WO2017067043A1 - 一种嵌合核酸分子及其在人源化抗体制备中的应用 - Google Patents

一种嵌合核酸分子及其在人源化抗体制备中的应用 Download PDF

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WO2017067043A1
WO2017067043A1 PCT/CN2015/095741 CN2015095741W WO2017067043A1 WO 2017067043 A1 WO2017067043 A1 WO 2017067043A1 CN 2015095741 W CN2015095741 W CN 2015095741W WO 2017067043 A1 WO2017067043 A1 WO 2017067043A1
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nucleic acid
acid molecule
gene
human
sequence
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PCT/CN2015/095741
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French (fr)
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邹贤刚
葛良鹏
刘作华
游小燕
吴梦
杨松全
林保忠
刘雪芹
黄勇
丁玉春
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重庆市畜牧科学院
邹贤刚
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    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • 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

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  • the invention belongs to the field of biotechnology, and mainly relates to a nucleic acid molecule and application thereof in the preparation of humanized antibody.
  • Antibodies are an important class of biomedical products that play an important role in the prevention and treatment of human diseases.
  • the development of therapeutic antibodies has undergone different stages of development, such as murine antibodies, chimeric antibodies, modified antibodies, surface remodeling antibodies, and fully humanized antibodies.
  • Full Humanized Antibody refers to an antibody that is identical to a human antibody protein sequence obtained by genetic modification or transgenic animal immunization. Since humanized antibodies do not contain animal protein, the side effects are lower and the effect is better. It has become the main research and development direction of current and future antibody engineering. At present, fully humanized antibodies are mainly produced by phage display technology and transgenic animal technology. In 2002, the first fully humanized antibody, Adalimumab (Humira), produced by phage display technology, was launched. In 2006, the first fully humanized antibody, Panitumumab, derived from genetically modified animals, was approved for marketing in the United States and Europe.
  • Phage Display-generating Antibody technology is relatively simple to produce fully humanized antibodies, and the technology is relatively mature. However, it is difficult to obtain high affinity antibodies by the effect of antibody library capacity and post-translational modification and efficient folding of antibodies. Very big.
  • Transgenic Humanized Antibody refers to the transfer of human immunoglobulin genes in whole or in part to the animal genome through transgenic or transgenic chromosome technology [animal endogenous antibody gene deletion (or inactivation)] The animal is allowed to express human antibodies to obtain fully humanized antibodies.
  • the greatest advantage of using transgenic animals to produce fully humanized antibodies is the greater chance of obtaining high-affinity antibodies, while using a transgenic animal strain to produce different types of antibodies against different antigens, without the need for phage display technology, The antigen, every time you have to rebuild the phage library.
  • the object of the present invention is to provide a nucleic acid molecule capable of efficiently preparing a fully humanized antibody, which overcomes the incompatibility problem of interaction between BCR and Ig ⁇ and Ig ⁇ of different species, and the method of human expression relative to Lee's method. There is no need for a secondary modification of the antibody.
  • the object of the invention is achieved by the following measures:
  • the present invention provides a nucleic acid molecule comprising a human immunoglobulin gene or a fragment thereof, and a gene fragment of a constant region of a host animal IgM (i.e., a gene fragment of IgH C ⁇ ).
  • the gene fragment of the above host animal IgM constant region is located (instead of) the corresponding constant region of the human immunoglobulin IgM heavy chain gene.
  • the above host IgM constant region gene fragment is the CH2 partial sequence, CH3, CH4, TM1 and TM2 sequences of the host animal IgH C ⁇ and the sequence therebetween and the related PolyA signal sequence.
  • the human CH1 sequence binds to the human Ig light chain to ensure antibody structure, and the CH2 partial region ensures normal transcription and translation of human and mouse C regions.
  • the murine IgM constant region gene fragment for substitution is shown in ⁇ 1> to ⁇ 4425> of SEQ ID NO. 1; the hIg sequence substituted with the murine IgM constant region gene fragment is SEQ ID NO. 2 is shown.
  • the porcine IgM constant region gene fragment for substitution is shown in ⁇ 1> to ⁇ 4386> of SEQ ID NO. 3, and the hIg sequence substituted by the porcine IgM constant region gene fragment is SEQ ID NO. 4 is shown.
