WO2019184015A1 - 一种核酸分子及其在人源化抗体中的应用 - Google Patents

一种核酸分子及其在人源化抗体中的应用 Download PDF

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WO2019184015A1
WO2019184015A1 PCT/CN2018/083163 CN2018083163W WO2019184015A1 WO 2019184015 A1 WO2019184015 A1 WO 2019184015A1 CN 2018083163 W CN2018083163 W CN 2018083163W WO 2019184015 A1 WO2019184015 A1 WO 2019184015A1
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nucleic acid
acid molecule
human
sequence
molecule according
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PCT/CN2018/083163
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English (en)
French (fr)
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刘作华
葛良鹏
丁玉春
邹贤刚
杨松全
游小燕
刘雪芹
吴梦
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重庆金迈博生物科技有限公司
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Priority to JP2020564297A priority Critical patent/JP2021512651A/ja
Priority to AU2018416757A priority patent/AU2018416757A1/en
Priority to EP18912455.5A priority patent/EP3760721A4/en
Priority to US17/041,460 priority patent/US20210009670A1/en
Publication of WO2019184015A1 publication Critical patent/WO2019184015A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • 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
    • 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
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/303—Liver or Pancreas
    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A01K2217/052—Animals comprising random inserted nucleic acids (transgenic) inducing gain of function
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/15—Animals comprising multiple alterations of the genome, by transgenesis or homologous recombination, e.g. obtained by cross-breeding
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/01—Animal expressing industrially exogenous proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
    • C07K2317/14—Specific host cells or culture conditions, e.g. components, pH or temperature
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • C12N2015/8518—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic expressing industrially exogenous proteins, e.g. for pharmaceutical use, human insulin, blood factors, immunoglobulins, pseudoparticles

Definitions

  • the invention belongs to the field of biotechnology, and mainly relates to a nucleic acid molecule and an application thereof.
  • Fully human antibodies also known as fully human monoclonal antibodies, Human (Monoclonal) Antibody, are a class of biotechnological products currently used in the treatment of tumors, autoimmune diseases, infectious diseases and transplant rejection.
  • 27 of the 72 monoclonal antibodies that have been marketed in Europe and the United States are human antibodies.
  • transgenic antibody animals can only produce human/mouse chimeric antibodies, that is, the VDJ of the heavy chain of the antibody is derived from human, and the C-region is derived from the animal, and further humanization is required in the drug development process. This process of humanization alters the affinity of the antibody and reduces the drug-forming properties of the antibody. Therefore, direct access to fully human antibodies from animals is of great significance.
  • CN105441455A adopts chimeric IgM sequence of human/host animal, which can realize the preparation of fully humanized antibody, but still has the following disadvantages: (1) The number of early B-cells in transgenic animals is less than that in normal mice; (2) The specificity and diversity of antibodies need to be improved.
  • the object of the present invention is to solve the above problems in the prior art and to provide a nucleic acid molecule which can express a fully human antibody in a transgenic animal, which reduces the artificial modification at the later stage and at the same time improves the drug-forming property of the antibody.
  • the object of the invention is achieved by the following measures:
  • a nucleic acid molecule comprising an immunoglobulin gene or a fragment thereof, comprising: an IgM gene (IgHC ⁇ ) and an IgM switch element (S ⁇ ), wherein the S ⁇ and IgHC ⁇ are all host animal sequences.
  • IgHC ⁇ immunoglobulin gene
  • S ⁇ IgM switch element
  • the IgHC ⁇ includes the CH1, CH2, CH3, CH4 exons and the sequence between them and the TM1, TM2, and polyA signal sequences.
  • the present invention ensures B-cell development and antibody maturation in host animals.
  • the above nucleic acid molecule also includes a 5'-end enhancer (5'-Enhancer) of the IgH heavy chain of the host animal. Its structure with S ⁇ and IgHC ⁇ is shown in Figure 1-1, and the construction process is shown in Figure 1.
  • 5'-Enhancer 5'-end enhancer
  • the above nucleic acid molecule also includes an IgG gene (i.e., Ig ⁇ ).
  • the IgG gene can be a host animal/human IgG chimeric element or a full human IgG sequence.
  • the above chimeric elements include a host animal Ig ⁇ conversion element (S ⁇ ) and TM1 and TM2, polyA and the like, and human CH1, Hinge, CH2, CH3 exons and sequences therebetween, etc., forming a host/human chimeric Ig ⁇ . Expression regulatory elements.
  • the structure of the Ig ⁇ chimeric element is as shown in FIG. 2-1, and the structure is as shown in FIG. 2 .
  • the above-described fully human Ig gamma sequence may further comprise a human transforming element (S ⁇ ) sequence and a sequence of CH1, Hinge, CH2 and CH3 exons of human Ig ⁇ and a sequence between them, and a termination signal (PolyA) and TM1, TM2 of human Ig ⁇ . Equal sequence.
  • the above Ig ⁇ sequence may include various subtypes of human Ig ⁇ and a conversion element (S ⁇ ) between each subtype of Ig ⁇ .
  • human Ig gamma subtypes include Ig gamma 3, Ig gamma 1, Ig gamma 2, and/or Ig gamma 4; mouse Ig gamma isoforms include Ig gamma 3, Ig gamma 1, Ig gamma 2a, and/or Ig gamma 2b, and the like.
  • the above nucleic acid molecules also include an IgH 3'-local expression region (LCR).
  • the LCR can be derived from a host animal sequence ( Figure 6) or a human sequence ( Figure 5).
  • the above nucleic acid molecules include human IgH heavy chain V-region sequences or fragments, human IgH D-region sequences or fragments, and human IgH J-region sequences or fragments.
  • the V-zone, D-zone, and J-zone of the heavy chain are all derived from humans as in Figures 5 and 6.
  • the above nucleic acid molecule contains multiple (or all) human immunoglobulin (IgH) V-regions, D-region and J-region sequences, linked 5'-enhancement of mouse immunoglobulin (IgH) 5'-Enhancer, the switch region (S ⁇ ) sequence and the IgM CH1, CH2, CH3, CH4 and PolyA, TM1 and TM2 sequences, and then link the human Ig ⁇ conversion element (S ⁇ ) sequence, followed by The CH1, Hinge, CH2 and CH3 sequences of human Ig ⁇ are linked, as well as sequences of human PolyA, TM1 and TM2, and finally the 3'-position expression regulation (LCR) sequence of human heavy chain IgH.
  • Transgenic mice express antibodies such as murine IgM and human IgG.
  • the transgenic vector contains multiple (or all) human immunoglobulin (IgH) V-regions, D-region and J-region sequences, linked to the 5'-enhancer of mouse immunoglobulin (IgH) (5' -Enhancer), the switch region (S ⁇ ) sequence and the IgM CH1, CH2, CH3, CH4 and PolyA, TM1 and TM2 sequences, and then link the mouse Ig ⁇ switch region (S ⁇ ) sequence, followed by The CH1, Hinge, CH2 and CH3 sequences of human Ig ⁇ are linked, as well as the sequences of PolyA, TM1 and TM2 of the mouse, and finally the 3'-position expression regulation (LCR) sequence of the mouse heavy chain IgH is linked.
  • Transgenic mice express antibodies such as murine IgM and human IgG.
  • a vector comprising the nucleic acid molecule described above.
