胞和10 7个骨髓瘤细胞混合在50mL离心管中(骨髓瘤细胞数可视收获细胞再提高)，加入30-40mL无血清的DMEM，充分混匀，1500rpm离心3min，去上清。
9)轻敲离心管的底部，使沉淀物充分打散。
10)将细胞放入37℃水浴，按下列方法缓慢滴入PEG。
11)用滴管沿管壁缓慢滴入1mL PEG溶液，持续时间1min，边加边轻轻振荡，100g，离心2min；
12)用同样的方法滴入4.5mL DMEM基础培养基(预热、无血清)，持续时间3min，边加边轻轻振荡；
13)用同样的方法滴入5mL DMEM基础培养基(预热、无血清)，持续时间2min，加完后轻轻振荡30s；
14)慢慢加入45mL DMEM基础培养基(预热、无血清)，
15)100g离心5min，去上清，轻敲底部。
16)加入含HAT的DMEM完全培养基(含20％FBS)，轻轻重悬，用台盼蓝进行细胞计数，100μL/孔，种入5块96孔板。
17)37℃，5％CO 2培养箱培养，培养期间不要晃动培养板。
18)融合后11-13天进行ELISA鉴定，并根据细胞生长情况进行测定，在测定之前的2-3天内不要换液，使上清中抗体的量充分蓄积。
4.有限稀释筛选单克隆
1)取出抗体阳性孔细胞，用HT培养液制成细胞悬液。并取样进行台盼兰染色，计数。
2)取320个细胞分散在6.4mL的HT培养液中，充分混匀后用排枪加入96孔板的第1-4列，每孔100μL，使每孔5个细胞；剩下的细胞液再加入3.2mL的HT培养液，充分混匀后，100μL每孔加入5-8列，使每孔2.5个细胞；取剩下的细胞液1mL，再加入2mL培养液，充分混匀后，100μL每孔加入9-12列，使每孔0.625个细胞。
3)将96孔板放入CO 2培养箱，37℃培养。
4)第五天，显微镜下观察细胞的生长情况，记录细胞生长的孔。
5)克隆大量繁殖后，布满孔底的1/3～1/2时，测培养液抗体。
6)在每孔0.625细胞的孔中选取Elisa阳性最高、生长状态良好的孔中的细胞，做下一轮有限稀释；同时选取相同情况的另一孔转移到含有饲养细胞的24孔板中的一孔扩增后冻存。
7)三轮有限稀释后将经过筛选并符合建株条件的细胞扩增培养冻存。
5.杂交瘤细胞产生小鼠腹水的制备方法
1)细胞注射7-10天之前，每只裸鼠腹腔注射0.5mL高压灭菌的降植烷(石蜡油)。
2)复苏建株细胞株培养传代，上清测ELISA，确定为阳性时，离心收集细胞，台盼蓝染色技术；按每只Balb/c 5×10 5个细胞，重悬在0.2mL PBS中。
3)腹腔注射8周雌性Balb/c小鼠。
4)1-2星期左右，腹水开始出现。当动物的体型像怀孕的雌鼠时，可开始抽取腹水。用5mL注射器从腹腔的外周抽取腹水，每只约2-3mL左右。
5)将腹水3000rpm(1500g)离心10分钟，上清分装后-70℃保存。
6)保持裸鼠健康的情况下，可根据腹水产生的情况，间隔1-3天反复抽取。
【实施例2】小鼠腹水纯化鉴定及ForteBio验证纯化抗体亲和力
1.小鼠腹水Mab Select Sure TM纯化
1)小鼠腹水在采集后立刻离心，上清冻存于-80℃。纯化前室温或4度解冻，离心取上清，加入1/10体积1M Tris-HCl，0.5M NaCl，pH 8.0调pH值，0.22μm过滤即为待上柱样品，取样S；
2)柱子用10CV的1M Tris-HCl，0.5M NaCl,pH 8.0平衡；
3)上样，收集所有流穿，取样FL；
4)Mab Select Sure TM用10CV的100mM Tris-HCl,50mM NaCl,pH 8.0洗涤；
5)Mab Select Sure TM用10CV的10mM Tris-HCl,5mM NaCl,pH 8.0洗涤；
6)收集管中预先加入0.1CV的1M Tris-HCl pH 9.0，洗脱时每次加入0.5CV的100mM NaCt/HCt pH3.0，并立即混合；洗脱Elute 1，2，3…管，分半取样制备样品。
2.纯化抗体SDS-PAGE及ELISA鉴定
1)SDS-PAGE鉴定
对纯化得到的腹水抗体CD271T-2进行SDS-PAGE蛋白电泳分析，具体结果见图2。
2)ELISA鉴定
收集所有收集管的蛋白，超滤离心后测定蛋白浓度，对CD271T-1，CD271T-2进行ELISA鉴定，底物为CD271全长蛋白，一抗为小鼠腹水纯化抗体，二抗为Goat Anti Mouse IgG(H+L)-HRP，具体结果见图3。
3.ForteBio验证纯化后抗体亲和力