  • the nucleic acid molecule may further comprise an FRT sequence, a human Ig intron sequence (eg, hIgM, hIgD); a gene fragment of the host animal IgM constant region in the nucleic acid molecule and a human immunoglobulin IgM gene or a fragment thereof, and
  • the structure of the connection with FRT and human Ig introns is shown in Figure 1-2.
  • the FRT sequence is SEQ ID NO.
  • the human Ig intron sequence includes SEQ ID NO. 1 ⁇ 4426> to ⁇ 4459>, and the human Ig intron sequence includes SEQ ID NO. 1 ⁇ 4460> to ⁇ 4717>; when the host is a pig, The FRT sequence is SEQ ID NO. 3 ⁇ 4387> to ⁇ 4420>, and the human Ig intron sequence includes SEQ ID NO. 3 ⁇ 4421> to ⁇ 4678>.
  • the regulatory region of IgHG3 (S ⁇ 3) and the IgHG3 CH1 sequence are sequentially linked to form a gene vector expressing chimeric human mouse IgM and human IgG (Fig. 1-3).
  • the CH1 and partial CH2 sequences of human IgM the CH2 partial sequence of the mouse, the CH3, CH4, TM1 and TM2 sequences and the sequence between them and the related PolyA signal sequence, and the FRT, IgM intron, human IgD3
  • the regulatory region (S ⁇ 3) is linked to the IgD3 CH1 sequence to form a gene vector expressing chimeric human mouse IgM and human IgD ( Figures 1-4).
  • the human immunoglobulin gene includes a V region gene, a D region gene, and a J region gene of a human immunoglobulin heavy chain gene.
  • the above human immunoglobulin heavy chain gene may further include hC ⁇ , hC ⁇ , hC ⁇ and/or hC ⁇ heavy chain gene and human 3'-regulatory region (hLCR) and the like.
  • the gene cluster of the above nucleic acid molecule is shown in Figure 2 (the black box position is the IgM constant region gene partial sequence (fragment) of the introduced host animal).
  • the above nucleic acid molecule expresses IgM homologous to the host animal in an early stage of development in the animal, and the IgM acts as a BCR, which facilitates interaction with the animal's own Ig ⁇ and Ig ⁇ , and promotes the conversion of the antibody to IgG, affinity. Mature and B cell development.
  • An expression vector comprising the above nucleic acid molecule.
  • a cell comprising the above nucleic acid molecule or the above vector.
  • An animal such as a pig or a mouse, comprises the above nucleic acid molecule.
  • a humanized antibody produced by rearranging and encoding the above nucleic acid molecule. Use of the above nucleic acid molecule or vector or cell for the preparation of a humanized antibody. Use of the above nucleic acid molecule or vector or cell in the preparation of a transgenic animal.
  • the human immunoglobulin gene transformed as described above is transferred into an animal to obtain a transgenic immunoglobulin transgenic animal, or further hybridized with an animal whose autoimmune globulin gene is not expressed to obtain a genetically engineered animal expressing only the human antibody protein.
  • a method of preparing a transgenic animal using the above nucleic acid molecule or vector or cell comprising the steps of:
  • the above host animals are mammals such as rats, rabbits, pigs, cows, sheep, chickens, horses and the like.
  • the above vectors are artificial chromosomes (such as yeast, bacteria), phage, plasmids and the like. Methods for introducing the vector into a host cell include electroporation, viral infection, lipofection, microinjection, and the like.
  • the gene fragment of the IgM constant region of the host animal of the present invention refers to a nucleotide sequence encoding a binding protein Ig ⁇ and/or Ig ⁇ that binds to the host animal.
  • the fully humanized antibody produced by the present invention has high affinity.
  • the present invention utilizes a transgenic animal strain to produce different types of various antibodies, and the invention is applicable to a wide variety of animals.
  • the host autoimmune globulin of the invention is not expressed or is below the detection limit.
  • the gene rearrangement efficiency of the invention is high, and the VDJC rearrangement, mutation and utilization rate are consistent with the human body.
  • the invention directly produces fully humanized antibodies, and does not require secondary modification, and the expression level in vivo can reach the level of healthy adults.
  • Figure 1-1 Structure of a gene fragment of the host animal IgM constant region after transformation and its linkage structure with a human immunoglobulin IgM constant region gene or a fragment thereof.