  • a prokaryote containing the above nucleic acid molecule or vector A cell comprising the above nucleic acid molecule or vector, including any of the cells of a transgenic animal derived from such a nucleic acid molecule, and including but not limited to lymphocytes derived from a transgenic animal, hybridomas, antibody-expressing cells, and the like.
  • a human antibody which is prepared by rearranging and encoding any of the above nucleic acid molecules; including any human antibody derived from the above nucleic acid molecule or nucleic acid molecule transgenic animal, including but not limited to antibody protein, antibody DNA, cDNA sequence And further modified, engineered antibodies, and the like.
  • a transgenic animal comprising the above nucleic acid molecule or vector, cell or antibody.
  • the animals may be mammals such as pigs, cows, horses, rats, rats, rabbits, chickens and sheep.
  • the nucleic acid molecule, vector, cell or animal is used for encoding DNA, cDNA, mRNA, expressing amino acid sequence, protein, vector, and culturing hybridoma, cell strain, transgenic animal.
  • a method of preparing a transgenic animal using the above nucleic acid molecule or vector comprises the following steps:
  • breeding heterozygous, homozygous transgenic animals including mating with animals whose host animal's own immunoglobulin genes are deleted.
  • the above host animal refers to a transgenic animal to which the above nucleic acid molecule can be applied, such as a mammal such as pig, cow, horse, mouse, rat, rabbit, chicken and sheep;
  • the above vector includes yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC) ), plasmids and DNA fragments, etc.
  • the above vector introduction methods include electroporation, viral infection, liposome-mediated and microinjection, and the like.
  • SEQ ID. NO1, SEQ ID. NO2, SEQ ID. NO3 as a specific embodiment, the sequence of the above nucleic acid molecule is as shown, but this is not intended to limit the scope of the present invention, and those skilled in the art can According to its non-essential improvements and adjustments to the nucleotide sequence, such as deletion, addition, substitution, etc. of nucleotides.
  • the transgenic animal produces various therapeutic whole human antibodies under the stimulation conditions of different antigens.
  • the invention directly obtains the whole human antibody, reduces the artificial modification in the later stage and improves the drug-forming property of the antibody.
  • the 5'-end enhancer and the IgM sequence of the IgH of the transgenic host animal are used to ensure the normal development of the early B-cell of the transgenic animal; and the transgenic host animal itself is also used.
  • the Ig ⁇ switch region (S ⁇ ) sequence and the Ig ⁇ termination signal (PolyA) and TM1, TM2 sequences of the transgenic host animal themselves control the expression of human Ig ⁇ , which facilitates DNA recombination, mutation and BCR (B- Signaling of cellular receptors causes human Ig gamma to mature under the stimulation of antigen.
  • the IgH transgenic originals of transgenic animals are human V-region, D-region, J-region and Ig ⁇ as well as human IgK and IgL sequences, expressing human IgG; all transgenic animals prepared with this vector are expressed.
  • the IgG antibody is a fully human antibody, which reduces the late artificial modification and improves the drug-forming properties of the antibody.
  • the advantages of the present invention are as follows: (1) The early development and maturation of transgenic B-cells and the number of B-cells are identical to those of normal mice, which is beneficial to the differentiation of B-cells; (2) increase the specificity of antibody production. And diversity; (3) improve the efficiency of screening therapeutic antibodies.
  • Figure 1 Schematic diagram of 5'-Enhancer and IgM expression elements: Replacement of human 5'-enhancer and IgM with human 5'-enhancer and IgM expression elements and human IgD expression elements: Homologous arms are used for homologous recombination (homologous arms of the same color indicate the same DNA sequence for homologous recombination), pGK-Puro is a bacterial and mammalian screening gene, and Loxp is 34 specific DNA sequences. Not1 and AsiS1 are cleavage sites (red shows the mouse sequence and green shows the mouse sequence).
  • FIG. 2 Schematic diagram of the construction and construction of host animal/human chimeric IgG expression elements: homology arms are used for homologous recombination, and human C ⁇ is substituted for CH1 of mouse C1 by Counter-selection recombineering (Counter-selection recombineering) , CH2 and CH3 sequences, the expression regulatory elements of murine/human chimeric Ig ⁇ 3 and Ig ⁇ 1 were established, and Not1 is a restriction site. (Red shows the human sequence and green shows the mouse sequence).
  • Figure 3 Schematic diagram of the construction of an IgG C-region of a nucleic acid molecule sequence: mouse 5'-enhancer (5'-En), mouse S ⁇ and mouse IgM, and human Ig ⁇ 3 and Ig ⁇ 1 and 3'-LCR Sequences and links.
  • the two screening genes, Puro and Zeo can be screened in bacteria and cells, and finally the Puro or Neo sequences are removed using CRE or Flpo expression plasmids or proteins, leaving only one Lox (34 bp) and Frt (34 bp) sequences.
  • This nucleic acid molecule is then linked to the human IgH V-region, D-region and J-region to form a transgenic vector (red shows the human sequence, green shows the mouse sequence).
  • Figure 4 Schematic diagram of the construction of an IgG C-region of a nucleic acid molecule sequence: mouse 5'-enhancer (5'-En), mouse S ⁇ and murine IgM, and mouse/human chimeric Ig ⁇ 3 and Ig ⁇ 1 expression regulation Component and mouse 3'-LCR sequences and links.
  • the Puro and Zeo screening genes can be screened in bacteria and cells, and finally the Puro or Neo sequences are removed using CRE or Flpo expression plasmids or proteins, leaving only one Lox (34 bp) and Frt (34 bp) sequences.
  • This nucleic acid molecule links to the V-region, D-region and J-region of human to form a transgenic vector (red shows the human sequence, green shows the human sequence).
  • transgenic vectors include: human IgH V-region, D-region and J-region sequences, mouse IgH5'-enhancer (Enhancer) and mouse IgM conversion Element (S ⁇ ) sequence and IgM all exon sequence and regulatory signal; human Ig ⁇ 3 conversion element (S ⁇ 3) sequence and human Ig ⁇ 3 CH1, Hinge, CH2 and CH3 sequences and human Ig ⁇ 3 PloyA, TM1 and TM2, etc.
  • Figure 6 Components of one of the IgH heavy chain transgenic nucleic acid molecules: human IgH V-region, D-region and J-region sequence, mouse IgH 5'-enhancer (5'-Enhancer) and mouse IgM Transformation element (S ⁇ ) sequence and IgM total exon sequence and regulatory signal; mouse Ig ⁇ 3 conversion element (S ⁇ 3) sequence and human Ig ⁇ 3 CH1, Hinge, CH2 and CH3 sequences and mouse Ig ⁇ 3 PloyA, TM1 And sequences such as TM2; the Ig ⁇ 1 conversion element (S ⁇ 1) sequence of mouse and the CH1, Hinge, CH2 and CH4 sequences of human Ig ⁇ 1 and the PloyA, TM1 and TM2 sequences of mouse Ig ⁇ 1, and then link the mouse IgH 3 '- positional expression regulatory sequence (LCR) (red is human sequence, green is mouse sequence).
  • LCR mouse IgH 3 '- positional expression regulatory sequence
  • Figure 1-6 is a key gene structure diagram of the transgene.
  • FIG. 7 J-region gene targeting of mouse immunoglobulin heavy chain gene IgH: The J-region of mouse IgH consists of J1, J2, J3 and J4, and the entire J-region sequence was excised by gene targeting techniques. Homologous mice without the J-region sequence are unable to produce murine Ig (including IgM and IgG, etc.).
  • Figure 8 Results of human IgHV2-26 PCR assay in transgenic mice (HGa).