用proA传感器固化全长抗原CD271，检测30，50，70，90，110，130nM CD271T-1，CD271T-2抗体亲和力。固化5分钟(固化高度1nm左右)结合5分钟，解离5分钟(解离拟合最前面1分钟)。再生5s，中和15s，循环三次。所有抗原抗体的稀释液为PBS，再生缓冲液为pH1.5 10mM甘氨酸缓冲液，平衡洗脱液为0.02％Tween PBS，中和为0.05％Tween PBS。固化物(即抗原)的浓度为10μg/mL。固化高度在1nm左右。具体参数见表1，曲线图见图4。
表1
  KD(M) Kon(1/Ms) Kdis(1/s) Full X 2 Full R 2
CD271T-2 3.37×10 -9 6.69×10 4 2.25×10 -4 4.4542 0.98420
【实施例3】抗人CD271的重轻链可变区基因克隆及嵌合抗体2的构建
1.RNA提取：采用Trizol一步法，取杂交瘤细胞CD271T-2约10 6个，加入Trizol，按照康为世纪Ultrapure RNA Kit CW0581S试剂盒提取RNA，核酸电泳鉴定RNA完整性；杂交瘤细胞CD271T-2的保藏号为CGMCC 21494。保藏于中国普通微生物菌种保藏管理中心，保藏地址为北京市朝阳区北辰西路1号院3号。保藏日期为2021年1月25日。分类命名为杂交瘤细胞株CD271T-2；
2.逆转录为cDNA(20μL)：取Oligo dT Primer(50μM)1μL，dNTP Mixture(10nM)1μL，模板RNA 5μg，加水至10μL，65℃保温5min，冰上迅速冷却；加入上述变性后反应液10μL，5x primerscript II Buffer 4μL，RNase Inhibitor(40U/μL)0.5μL，PrimeScirpt II RTase(200U/μL)1μL，加水至20μL，42℃反应60min，70℃，15min失活；
3.PCR扩增抗CD271抗体的重轻链可变区基因：轻、重链可变区基因PCR扩增于苏州泓迅生物技术公司操作，PCR结果见图5A&5B；
4.测序载体的构建：PMD19-T载体购自takara，将重轻链可变区基因pcr产物回收，与T载体连接后，进行DH5α转化，以100μg/mL氨苄浓度筛选阳性克隆，送测序，与蛋白数据库所具有的若干保守的框架氨基酸比对。所得单克隆抗体包括轻链和重链，轻链包括LCDR1、LCDR2和LCDR3，所述LCDR1的氨基酸序列如SEQ ID NO.1所示，所述LCDR2的氨基酸序列如SEQ ID NO.2所示，所述LCDR3的氨基酸序列如SEQ ID NO.3所示；所述重链包括HCDR1、HCDR2和HCDR3，所述HCDR1的氨基酸序列如SEQ ID NO.4所示，所述HCDR2的氨基酸序列如SEQ ID NO.5所示，所述HCDR3的氨基酸序列如SEQ ID NO.6所示。轻链可变区氨基酸序列如SEQ ID NO.7所示。重链可变区氨基酸序列如SEQ ID NO.8所示。编码轻链互补决定区LCDR1的核苷酸序列如SEQ ID NO.9所示；编码轻链互补决定区LCDR2的核苷酸序列如SEQ ID NO.10所示；编码轻链互补决定区LCDR3的核苷酸序列如SEQ ID NO.11所示；编码重链互补决定区HCDR1的核苷酸序列如SEQ ID NO.12所示；编码重链互补决定区HCDR2的核苷酸序列如SEQ ID NO.13所示；编码重链互补决定区HCDR3的核苷酸序列如SEQ ID NO.14所示。轻链可变区的核苷酸序列如SEQ ID NO.15所示，重链可变区的核苷酸序列如SEQ ID NO.16所示。
【实施例4】抗人CD271腹水抗体与Hu-MSC流式亲和表达鉴定
取适当生长良好的Hu-MSC细胞胰酶消化离心收集，用FACS(PBS+2％FBS)重悬洗涤，1000rpm，3min离心，弃上清；按表3加入一抗，室温避光孵育30min，用FACS (PBS+2％FBS)重悬洗涤，离心1000rpm，3min，重复3次，弃上清，按表1加入二抗，室温避光30min，用FACS(PBS+2％FBS)重悬洗涤，离心1000rpm，3min，重复3次，加入300μL FACS重悬细胞，流式细胞仪检测，具体结果见图6。图中表明，来源于Biolegend的APC mouse anti human CD271的阳性抗体，免疫原来自黑色素瘤细胞，其细胞表面高表达CD271分子，此抗体与阴性对照相比有略微迁移(图6A)；来源于Abcam的Rabbit anti human CD271阳性对照抗体，免疫原来自人CD271细胞膜内350-450氨基酸区域，与阴性对照相比，也无任何偏移(图6B)；CD271T-2的腹水纯化抗体与阳性和阴性对照相比，其偏移程度较高，说明制备的抗人CD271单克隆抗体具有非常高的亲和力和特异性(图6C)。