  • Figure 1-2 Vector for transformation: a gene fragment comprising a constant region of a host animal IgM and a human immunoglobulin IgM constant region gene or a fragment thereof, and a FRT, a screening gene, a human Ig intron (such as IgM, IgD) And other connection structures.
  • FIG. 1 Structure of the IgM constant region gene fragment of the host animal after genetic engineering and its linkage structure with the human IgM gene fragment and its linkage structure with other human Ig, such as human IgG3 gene or fragment
  • FIG. 1-4 Structure of IgM constant region gene fragment of host animal after genetic engineering and its linkage structure with human IgM gene fragment and its linkage structure with other human Ig, such as hIGD gene or fragment
  • Figure 2 is a structural diagram of the human immunoglobulin heavy chain gene cluster after modification in Example 1.
  • the engineered human immunoglobulin gene is transferred into a mouse, and the mouse containing the human immunoglobulin gene is then immunized to obtain a fully humanized antibody.
  • the specific steps are as follows:
  • the host animal IgM constant region gene fragment was transformed into the YAC or BAC vector containing human Ig by homologous recombination, and the gene cluster of the human immunoglobulin heavy chain gene was constructed as shown in FIG. 2 (the black frame position is the introduced mouse IgM). Gene fragment of the constant region).
  • V region, D region and J region which are human immunoglobulin heavy chain genes, hIgHC ⁇ , hIgHC ⁇ 3, hIgHC ⁇ 1, hIgHC ⁇ 1, hLCR; wherein, hIgHC ⁇ is genetically modified, CH2 partial sequence of mouse IgHC ⁇ , CH3, CH4, TM1 And the TM2 sequence and the sequence therebetween and the related PolyA signal sequence, etc.
  • DNA sequence is SEQ ID NO. 1
  • DNA sequence is SEQ ID NO. 2
  • the human immunoglobulin kappa light chain gene includes all or part of the human immunoglobulin kappa light chain gene V region, J region, C region, and KDE region.
  • the gene cluster is shown in Figure 3.
  • the human immunoglobulin lambda light chain gene includes all or part of the human immunoglobulin lambda light chain gene V region, J region and C region, and includes the terminal enhancer structure.
  • the gene cluster is shown in Figure 4.
  • the human immunoglobulin heavy chain gene constructed in 1) of step 1 was transferred into a mouse using an existing conventional transgenic technique.
  • Transgenic human immunoglobulin heavy chain transgenic mice obtained by double standard screening were detected by PCR detection and ELISA.
  • PCR identification primers used were:
  • the antibodies identified by ELISA were: sigma (I1886) and sigma (A8792), with healthy adult and healthy mouse serum as controls.
  • the human immunoglobulin kappa light chain gene constructed in 2) of step 1 was transferred into mice using conventional conventional transgenic techniques.
  • Transgenic human immunoglobulin kappa light chain transgenic mice obtained by double standard screening were detected by PCR detection and ELISA.
  • PCR identification primers used were:
  • the antibodies identified by ELISA were: sigma (K3502) and sigma (A7164) with healthy adult and healthy mouse serum as controls.
  • the human immunoglobulin lambda light chain gene constructed in step 3 of step 1 was transferred into mice using conventional conventional transgenic techniques.
  • Transgenic human immunoglobulin lambda light chain transgenic mice obtained by double standard screening were detected by PCR detection and ELISA.
  • PCR identification primers used were:
  • the antibodies identified by ELISA were: sigma (L1645) and sigma (A5175), with healthy adult and healthy mouse serum as controls.
  • Immunoglobulin heavy chain knockout mice were constructed using Crispr/Cas9 technology. IgHC ⁇ of the mouse immunoglobulin heavy chain gene was selected as a knockout site (knockout site and gene knockout effect is shown in Fig. 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 760 bp. Then, it was digested with BstEII, and the gene was targeted at 753 bp, and the two targets with no gene targeting were 500 bp and 260 bp.
  • the antibodies identified by ELISA were: sigma (M8644) and sigma (A8786) with healthy adult and healthy mouse serum as controls.
  • the immunoglobulin kappa light chain knockout mouse was obtained by knocking out the entire constant region (C region) of the mouse immunoglobulin kappa light chain gene using a conventional gene knockout technique.
  • Mouse immunoglobulin kappa light chain knockout mice obtained by double standard screening were detected by PCR detection and ELISA.