  • Figure 9 Elisa detection results of transgenic mouse (HGa) serum.
  • Figure 10 Results of human IgHV2-26 PCR assay in transgenic mice (HGb).
  • Figure 11 Elisa detection results of transgenic mouse (HGb) serum.
  • Figure 12 Human IgG content in serum of transgenic antibody mice (under different dilution conditions).
  • FIG 13 Elisa after OVA immunized mice (OD 450) the detection result.
  • Figure 14 Statistical results of hybridoma IgH V-region sequence analysis (number and location of mutations) after OVA immunization of mice.
  • Figure 15 Elisa analysis results of supernatants of some hybridoma cell lines.
  • Figure 16 Specific binding of antibodies to HepG2 cells.
  • Figure 17 Statistical table of antigen-specific antibodies produced by transgenic mice.
  • FIG. 18 Photograph of spleen appearance after immunization of transgenic mice [A, transgenic mice (HGa), B, transgenic mice (HGb), C, normal mice)].
  • the engineered human immunoglobulin gene heavy chain vector is transferred into a mouse, and the transgenic mouse containing the human immunoglobulin gene is immunized to obtain a fully human antibody, and the specific steps are as follows:
  • the 5'-enhancer sequence of mouse IgM, all expression regulatory sequences of IgM, and homology arms and screening genes were obtained by PCR and gene synthesis (Fig. 1).
  • Human Ig gamma 3 and Ig gamma 1 and 3'-LCR sequences as well as homology arms and selection genes were obtained (Fig. 3).
  • all the DNA fragments are homologously recombined to obtain a new DNA nucleic acid molecule, and then the above modified DNA fragment is transferred into a YAC or BAC vector containing a human immunoglobulin heavy chain gene (Ig) to construct an immunoglobulin weight.
  • the gene cluster of the strand gene is shown in Figure 5 (green is the mouse sequence and red is the human sequence).
  • the V region, the D region, the J region, the mouse IgH 5'-enhancer, the mouse IgM, the human Ig gamma 3, the human Ig gamma 1 and the human LCR are included in the human immunoglobulin heavy chain gene.
  • the 5'-enhancer sequence of mouse IgM, all expression regulatory sequences of IgM, and homology arms and screening genes were obtained by PCR and gene synthesis (Fig. 1). Substituting CH1, Hinge, CH2 and CH3 of human Ig ⁇ 3 for the sequences of CH1, Hinge, CH2 and CH3 of mouse Ig ⁇ 3 by homologous recombination and reverse sieving recombination techniques; replacing CH1, Hinge, CH2 and CH3 of human Ig ⁇ 1 The sequences of CH1, Hinge, CH2 and CH3 of murine Ig ⁇ 1. The 3'-LCR sequence of the mouse as well as the homology arm and the selection gene were obtained (Fig. 3).
  • the gene cluster of the immunoglobulin heavy chain gene is constructed by homologous recombination technology, and then the above modified DNA fragment is sequentially transferred into a YAC or BAC vector containing the human immunoglobulin heavy chain gene (Ig). Shown (green is the mouse sequence, red is the human sequence). Including V-region, D-region, J-region, human IgH 5'-enhancer, mouse IgM, human/mouse Ig ⁇ 3, human/mouse Ig ⁇ 1 and mouse 3'-LCR.
  • the human immunoglobulin heavy chain gene constructed in 1) of step 1 (Fig. 5) was transferred into mice using conventional conventional transgenic techniques.
  • a transgenic mouse (HGa) transfected with a human immunoglobulin heavy chain vector was obtained by PCR detection and ELISA detection by double standard screening.
  • PCR identification primers used were:
  • the size of the PCR product is: 433 bp
  • Elisa detects transgenic mice: Serum from healthy adults and healthy mice was used as a control. See Figure 9. Description: Serum was tested for human IgG Elisa at a 1:100 dilution. Transgenic mice were positive for human IgG and were compared to human serum, normal mouse serum and blanks. The antibodies identified by ELISA used were: Goat Anti Human IgG Fc (abcam, ab97221), Goat Anti-Human IgG Fc (HRP) (ab97225; abcam).
  • the human immunoglobulin heavy chain gene constructed in 2) of step 1 was transferred into mice using conventional conventional transgenic techniques.
  • Transgenic mice (HGb) transfected with human immunoglobulin heavy chain vector were obtained by double-standard screening by PCR detection and ELISA detection.
  • PCR identification primers used were:
  • the size of the PCR product is: 433 bp
  • Elisa detects transgenic mice: Serum from healthy adults and healthy mice was used as a control. See Figure 11. Description: Serum was tested for human IgG Elisa at a 1:100 dilution. Transgenic mice were positive for human IgG and were compared to human serum, normal mouse serum and blanks. The antibodies identified by ELISA used were: Goat Anti Human IgG Fc (abcam, ab97221), Goat Anti-Human IgG Fc (HRP) (ab97225; abcam).
  • Immunoglobulin heavy chain knockout mice were constructed using gene targeting technology. The IgH J-region of the mouse immunoglobulin heavy chain gene was selected as a knockout site (knockout site and gene knockout effect is shown in Figure 6), and immunoglobulin heavy chain knockout mice were obtained. Immunoglobulin heavy chain knockout mice obtained by double standard screening were detected by PCR detection and ELISA.
  • PCR identification primers used were:
  • PCR product The size of the PCR product after the J-region gene targeting was 732 bp, and the size of the normal J-region PCR product was 2422 bp.
  • HGa or HGb transgenic mice of the second step were hybridized with the mice obtained in steps 2), 3), 4) and 5), respectively, and PCR and ELISA were used to obtain high-expression human IgG antibody mice (HGa). + HK + HL + mK -- mH -- and HGb + HK + HL + mK -- mH - ) Five-gene mice. Transgenic mice that do not express (or underexpress) murine immunoglobulin.
  • Transgenic mice have lower IgM and IgG levels than normal human IgM and IgG levels because transgenic mice are housed in a clean, sterile environment.
  • the human antibody transgenic mice are immunized to obtain B-cells, and then combined with hybridoma technology and cell culture technology to obtain therapeutic human antibodies.
  • Humanized antibody transgenic mice obtained by OVA immunization Eight-week-old mice were selected for immunization.
  • OVA Dilute OVA (Sigma A7641) antigen with PBS to a final concentration of 2 mg/ml, add 20 ug of CpG (ODN1826, tlrl-1826, Invivogen), add appropriate amount of aluminum hydroxide (vac-alu-50, Invivogen), and make aluminum hydroxide. The concentration is 1%.
  • the antigens are prepared with CFA 0.75mL adjuvant (Sigma F5881) by 1:1 mixed with MIXPAC TM syringe emulsified, each mouse was injected dose 200ul (0.2mg), were injected subcutaneously immunized .
  • the second dose was performed, and the antigen was diluted with PBS to a final concentration of 1.0 mg/ml, 10 ug of CpG was added, and an appropriate amount of aluminum hydroxide was added to make the aluminum hydroxide concentration 1%.
  • the 1 2 prepared in IFA adjuvant and antigen 0.75mL 1:1 by mixing, with a MIXPAC TM syringe emulsified, injected dose per mouse 200ul (0.1mg), injected intraperitoneally immunized.
  • the third immunization was performed, and the antigen was diluted with PBS to a final concentration of 1.0 mg/ml, 10 ug of CpG was added, and an appropriate amount of aluminum hydroxide was added to make the aluminum hydroxide concentration 1%.