表2
Figure PCTCN2022074522-appb-000006
以上所述，仅为本发明的较佳实施例，并非对本发明任何形式上和实质上的限制，应当指出，对于本技术领域的普通技术人员，在不脱离本发明方法的前提下，还将可以做出若干改进和补充，这些改进和补充也应视为本发明的保护范围。凡熟悉本专业的技术人员，在不脱离本发明的精神和范围的情况下，当可利用以上所揭示的技术内容而做出的些许更动、修饰与演变的等同变化，均为本发明的等效实施例；同时，凡依据本发明的实质技术对上述实施例所作的任何等同变化的更动、修饰与演变，均仍属于本发明的技术方案的范围内。

Claims (11)

  1. 一种分泌抗人CD271单克隆抗体的杂交瘤细胞株CD271T-2，所述杂交瘤细胞株的保藏号为CGMCC 21494。
  2. 一种抗人CD271单克隆抗体，其特征在于，所述抗人CD271的单克隆抗体由权利要求1所述的CD271T-2杂交瘤细胞株分泌。
  3. 一种抗人CD271的工程抗体，其特征在于，所述抗人CD271的工程抗体包括轻链和重链，所述轻链互补决定区包括LCDR1、LCDR2和LCDR3，所述LCDR1的氨基酸序列如SEQ ID NO.1所示，所述LCDR2的氨基酸序列如SEQ ID NO.2所示，所述LCDR3的氨基酸序列如SEQ ID NO.3所示；所述重链互补决定区包括HCDR1、HCDR2和HCDR3，所述HCDR1的氨基酸序列如SEQ ID NO.4所示，所述HCDR2的氨基酸序列如SEQ ID NO.5所示，所述HCDR3的氨基酸序列如SEQ ID NO.6所示。
  4. 如权利要求3所述的抗人CD271的工程抗体，其特征在于，包括以下特征中的任一项或多项：(1)所述抗人CD271的工程抗体的轻链可变区氨基酸序列如SEQ ID NO.7所示；或为对SEQ ID NO.7进行一定的取代、添加和/或缺失一个或几个氨基酸获得具有同等功能的氨基酸序列；(2)重链可变区氨基酸序列如SEQ ID NO.8所示，或为对SEQ ID NO.8进行一定的取代、添加和/或缺失一个或几个氨基酸获得具有同等功能的氨基酸序列。
  5. 如权利要求3-4任一所述的抗人CD271的工程抗体，其特征在于，所述抗体由CD271T-2杂交瘤细胞株经反转录-聚合酶链式合成反应制备产生。
  6. 一种多核苷酸，编码如权利要求3-5任一所述的抗人CD271抗体的重链可变区和/或轻链可变区或全长氨基酸。
  7. 如权利要求6所述的多核苷酸，其特征在于，还包括以下特征中的任一项或多项：(1)编码轻链互补决定区LCDR1的核苷酸序列如SEQ ID NO.9所示；编码轻链互补决定区LCDR2的核苷酸序列如SEQ ID NO.10所示；编码轻链互补决定区LCDR3的核苷酸序列如SEQ ID NO.11所示；(2)编码重链互补决定区HCDR1的核苷酸序列如SEQ ID NO.12所示；编码重链互补决定区HCDR2的核苷酸序列如SEQ ID NO.13所示；编码重链互补决定区HCDR3的核苷酸序列如SEQ ID NO.14所示。
  8. 一种多核苷酸，其特征在于，所述多核苷酸编码如权利要求3-5任一所述的抗CD271抗体的Fab段的轻链可变区和重链可变区的重组序列，能够获得分子量更小的单链抗体。
  9. 一种构建体，含有如权利要求6-8任一所述的多核苷酸。
  10. 一种宿主细胞，所述宿主细胞含有如权利要求9所述的构建体或基因组中整合有外源的如权利要求6-8任一所述的多核苷酸。
  11. 如权利要求1所述的细胞株，或权利要求2所述的抗人CD271单克隆抗体，或权利要求3-5中任一项所述的抗人CD271的工程抗体，或权利要求6-8中任一项所述的多核苷酸，或权利要求9所述的构建体，或权利要求10所述的抗体的宿主细胞的用途，选自以下任一种：
    a)表达CD271的细胞，所述细胞包括但不限于间充质干细胞、肿瘤干细胞、神经系统细胞，其体外的检测、鉴定、分离、富集、靶向；
    b)介导间充质干细胞靶向修复组织；
    c)干细胞、肿瘤干细胞、神经系统细胞的检测及示踪；
    d)制备神经系统靶向药物；
    e)制备肿瘤治疗药物；
    f)与其他化学小分子、生物大分子、放射性分子、纳米材料等偶联，用于上述a)-e)的各类用途。
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