  • PCR identification primers used were:
  • the antibodies identified by ELISA were: Southern Biotech (1170-01) and Southern Biotech (1170-05, with healthy adult and healthy mouse serum as controls.
  • mice obtained in the second step 1), 2), 3), 4), 5) were cross-breeded, and subjected to PCR and ELISA. Detection, finally obtaining a humanized antibody transgenic mouse that highly expresses human immunoglobulin without expressing (or underexpressing) murine immunoglobulin was used for the next study.
  • mice Eight weeks old mice were selected for immunization.
  • the second dose was performed, and the antigen was diluted with PBS to a final concentration of 2.5 mg/ml, 25 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 (0.25mg), injected intraperitoneally immunized.
  • the third dose was performed, and the antigen was diluted with PBS to a final concentration of 1.25 mg/ml. 12.5 ug of CpG was added, and an appropriate amount of aluminum hydroxide was added to make the aluminum hydroxide concentration 1%.
  • mice with numbers 3554, 3555, and 3556 were taken for ELISA, and healthy adults and wild-type mice were used as controls to detect the content of murine IgG in the serum of immunized mice. And human IgG content, the results are as follows:
  • the kit is a mouse IgG ELISA kit (Abeam, ab157719)
  • kit as a human IgG ELISA kit (Abeam, ab100547)
  • Mouse 3556 was selected, and the spleen cells and hybridoma cells were fused, and monoclonal antibodies were screened. Three cell clones with the highest expression were screened by ELISA, and anti-OVA antibodies were performed by competitive ELISA assay (CEB459Ge, Cloud-Clone Corp). Affinity assay showed that the antibody with the highest affinity was 230 pM.
  • the modification scheme of the human immunoglobulin gene of the present invention is equally applicable to other mammals.
  • a mammal such as a pig as a host animal can also achieve the beneficial effects of the expression of the fully humanized antibody of the present invention.

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Abstract