  • the IV was diluted, the antigen was diluted with PBS to a final concentration of 1.0 mg/ml, 10 ug of CpG was added, and an appropriate amount of aluminum hydroxide was added to make the aluminum hydroxide concentration 1%.
  • mice in which the Elisa results were satisfactory were boosted, and then B-cells of the spleen were obtained for hybridoma fusion, culture, and screening.
  • mice were taken for blood ELISA, and the pre-immune mice were used as controls to detect the content of mouse IgG and human IgG in the serum of the immunized mice.
  • the results are as follows:
  • Detection of human IgG in mouse serum 96-well plates were embedded with antigen OVA, and the specific antibody content in the diluted immune serum was measured by hIgG-HRP (Millipore, AP113P). Kunming white mice (control) were tested for mouse IgG.
  • Elisa (OD 450) the detection result of OVA immunized mice (mice transfected gene HGa + HK + HL + mK - mH - and HGb + HK + HL + mK - mH -) shown in Figure 13.
  • HGa transgenic mice are HGa + HK + HL + mK -- mH -- five gene mice
  • HGb transgenic mice are HGb + HK + HL + mK -- mH -- five gene mice
  • Kunming white mice are normal control mice .
  • the transgenic mice (HGb + HK + HL + mK - mH -- ) were immunized with GPC3, and the spleen cells of the mice were collected and fused with Sp2/0 cells to prepare a monoclonal antibody, that is, a fully human antibody.
  • the hybridoma test was carried out after immunization of the transgenic animal antigen, and then screened in a semi-solid medium, and the hybridoma clone was picked and cultured in a 96-well plate, and the culture supernatant was subjected to Elisa-specific antibody analysis.
  • Figure 16 shows that both 4-10F-4G and 6-7A-2E are positive for Elisa detection after GPC3 polypeptide immunization, but when combined with HepG2 (human cancer cells, expressing GPC3), 4-10F-4G does not.
  • HepG2 human cancer cells, expressing GPC3
  • 6-7A-2E specifically binds to HepG2 cells, demonstrating that transgenic antibody mice can be antibody specific for anti-GPC3 antibodies (6-7A-2E).
  • Fig. 17 is a statistical table of antigen-specific antibodies produced by transgenic mice, and is a statistical table showing the affinity of antibodies for hybridomas obtained by immunizing and fusing HGa and HGb transgenic mice with OVA, GPC3 polypeptide and GPC3 protein. Both transgenic mice have high affinity human antibodies.
  • the size of the spleen of the mouse obtained by the present invention is the same as that of the normal mouse, and is larger than that of the transgenic mouse of CN105441455A;
  • the amount of IgM in the transgenic mouse of the present invention is higher than that in the serum of the transgenic mouse of CN105441455A;
  • the GM amount of the transgenic mouse of the present invention is high, and the IgG content in the serum of the transgenic mouse of CN105441455A is higher in the same clean and sterile environment;
  • the transgenic IgH heavy chain CDR mutation of the present invention is more: under the same antigen immunization condition, the hybridoma IgG V-sequence mutation obtained by the transgenic mouse of the present invention has more mutations than the hybridoma IgGV-sequence obtained by the transgenic mouse of CN105441455A. .
  • the number of B-cells in the spleen after immunization in normal mice 1.5 ⁇ 10 8 , 1.8 ⁇ 10 8 , 1.9 ⁇ 10 8 .