一种核酸分子，包括了人免疫球蛋白基因或其片段，其特征在于：还包括宿主动物IgM恒定区基因片段。

Description

一种嵌合核酸分子及其在人源化抗体制备中的应用 技术领域
本发明属于生物技术领域，主要涉及一种核酸分子及其在人源化抗体制备中的应用。
背景技术
抗体是一类重要的生物医药制品，在人类疾病的预防和治疗过程中发挥了重要的作用。治疗性抗体的发展经历了鼠源抗体、嵌合抗体、改型抗体、表面重塑抗体和全人源化抗体等不同的发展阶段。
全人源化抗体(Full Humanized Antibody)，是指经过基因改造或转基因动物免疫而获得的与人源抗体蛋白质序列完全一致的抗体。全人源化抗体由于不含动物蛋白，因此副作用较低，作用效果更好，已成为当前和未来抗体工程的主要研究和发展方向。目前全人源化抗体主要由噬菌体展示技术和转基因动物技术生产而来。2002年，第一个由噬菌体展示技术生产的全人源化抗体Adalimumab(Humira)上市。2006年，第一个由转基因动物来源的全人源化抗体Panitumumab在美国和欧洲批准上市。
噬菌体展示抗体(Phage Display-generating Antibody)技术生产全人源化抗体相对简单，技术相对成熟，但受抗体库容量和抗体翻译后修饰及有效折叠的影响，利用该技术获得高亲合力抗体的难度很大。
转基因动物全人源化抗体(Transgenic Humanized Antibody)是指将人类免疫球蛋白基因全部或部分通过转基因或转人工染色体技术，转移至动物基因组中[动物内源的抗体基因缺失(或失活)]，使动物表达人类抗体，从而获得全人源化抗体。利用转基因动物生产全人源化抗体的最大优势是获得高亲合力抗体的机率较大，同时利用一种转基因动物品系可针对不同的抗原生产不同类型的抗体，无需像噬菌体展示技术，针对不同的抗原，每次都要重新构建噬菌体库。但随着研究的深入，发现转入的人类免疫球蛋白基因片段虽然能在动物体内重排和表达，但其产生人抗体蛋白的性能要低于未经基因改造前的动物自身抗体生产效果。以小鼠为例，其原因主要是当抗体在B细胞发育的初级阶段作为B细胞表面受体(BCR)时，其与鼠源的信号蛋白Igα和Igβ的相互作用并不是最优的(即鼠源BCR与鼠源的Igα和Igβ或人BCR与人的Igα和Igβ相互作用效果是最好的)，因此影响了抗体的类型转换、亲和力成熟和B细胞发育为成熟的生产抗体 的浆细胞。为克服这一难题，Lee等(Nature biotechnology，2014；32(4)：356-363)将三个人免疫球蛋白基因(IgH，Igκ和Igλ)的可变区替代小鼠的免疫球蛋白可变区，而恒定区使用小鼠免疫球蛋白的相应片段，利用这种策略可克服上述提到的问题，获得可变区为人源，恒定区为鼠源的嵌合抗体，再通过下游的改造将鼠源的恒定区改造为人源的恒定区，得到全人源化抗体。这种方法存在的缺点是需要进行二次改造才能获得全人源化抗体。
发明内容
本发明的目的在于提供一种核酸分子，可以高效的制备全人源化抗体，其克服了不同物种BCR与Igα和Igβ相互作用的不兼容性问题，同时相对Lee的方法，其表达的人源化抗体无需进行二次改造。
本发明的目的是通过以下措施实现的：
本发明提供了一种核酸分子，包括了人免疫球蛋白基因或其片段，以及宿主动物IgM恒定区的基因片段(即IgH Cμ的基因片段)。
优选的，上述宿主动物IgM恒定区的基因片段位于(替代)人免疫球蛋白IgM重链基因的相应恒定区。
上述宿主IgM恒定区基因片段为宿主动物IgH Cμ的CH2部分序列、CH3、CH4、TM1和TM2序列以及其之间的序列和相关PolyA信号序列。人CH1序列与人Ig轻链结合，保证抗体结构，CH2部分区又保证人和小鼠C区正常转录和翻译。当宿主为鼠时，用于取代的鼠IgM恒定区基因片段如SEQ ID NO.1的<1>～<4425>所示；被鼠源IgM恒定区基因片段替代的hIg序列如SEQ ID NO.2所示。当宿主为猪时，用于取代的猪IgM恒定区基因片段如SEQ ID NO.3的<1>～<4386>所示，被猪源IgM恒定区基因片段替代的hIg序列如SEQ ID NO.4所示。
上述核酸分子中宿主动物IgM恒定区的基因片段的结构及其与人免疫球蛋白IgM基因或其片段的连接结构如图1-1所示。进一步地，上述核酸分子还可以包括FRT序列、人Ig内含子序列(如hIgM、hIgD)；所述核酸分子中宿主动物IgM恒定区的基因片段与人免疫球蛋白IgM基因或其片段，以及与FRT、人Ig内含子(如IgM、IgD)的连接结构如图1-2所示。当宿主为鼠时，所述FRT序列如SEQ ID NO.1<4426>～<4459>，人Ig内含子序列包括SEQ ID NO.1<4460>～<4717>；当宿主为猪时，FRT序列如SEQ ID NO.3<4387>～<4420>，人Ig内含子序列包括SEQ ID NO.3<4421>～<4678>。