  • Figure 18 is a photograph of the appearance of spleen after immunization of transgenic mice:
  • Figure 18-A shows the appearance of spleen in transgenic mice HGa + HK + HL + mK - mH - and
  • Figure 18-B shows transgenic mice HGb + HK + HL + mK -- mH -- Photograph of the appearance of the spleen after immunization.
  • Figure 18-C shows the spleen after immunization in normal mice.

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Abstract

本发明提供了一种核酸分子，包括了免疫球蛋白基因或其片段，其特征在于：包括了IgM基因(IgHCμ)和IgM转换元件(switch region，Sμ)，所述Sμ和IgHCμ全部为宿主动物序列。本发明可以直接获得全人抗体，减少后期的人工改造并提高了抗体的成药性。本发明转基因的B-细胞的早期发育和成熟以及B-细胞的数量与正常小鼠完全一致，有利于B-细胞的分化；增加生产抗体的特异性和多样性；提高筛选治疗性抗体的效率。

Description

一种核酸分子及其在人源化抗体中的应用 技术领域
本发明属于生物技术领域，主要是一种核酸分子及其应用。
背景技术
全人抗体，也又称全人源单克隆抗体，Human(Monoclonal)Antibody，是当今应用于治疗肿瘤、自身免疫性疾病、感染性疾病和移植排斥反应等最重要疾病的一类生物技术产品。在全球治疗性单抗中，已在欧美上市的72个单抗中，有27个是人抗体。人抗体的来源有两个：第一：建立人的B细胞噬菌体抗体文库，并结合噬菌体展示技术提高亲和力而获得；第二：利用转基因动物，将动物本身的免疫球蛋白基因失活，然后转入经过改造的部分(或全部)人的免疫球蛋白基因，利用动物的免疫机制，生产人(或部分人源)的抗体。随着转基因技术和B-细胞发育机制的研究清楚，人源化转基因动物的制备更合理化，转基因动物的B-细胞发育正常，DNA重组和突变，抗体成熟和特异性都很高。
现有的大部分转基因抗体动物都只能产生人/鼠嵌合抗体，即抗体的重链的VDJ来源于人，而C-区来源于动物，在药物研发过程中，需要进一步人源化。这个人源化的过程改变了抗体的亲和力和降低抗体的成药性。因此，从动物中直接获得全人抗体的意义重大。
CN105441455A采用了人/宿主动物的嵌合的IgM序列，可以实现全人源化抗体的制备，但仍存在以下不足：(1)转基因动物的早期B-细胞数量比正常小鼠少；(2)抗体的特异性和多样性有待提高。
发明内容
本发明的目的在于解决现有技术存在的上述问题，提供一种核酸分子，其可在转基因动物中表达全人抗体，减少后期的人工改造，同时提高了抗体的成药性。
本发明的目的是通过以下措施实现的：
一种核酸分子，包括了免疫球蛋白基因或其片段，其特征在于：包括了IgM基因(IgHCμ)和IgM转换元件(switch region，Sμ)，所述Sμ和IgHCμ全部为宿主动物序列。
进一步的，所述IgHCμ，包括CH1、CH2、CH3、CH4外显子以及之间的序列和TM1、TM2、polyA信号序列。
本发明保证宿主动物的B-细胞发育和抗体成熟。
上述核酸分子，还包括了宿主动物IgH重链的5′-端的增强子(5’-Enhancer)。其与Sμ和IgHCμ的结构如图1-1所示，构建过程如图1所示。
上述核酸分子还包括IgG基因(即Igγ)。IgG基因可以为宿主动物/人IgG嵌合元件或全人IgG序列。上述嵌合元件包括宿主动物Igγ的转换元件(Sγ)和TM1和TM2、polyA等序列，以及人的CH1、Hinge、CH2、CH3外显子以及之间的序列等，形成宿主/人嵌合Igγ表达调控元件。具体地，所述Igγ嵌合元件的结构如图2-1所示，构建如图2所示。
上述全人Igγ序列还可以包括人的转换元件(Sγ)序列和人的Igγ的CH1、Hinge、CH2和CH3外显子以及之间的序列，和人Igγ的终止信号(PolyA)和TM1，TM2等序列。
上述Igγ序列，可以包括人Igγ的各种亚型以及Igγ各亚型间的转换元件(Sγ)。比如，人Igγ亚型包括Igγ3、Igγ1、Igγ2和/或Igγ4；小鼠Igγ亚型包括Igγ3、Igγ1、Igγ2a和/或Igγ2b等。
上述核酸分子还包括IgH 3’-位置表达调控序列(Local Control Region，LCR)。LCR可以来源于宿主动物序列(如图6)或人的序列(如图5)。
上述核酸分子包括了人IgH重链V-区序列或片段，人IgH D-区序列或片段，和人IgH J-区序列或片段。重链的V-区、D-区和J-区都来源于人如图5和图6。
例如：具体地，上述核酸分子含有多个(或全部)人的免疫球蛋白(IgH)V-区，D-区和J-区序列，链接小鼠免疫球蛋白(IgH)的5’-增强子(5’-Enhancer)，转换元件(switch region，Sμ)序列和IgM的CH1、CH2，CH3，CH4以及PolyA，TM1和TM2等序列，再链接人的Igγ的转换元件(Sγ)序列，其次链接人Igγ的CH1、Hinge，CH2和CH3序列，以及人的PolyA，TM1和TM2等序列，最后链接人重链IgH的3’-位置表达调控(LCR)序列。如图5所示。转基因小鼠表达鼠的IgM和人的IgG等抗体。
或者，转基因载体含有多个(或全部)人的免疫球蛋白(IgH)V-区，D-区和J-区序列，链接小鼠免疫球蛋白(IgH)的5’-增强子(5’-Enhancer)，转换元件(switch region，Sμ)序列和IgM的CH1、CH2，CH3，CH4以及PolyA，TM1和TM2等序列，再链接小鼠的Igγ的转换元件(switch region，Sγ)序列，其次链接人Igγ的CH1、Hinge，CH2和CH3序列，以及小鼠的PolyA，TM1和TM2等序列，最后链接小鼠重链 IgH的3’-位置表达调控(LCR)序列。如图6所示。转基因小鼠表达鼠的IgM和人的IgG等抗体。
一种载体，包含上述的核酸分子。
一种含有上述核酸分子或载体的原核生物。一种含有上述核酸分子或载体的细胞，包括任何一种来源于这些核酸分子的转基因动物的细胞，还包括但不限于来源于转基因动物的淋巴细胞、杂交瘤、抗体表达细胞等。
一种人源抗体，由上述任一核酸分子重排、编码制得；包括任何一种来源于上述核酸分子或核酸分子转基因动物的人源抗体，包括但不限于抗体蛋白质、抗体DNA、cDNA序列和经过进一步修饰，改造的抗体等。
一种含有上述核酸分子或载体、细胞或抗体的转基因动物。动物可以是猪、牛、马、鼠、大鼠、兔、鸡和羊等哺乳动物。
上述核酸分子、载体、细胞或动物在编码DNA、cDNA、mRNA，表达氨基酸序列、蛋白质、载体，培养杂交瘤、细胞株、转基因动物中的应用。
尤其是，将上述转基因载体转入动物基因组内获得的转基因动物，或者与宿主动物自身免疫球蛋白基因缺失的动物进行杂交的后代，宿主动物本身的重链和轻链被失活，只表达人抗体蛋白的基因工程动物。利用抗原免疫转入经过上述改造后的人免疫球蛋白基因的动物，获得的全人抗体。
采用上述核酸分子或载体制备转基因动物的方法，包括以下一些步骤：
(1)所述核酸分子的获得；
(2)将所述核酸分子构建载体；
(3)向宿主动物细胞(包括干细胞，诱导干细胞和体细胞)或胚胎导入上述载体；
(4)将含有上述载体的细胞植入宿主动物的胚胎内(嵌合体的制备)或体细胞克隆；
(5)繁殖杂合，纯合的转基因动物(包括与宿主动物自身免疫球蛋白基因缺失的动物交配)。