例如，具体地，人IgM的CH1和部分CH2序列，小鼠的CH2部分序列、CH3、CH4、TM1和TM2序列以及其之间的序列和相关PolyA信号序列，与FRT、IgD内含子、人的IgHG3的调控区(Sγ3)和IgHG3 CH1序列依次链接，形成一个表达嵌合人小鼠IgM和人IgG的基因载体(如图1-3)。或者，人IgM的CH1和部分CH2序列，小鼠的CH2部分序列、CH3、CH4、TM1和TM2序列以及其之间的序列和相关PolyA信号序列，与FRT、IgM内含子、人的IgD3的调控区(Sδ3)和IgD3 CH1序列链接，形成一个表达嵌合人小鼠IgM和人IgD的基因载体(图1-4)。
上述人免疫球蛋白基因包括了人免疫球蛋白重链基因的V区基因、D区基因、J区基因。上述人免疫球蛋白重链基因还可以包括hCγ、hCα、hCδ和/或hCξ重链基因和人的3’-调控区(hLCR)等。
例如，上述核酸分子的基因簇如图2所示(黑框位置为导入的宿主动物的IgM恒定区基因部分序列(片段))。
通过以上的改造，上述核酸分子在动物体内发育的早期阶段表达与宿主动物同源的IgM，该IgM作为BCR，有利于与动物自身的Igα和Igβ相互作用，促进抗体的类型转换为IgG、亲和力成熟和B细胞发育。
一种表达载体，包含上述核酸分子。一种细胞，包含上述核酸分子或上述载体。一种动物，如猪、鼠，包含上述核酸分子。一种人源化抗体，由上述核酸分子重排、编码制得。上述核酸分子或载体或细胞在制备人源化抗体中的应用。上述核酸分子或载体或细胞在制备转基因动物中的应用。
将经过上述改造的人免疫球蛋白基因转入动物体内获得转人免疫球蛋白转基因动物，或进一步与自身免疫球蛋白基因不表达的动物进行杂交获得只表达人抗体蛋白的基因工程动物。利用抗原免疫转入人免疫球蛋白基因(重链、轻链)的动物，获得全人源化抗体。例如：
采用上述核酸分子或载体或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)向宿主细胞(包括干细胞，诱导干细胞和体细胞)或胚胎导入含有所述核酸分子的载体；
(4)将含有人Ig的细胞植入宿主动物的胚胎内(嵌合体制备)或体细胞克隆；
(5)繁育杂合、纯合的转人Ig基因的动物。(包括与宿主内源免疫球蛋白基因失活的动物杂交)。
上述宿主动物为哺乳动物，如鼠、兔、猪、牛、羊、鸡、马等。上述载体为人工染色体(如酵母，细菌)、噬菌体、质粒等。上述载体导入宿主细胞的方法，包括电穿孔、病毒感染、脂质体转染和显微注射等。
本发明所述宿主动物的IgM恒定区的基因片段、CH2部分序列是指编码可结合宿主动物信号蛋白Igα和/或Igβ的核苷酸序列。
有益效果
1.本发明生产的全人源化抗体具有高亲和力。
2.本发明可利用一种转基因动物品系产生不同类型的各种抗体，且本发明适用于多种动物。
3.本发明的宿主自身免疫球蛋白不表达或在检测限度以下。
4.本发明基因重排效率高，VDJC重排、突变和利用率与人体一致。
5.本发明直接生产全人源化抗体，不需进行二次改造，体内表达量可达到健康成人水平。
附图说明
图1-1改造后宿主动物IgM恒定区的基因片段的结构及其与人免疫球蛋白IgM恒定区基因或其片段的连接结构。
图1-2改造用的载体：包括宿主动物IgM恒定区的基因片段与人免疫球蛋白IgM恒定区基因或其片段，以及与FRT、筛选基因、人Ig内含子(如IgM、IgD)的等连接结构。
图1-3基因改造后宿主动物IgM恒定区基因片段结构及其与人IgM基因片段连接结构以及其与其它的人Ig，如人IgG3基因或片段的连接结构
图1-4基因改造后宿主动物IgM恒定区基因片段结构及其与人IgM基因片段连接结构以及其与其它的人Ig，如hIGD基因或片段的连接结构
图2实施例1的改造后人免疫球蛋白重链基因簇结构图
图3实施例1人免疫球蛋白Kappa轻链基因簇结构图
图4实施例1人免疫球蛋白lambda轻链基因簇结构图
图5实施例1小鼠免疫球蛋白重链基因簇
图6实施例1敲除小鼠免疫球蛋白重链基因的基因打靶
图7实施例1小鼠免疫球蛋白轻链基因簇
图8实施例1敲除小鼠免疫球蛋白轻链基因的基因打靶
具体实施方式
下面通过具体实施例对本发明进行具体描述，在此指出以下实施例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1
将改造的人免疫球蛋白基因转入小鼠体内，再免疫含有人免疫球蛋白基因的小鼠获得全人源化抗体，具体步骤如下：
1.人免疫球蛋白基因的优化改造
1)人免疫球蛋白重链基因的改造
将宿主动物IgM恒定区基因片段经同源重组转入含人Ig的YAC或BAC载体上，构建人免疫球蛋白重链基因的基因簇如图2所示(黑框位置为导入的鼠的IgM恒定区的基因片段)。包括了依次为人免疫球蛋白重链基因的V区、D区、J区，hIgHCμ、hIgHCγ3、hIgHCγ1、hIgHCα1、hLCR；其中，hIgHCμ经基因改造，鼠源IgHCμ的CH2部分序列、CH3、CH4、TM1和TM2序列以及其之间的序列和相关PolyA信号序列等(DNA序列为SEQ ID NO.1)取代了部分原hIgHCμ和IgHCδ的相应区域(DNA序列为SEQ ID NO.2)，其改造后的结构如图1-1和图1-3所示。