上述宿主动物是指上述核酸分子可以应用的转基因动物，比如猪、牛、马、鼠、大鼠、兔、鸡和羊等哺乳动物；上述载体包括酵母人工染色体(YAC)，细菌人工染色体(BAC)，质粒和DNA片段等，上述载体导入方法包括：电穿孔、病毒感染、脂质体介导和显微注射等。
SEQ ID.NO1、SEQ ID.NO2、SEQ ID.NO3作为某(几)次具体的实施方式，上述核酸分子的序列如所示，但这并不作为对本发明范围的限制，本领域技术人员可以根据其对核苷酸序列作出一些非本质的改进和调整，如对核苷酸的删除、增加、替换等。
有益效果
1.本转基因动物在不同抗原的刺激条件下，生产各种治疗性全人抗体。
2.本发明直接获得全人抗体，减少后期的人工改造和提高了抗体的成药性。
3.本发明转基因动物的IgH的转基因载体中，采用转基因宿主动物本身IgH的5’-端增强子和IgM序列，保证了转基因动物的早期B-细胞的正常发育；同时还采用转基因宿主动物本身的Igγ的转换元件(switch region，Sγ)序列和转基因宿主动物本身Igγ的终止信号(PolyA)和TM1，TM2序列来控制人的Igγ的表达，这样有利于DNA的重组、突变和BCR(B-细胞受体)的信号传递，使得人的Igγ在抗原的刺激下成熟。转基因动物的IgH转基因原件中是人的V-区、D-区、J-区和Igγ以及人的IgK和IgL序列，表达的是全人的IgG；所有用这种载体制备的转基因动物表达的IgG抗体为全人抗体，减少后期的人工改造和提高了抗体的成药性。
4.本发明优点在于：(1)转基因的B-细胞的早期发育和成熟以及B-细胞的数量与正常小鼠完全一致，有利于B-细胞的分化；(2)增加生产抗体的特异性和多样性；(3)提高筛选治疗性抗体的效率。
附图说明：
图1：5’-增强子(5’-Enhancer)和IgM表达元件构建示意图：用小鼠5’-增强子和IgM表达元件取代人的5’-增强子和IgM以及人的IgD表达元件：同源臂用于同源重组(相同颜色的同源臂字表示DNA序列相同，进行同源重组)，pGK-Puro是细菌和哺乳动物的筛选基因，Loxp是34个特异DNA序列。Not1和AsiS1是酶切位点(红色显示小鼠序列，绿色显示小鼠的序列)。
图2：宿主动物/人嵌合IgG表达元件组成结构和构建示意图：同源臂用于同源重组，人的Cγ是用反筛重组(Counter-selection recombineering)取代小鼠的Cγ的CH1、Hinge、CH2和CH3序列，建立鼠/人嵌合Igγ3和Igγ1的表达调控元件，Not1是酶切位点。(红色显示人的序列，绿色显示小鼠序列)。
图3：一种核酸分子序列IgG C-区的构建示意图：小鼠的5’-增强子(5’-En)、小鼠的Sμ和小鼠IgM以及人的Igγ3和Igγ1以及3’-LCR序列和链接。Puro和Zeo两 个筛选基因可以在细菌和细胞中进行筛选，最后用CRE或Flpo表达质粒或蛋白质去除Puro或Neo序列，只留下一个Lox(34bp)和Frt(34bp)序列。这个核酸分子再链接到人的IgH V-区、D-区和J-区后组成一个转基因载体(红色显示人的序列，绿色显示鼠的序列)。
图4：一种核酸分子序列IgG C-区的构建示意图：小鼠的5’-增强子(5’-En)、小鼠的Sμ和鼠IgM以及鼠/人嵌合的Igγ3和Igγ1表达调控元件和鼠的3’-LCR序列和链接。Puro和Zeo两个筛选基因可以在细菌和细胞中进行筛选，最后用CRE或Flpo表达质粒或蛋白质去除Puro或Neo序列，只留下一个Lox(34bp)和Frt(34bp)序列。这个核酸分子链接在人的V-区、D-区和J-区后组成一个转基因载体(红色显示人的序列，绿色显示人的序列)。
图5：其中一个IgH重链转基因核酸分子的组成元件：转基因载体包括：人的IgH V-区，D-区和J-区序列，小鼠IgH5’-增强子(Enhancer)和小鼠IgM转换元件(Sμ)序列和IgM全部外显子序列和调控信号；人的Igγ3的转换元件(Sγ3)序列和人的Igγ3的CH1、Hinge、CH2和CH3序列以及人的Igγ3的PloyA、TM1和TM2等序列；人的Igγ1的转换元件(Sγ1)序列和人的Igγ1的CH1、Hinge、CH2和CH3序列以及人的Igγ1的PloyA、TM1和TM2等序列，然后链接人的IgH 3’-位置表达调控序列(LCR)(红色为人的序列，绿色为小鼠序列)。
图6：其中一个的IgH重链转基因核酸分子的组成元件：人的IgH V-区，D-区和J-区序列，小鼠IgH 5’-增强子(5’-Enhancer)和小鼠IgM转换元件(Sμ)序列和IgM全部外显子序列和调控信号；小鼠的Igγ3的转换元件(Sγ3)序列和人的Igγ3的CH1、Hinge、CH2和CH3序列以及小鼠的Igγ3的PloyA、TM1和TM2等序列；小鼠的Igγ1的转换元件(Sγ1)序列和人的Igγ1的CH1、Hinge、CH2和CH4序列以及小鼠的Igγ1的PloyA、TM1和TM2等序列，然后链接小鼠的IgH 3’-位置表达调控序列(LCR)(红色为人的序列，绿色为小鼠序列)。
图1-6是转基因的关键基因结构图。
图7：小鼠免疫球蛋白重链基因IgH的J-区基因打靶：小鼠IgH的J-区由J1、J2、J3和J4组成，用基因打靶技术，将整个J-区的序列切除。没有J-区序列的同源小鼠就不能产生鼠源的Ig(包括IgM和IgG等)。
图8：转基因小鼠(HGa)的人IgHV2-26 PCR检测结果。
图9：转基因小鼠(HGa)血清的Elisa检测结果。
图10：转基因小鼠(HGb)的人IgHV2-26 PCR检测结果。
图11：转基因小鼠(HGb)血清的Elisa检测结果。
图12：转基因抗体小鼠血清中的人IgG含量(在不同稀释条件下)。
图13：OVA免疫小鼠后的Elisa(OD 450)检测结果。
图14：OVA免疫小鼠后的杂交瘤IgH V-区序列分析(突变数量和位置)统计结果。
图15：部分杂交瘤细胞株上清液的Elisa分析结果。
图16：抗体与HepG2细胞特异性结合情况。
图17：转基因小鼠产生的抗原特异性抗体的统计表。
图18：转基因小鼠免疫后脾脏外观照片【A、转基因小鼠(HGa)，B、转基因小鼠(HGb)，C、正常小鼠)】。
具体实施方式
下面是具体实施例证对本发明进行具体描述，在此指出以下实施例证只用于对本发明进行进一步说明，不能理解为对本发明保护范围的限制，本领域的技术是由熟练人员可以根据上述发明内容对本发明作出一些非本质的改进和调整。
实施例
将改造的人免疫球蛋白基因重链载体转入小鼠体内，再免疫含有人免疫球蛋白基因的转基因小鼠获得全人抗体，具体步骤如下：
1.免疫球蛋白基因的载体的构建
1)免疫球蛋白重链基因的构建(如图5)
首先用PCR和基因合成获得小鼠IgM的5’-增强子序列，IgM的所有表达调控序列以及同源臂和筛选基因(如图1)。再获得人的Igγ3和Igγ1以及3’-LCR序列以及同源臂和筛选基因(如图3)。然后将所有的DNA片段同源重组获得新的DNA核酸分子，然后将上述改造好的DNA片段转入含人免疫球蛋白重链基因(Ig)的YAC或BAC载体等上，构建免疫球蛋白重链基因的基因簇如图5所示(绿色为小鼠序列，红色为人的序列)。包括了依次为人免疫球蛋白重链基因的V区、D区、J区、小鼠的IgH 5’-增强子、小鼠的IgM、人Igγ3，人Igγ1和人的LCR。
2)免疫球蛋白重链基因的改造(如图6)
首先用PCR和基因合成获得小鼠IgM的5’-增强子序列，IgM的所有表达调控序列 以及同源臂和筛选基因(如图1)。通过同源重组和反筛重组技术，将人的Igγ3的CH1、Hinge、CH2和CH3取代鼠的Igγ3的CH1、Hinge、CH2和CH3的序列；将人的Igγ1的CH1、Hinge、CH2和CH3取代鼠的Igγ1的CH1、Hinge、CH2和CH3的序列。再获得鼠的3’-LCR序列以及同源臂和筛选基因(如图3)。通过同源重组技术，然后将依次将上述改造好的DNA片段转入含人免疫球蛋白重链基因(Ig)的YAC或BAC载体上，构建免疫球蛋白重链基因的基因簇如图6所示(绿色为小鼠序列，红色为人的序列)。包括了依次为人免疫球蛋白重链基因的V-区、D-区、J-区、小鼠的IgH 5’-增强子、小鼠的IgM、人/鼠Igγ3，人/鼠Igγ1和小鼠的3’-LCR。
2.人抗体转基因小鼠的培育
1)转人免疫球蛋白重链基因小鼠的培育
A.转人免疫球蛋白重链基因小鼠(抗体转基因鼠HGa)的培育
利用已有的常规转基因技术将步骤1的1)(如图5)中构建的人免疫球蛋白重链基因转入小鼠体内。通过PCR检测和ELISA检测双标准筛选获得转人免疫球蛋白重链载体的转基因小鼠(HGa)。
使用的PCR鉴定引物为：
人的IgHV2-26 PCR：
For：GACACACTTTGCTACACACTCCTG
Rev：GCACAGTAATATGTGGCTGTGTCC
PCR产物大小为：433bp
PCR检测结果如图8所示。说明：人IgHV2-26转基因小鼠DNA PCR结果，阳性小鼠有433bp的PCR带(1％凝胶电泳)
人的Igγ1 PCR：
For：TCTCTCCTCCCAGATTCCAGTAAC
Rev：GTACGTGCTGTTGTACTGCTCCTC
PCR产物大小为：417bp
Elisa检测转基因小鼠：以健康成年人和健康小鼠血清作为对照。见图9。说明：血清在1∶100稀释下进行人IgG Elisa检测。转基因小鼠是人IgG阳性，并与人血清、正常小鼠血清和空白进行对照。使用的ELISA鉴定的抗体为：Goat Anti Human IgG Fc(abcam，ab97221)，Goat Anti-Human IgG Fc(HRP)(ab97225；abcam)。
B.转人免疫球蛋白重链基因小鼠(抗体转基因鼠HGb)的培育
利用已有的常规转基因技术将步骤1的2)(如图6)中构建的人免疫球蛋白重链基因转入小鼠体内。通过PCR检测和ELISA检测双标准筛选获得转人免疫球蛋白重链载体的转基因小鼠(HGb)。
使用的PCR鉴定引物为：
人的IgHV2-26 PCR：
For：GACACACTTTGCTACACACTCCTG
Rev：GCACAGTAATATGTGGCTGTGTCC
PCR产物大小为：433bp
PCR检测结果如图10所示。说明：人IgHV2-26转基因小鼠DNA PCR结果，阳性小鼠有433bp的PCR带(1％凝胶电泳)
人Igγ1 PCR：
For：TCTCTCCTCCCAGATTCCAGTAAC
Rev：GTACGTGCTGTTGTACTGCTCCTC
PCR产物大小为：417bp
Elisa检测转基因小鼠：以健康成年人和健康小鼠血清作为对照。见图11。说明：血清在1∶100稀释下进行人IgG Elisa检测。转基因小鼠是人IgG阳性，并与人血清、正常小鼠血清和空白进行对照。使用的ELISA鉴定的抗体为：Goat Anti Human IgG Fc(abcam，ab97221)，Goat Anti-Human IgG Fc(HRP)(ab97225；abcam)。
2)免疫球蛋白重链基因敲除小鼠的培育(mH --小鼠图7)
利用基因打靶技术，构建免疫球蛋白重链基因敲除小鼠。选择小鼠免疫球蛋白重链基因的IgH J-区作为基因敲除位点(敲除位点及基因敲除效果见图六)，获得免疫球蛋白重链基因敲除小鼠。通过PCR检测和ELISA检测双标准筛选获得的免疫球蛋白重链基因敲除小鼠。
使用的PCR鉴定引物为：
For：GGGAAAGAATGAGCAAATGCAAGC
Rev：TTCTGTGTTCCTTTGAAAGCTGGAC
PCR产物：J-区基因打靶后PCR产物大小为732bp，正常J-区PCR产物的大小为2422bp。
3)免疫球蛋白kappa轻链基因敲除小鼠的培育(mK --小鼠。参考CN105441455A)
4)人免疫球蛋白kappa轻链转基因小鼠的培育(HK +小鼠。参考CN105441455A)
5)人免疫球蛋白Lambda轻链转基因小鼠的培育(HL +小鼠。参考CN105441455A)
6)杂交组合获得人源化抗体转基因小鼠
将第二步的HGa或HGb转基因小鼠分别与2)、3)、4)和5)步获得的小鼠进行杂交繁育，经过PCR和ELISA检测，最终获得高表达人IgG抗体小鼠(HGa +HK +HL +mK --mH --和HGb +HK +HL +mK --mH -)五基因小鼠。而不表达(或低表达)鼠免疫球蛋白的转基因小鼠。
转基因小鼠的鉴定：
A.五基因转基因小鼠血清中小鼠IgM和人IgG含量的Elisa检测结果：
结果：正常小鼠血清的IgM含量：0.8-6.5mg/ml
转基因小鼠(HGa+HK+HL+mK--mH--，重链图5)血清中小鼠IgM含量：0.1-2.8mg/ml
转基因小鼠(HGb+HK+HL+mK--mH--，重链图6)血清中小鼠IgM含量：0.4-2.5mg/ml
正常人血清的IgG含量：3.5-15mg/ml
转基因小鼠(HGa +HK +HL +mK --mH --，重链图5)血清中人IgG含量：0.08-1.2mg/ml
转基因小鼠(HGb +HK +HL +mK --mH --，重链图6)血清中人IgG含量：0.5-2.5mg/ml
转基因小鼠的IgM和IgG含量要比正常人的IgM和IgG含量低一点，是因为转基因小鼠饲养在清洁无菌的环境中。
B.转基因抗体小鼠血清中的人IgG含量(与CN10544145A转基因小鼠比较)，见图12。
说明：转基因抗体小鼠HGa +HK +HL +mK --mH -(HGa五基因小鼠)、HGb +HK +HL +mK --mH --(HGb五基因小鼠)和HG +HK +HL +mK --mH -(HG五基因小鼠，CN105441455A)以及人血清中含人IgG的Elisa结果，血清稀释为(1∶10，1∶100，1∶1000，1∶10000，1∶100000，1∶1000000)(3-5只五杂小鼠的平均值)。
3.全人抗体的获得
对全人抗体转基因小鼠进行免疫，获得B-细胞，再结合杂交瘤技术，细胞培养技术，获得治疗性人抗体。
OVA免疫获得的人源化抗体转基因小鼠选择8周龄的小鼠进行免疫。
初免：
①用PBS稀释OVA(Sigma A7641)抗原，终浓度为2mg/ml，加入20ug CpG(ODN1826，tlrl-1826，Invivogen)，加入适量氢氧化铝(vac-alu-50，Invivogen)，使氢氧化铝浓度为1％。
②将①中准备好的抗原0.75mL与CFA佐剂(Sigma F5881)按1∶1混合，用MIXPAC TM注射器使之乳化，每只小鼠，注射剂量为200ul(0.2mg)，进行皮下注射免疫。
二免：
①免疫后第21天进行二免，用PBS稀释抗原，终浓度为1.0mg/ml，加入10ug CpG，加入适量氢氧化铝，使氢氧化铝浓度为1％。
②将①中准备好的抗原0.75mL与IFA佐剂按1∶1混合，用MIXPAC TM注射器使之乳化，每只小鼠注射剂量为200ul(0.1mg)，进行腹腔注射免疫。
三免：
①免疫后第21天后进行三免，用PBS稀释抗原，终浓度为1.0mg/ml，加入10ug CpG，加入适量氢氧化铝，使氢氧化铝浓度为1％。
②直接注射抗原蛋白，按①中方法配制，每只小鼠注射剂量为200ul(0.1mg)进行腹腔注射免疫。
四免：
①免疫后第21天后进行四免，用PBS稀释抗原，终浓度为1.0mg/ml，加入10ug CpG，加入适量氢氧化铝，使氢氧化铝浓度为1％。
②直接注射抗原蛋白，按①中方法配制，每只小鼠注射剂量为200ul(0.1mg)进行腹腔注射免疫。
加强免疫：
在第四免疫后21天，对Elisa结果符合要求的小鼠，开展加强免疫，然后获得脾脏的B-细胞进行杂交瘤融合、培养和筛选。
2)小鼠免疫检测
第4次免疫后10天，分别取小鼠取血进行ELISA检测，以免疫前小鼠作为对照，分别检测免疫后小鼠血清中鼠IgG的含量和人IgG的含量，结果如下：
小鼠血清中人IgG的检测：用抗原OVA包埋96孔板，用hIgG-HRP(Millipore，AP113P))检测经过稀释后免疫血清中的特异抗体含量。昆明白小鼠(对照)是检测小鼠IgG。
结果显示：免疫后的人源化抗体小鼠的人源IgG表达量较高。免疫Elisa分析结果，将血清稀释1∶8000，OD 450大于1的小鼠脾脏B-细胞用于杂交瘤融合。
OVA免疫小鼠后的Elisa(OD 450)检测结果(转基因小鼠HGa +HK +HL +mK --mH --和HGb +HK +HL +mK --mH --)见图13。说明：HGa转基因鼠是HGa +HK +HL +mK --mH --五基因小鼠；HGb转基因鼠是HGb +HK +HL +mK --mH --五基因小鼠；昆明白鼠是正常对照鼠。小鼠在经过4次OVA免疫后，血清中抗OVA特异抗体的Elisa检测(不同稀释浓度)。
OVA免疫小鼠后的杂交瘤IgH V-区序列分析(突变数量和位置)统计结果见图14。
3)全源抗体的获得：
用GPC3免疫转基因小鼠(HGb +HK +HL +mK --mH --)，收集小鼠的脾细胞，与Sp2/0细胞融合，制备单克隆抗体，即全人抗体。
转基因动物抗原免疫后进行杂交瘤试验，然后在半固体培养基中筛选，再将杂交瘤克隆挑入96孔板中培养，培养的上清液做Elisa特异抗体分析。
杂交瘤上清液的Elisa结果见图15：(一次杂交瘤融合固体培养基筛选后，其中2个杂交瘤96孔板的Elisa结果，红色孔表达抗GPC3的全人抗体，GPC3多肽免疫)(抗原包埋96孔板，加上杂交瘤上清液，再用人IgG-HRP抗体和TMB显色)。
图16表明，4-10F-4G和6-7A-2E都是GPC3多肽免疫后Elisa检测阳性抗体，但在与HepG2(人的癌细胞，表达GPC3)结合分析时，4-10F-4G就没有任何功能，6-7A-2E能特异地与HepG2细胞结合，证明转基因抗体小鼠可以抗体特异性抗GPC3的抗体(6-7A-2E)。
图17为转基因小鼠产生的抗原特异性抗体的统计表，是用OVA、GPC3多肽和GPC3蛋白质对HGa和HGb转基因小鼠的进行免疫，融合，获得杂交瘤的抗体的亲和力的统计表。两种转基因小鼠都能获得高亲和力的人源抗体。
(1)本发明所得小鼠的脾的大小与正常小鼠一致，比CN105441455A的转基因小鼠的脾大；
(2)本发明转基因小鼠骨髓和脾中的B-细胞更多，多于CN105441455A的转基因小鼠的B-细胞；
(3)本发明转基因小鼠的IgM量高，多于CN105441455A的转基因小鼠血清中的IgM含量；
(4)本发明转基因小鼠的IgG量高，在同样的清洁无菌的环境中，比CN105441455A的转基因小鼠血清中的IgG含量高；
(5)本发明转基因IgH重链CDR突变更多：在相同抗原免疫条件下，本发明转基因小鼠获得的杂交瘤IgG V-序列突变比CN105441455A的转基因小鼠获得的杂交瘤IgGV-序列突变多。
转基因小鼠HGa +HK +HL +mK --mH --免疫后脾的B-细胞数量：6.2X10 7，1.0X10 8，9.5X10 7。
转基因小鼠HGb +HK +HL +mK --mH --免疫后脾的B-细胞数量：1.2X10 8，1.02X10 8，1.25X10 8。
普通小鼠免疫后脾脏的B-细胞数量：1.5X10 8，1.8X10 8，1.9X10 8。
图18为转基因小鼠免疫后脾脏外观照片：图18-A为转基因小鼠HGa +HK +HL +mK --mH --免疫后脾脏外观照片，图18-B为转基因小鼠HGb +HK +HL +mK --mH --免疫后脾脏外观照片，图18-C为普通小鼠免疫后的脾脏。
Figure PCTCN2018083163-appb-000001
Figure PCTCN2018083163-appb-000002
Figure PCTCN2018083163-appb-000003
Figure PCTCN2018083163-appb-000004
Figure PCTCN2018083163-appb-000005
Figure PCTCN2018083163-appb-000006
Figure PCTCN2018083163-appb-000007
Figure PCTCN2018083163-appb-000008
Figure PCTCN2018083163-appb-000009

Claims (21)

  1. 一种核酸分子，包括了免疫球蛋白基因或其片段，其特征在于：包括了IgM基因(IgHCμ)和IgM转换元件(switch region，Sμ)，所述Sμ和IgHCμ全部为宿主动物序列。
  2. 如权利要求1所述的核酸分子，所述IgHCμ包括CH1、CH2、CH3、CH4外显子以及之间的序列，和TM1、TM2、polyA信号序列。
  3. 如权利要求1或2所述的核酸分子，所述Sμ的核苷酸序列如SEQ ID NO.1中(2550)..(4451)所示。
  4. 如权利要求1-3任一所述的核酸分子，其特征在于：包括了宿主动物IgH重链的5′-端的增强子(5’-Enhancer)。
  5. 如权利要求4所述的核酸分子，5’-Enhancer的核苷酸序列为SEQ ID NO.1(433)..(1444)所示。
  6. 如权利要求1-5任一所述的核酸分子，其结构如图1-1所示。
  7. 如权利要求1-6任一所述的核酸分子，其特征在于：包括IgG基因(Igγ)。
  8. 如权利要求7所述的核酸分子，IgG基因为宿主动物/人IgG嵌合元件或全人IgG序列。
  9. 如权利要求8所述的核酸分子，所述嵌合元件包括宿主动物Igγ的转换元件(Sγ)和TM1和TM2、polyA等序列，以及人的CH1、Hinge、CH2、CH3外显子以及之间的序列等。
  10. 如权利要求8或9所述的核酸分子，IgG基因结构如图2-1所示。
  11. 如权利要求8所述的核酸分子，所述全人IgG序列包括：人的转换元件(Sγ)、人Igγ的CH1、Hinge、CH2和CH3外显子以及之间的序列，和人Igγ的PolyA、TM1、TM2等序列。
  12. 如权利要求8-11任一所述的核酸分子，所述Igγ序列包括Igγ的单种或多种亚型，多种Igγ亚型间的转换元件(Sγ)。
  13. 如权利要求8-12任一所述的核酸分子，人Igγ亚型包括Igγ3、Igγ1、Igγ2和/或Igγ4；小鼠Igγ亚型包括Igγ3、Igγ1、Igγ2a和/或Igγ2b等。
  14. 如权利要求8-13任一所述的核酸分子，包括人或宿主动物IgH重链的3’-位置表达调控序列(Local Control Region)。
  15. 如权利要求8-14任一所述的核酸分子，包括了人IgH重链V-区序列或片段，人IgH D-区序列或片段，和人IgH J-区序列或片段。
  16. 如权利要求1-15所述的核酸分子，其结构组成如图5或图6所示。
  17. 一种载体，包含如权利要求1-16所述的核酸分子。
  18. 一种细胞，包含如权利要求1-16任一所述核酸分子或权利要求17所述载体。
  19. 一种人源抗体，来源于权利要求1-16任一核酸分子或权利要求17所述载体或或权利要求18所述细胞。
  20. 权利要求1-16任一所述核酸分子或权利要求17所述载体或权利要求18所述细胞在编码DNA、cDNA、mRNA，表达氨基酸序列、蛋白质、载体，培养杂交瘤、细胞株、转基因动物和/或制备人源抗体中的应用。
  21. 采用权利要求1-16任一所述核酸分子或权利要求17所述载体或权利要求18所述细胞制备转基因动物的方法，包括以下步骤：
    (1)所述核酸分子的获得；
    (2)将所述核酸分子构建入载体；
    (3)向宿主动物细胞或胚胎导入所述载体；
    (4)将含有上述载体的细胞植入宿主动物的胚胎内或体细胞克隆；
    (5)繁殖杂合、纯合的转基因动物。
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