2)人免疫球蛋白Kappa轻链基因的改造
人免疫球蛋白kappa轻链基因包括了人免疫球蛋白kappa轻链基因全部或部分V区、J区、C区，以及KDE区。基因簇如图3所示。
3)人免疫球蛋白lambda轻链基因的改造
人免疫球蛋白lambda轻链基因包括了人免疫球蛋白lambda轻链基因全部或部分V区、J区和C区，同时包括末端的增强子结构。基因簇如图4所示。
2.人源化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
利用已有的常规转基因技术将步骤1的1)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的转人免疫球蛋白重链转基因小鼠。
使用的PCR鉴定引物为：
PCR 1
For：TGCTTGGAACTGGATCAGGCAGTC
Rev：TTGCTTAACTCCACACCTGCTCCTG
PCR product size：329bp
PCR2
For：TTGAGGAGACTGTCCATCCTTCAC
Rev：GAGAGGGCATCTTGGTCTTCTTTC
PCR product size：471bp
使用的ELISA鉴定的抗体为：sigma(I1886)和sigma(A8792)，以健康成年人和健康小鼠血清作为对照。
2)转人免疫球蛋白kappa轻链基因小鼠的培育
利用已有的常规转基因技术将步骤1的2)中构建的人免疫球蛋白kappa轻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的转人免疫球蛋白kappa轻链转基因小鼠。
使用的PCR鉴定引物为：
PCR 1：
FOR：TGCTCTGACCTCTGAGGACCTGTCTGTA
Rev：TTCAGGCAGGCTCTTACCAGGACTCA
PCR product size：616bp
PCR 2：
For：CACCCAAGGGCAGAACTTTGTTACT
Rev：GAGGAAAGAGAGAAACCACAGGTGC
PCR product size：596bp
使用的ELISA鉴定的抗体为：sigma(K3502)和sigma(A7164)，以健康成年人和健康小鼠血清作为对照。
3)转人免疫球蛋白lambda轻链基因小鼠的培育
利用已有的常规转基因技术将步骤1的3)中构建的人免疫球蛋白lambda轻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的转人免疫球蛋白lambda轻链转基因小鼠。
使用的PCR鉴定引物为：
PCR 1：
For：AGCACAATGCTGAGGATGTTGCTCC
Rev：ACTGACCCTGATCCTGACCCTACTGC
PCR product size：562bp
PCR 2：
FOR：CTCTGCTGCTCCTCACCCTCCTCACTCAGG
REV：GAGAGTGCTGCTGCTTGTATATGAGCTGCA
PCR product size：462bp
使用的ELISA鉴定的抗体为：sigma(L1645)和sigma(A5175)，以健康成年人和健康小鼠血清作为对照。
4)免疫球蛋白重链基因敲除小鼠的培育(图5、图6)
利用Crispr/Cas9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IgHCμ作为基因敲除位点(敲除位点及基因敲除效果见图6)，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：AGCACCATTTCCTTCACCTGGAAC
Rev：CAAGGAGCAAATGACCATGTCTGG
PCR产物：760bp。然后用BstEII酶切，基因打靶的是753bp，没有基因打靶的是500bp和260bp的两条带。
使用的ELISA鉴定的抗体为：sigma(M8644)和sigma(A8786)，以健康成年人和健康小鼠血清作为对照。
5)免疫球蛋白kappa轻链基因敲除小鼠的培育(图7、图8)
利用常规基因敲除技术，敲除小鼠免疫球蛋白kappa轻链基因的整个恒定区(C区)，获得免疫球蛋白kappa轻链基因敲除小鼠。通过PCR检测和ELISA检测双标准筛选获得的小鼠免疫球蛋白kappa轻链基因敲除小鼠。
使用的PCR鉴定引物为：
PCR1：
For：CCCTTCCCTAGCCAAAGGCAACTA
Rev：CACAACGGGTTCTTCTGTTAGTCC
PCR product size：466
PCR 2：
For：CACACCTCCCCCTGAACCTGAAAC
Rev：GTTGTGGGTAGTGCCCAGCCTTGC
PCR product size：464bp
使用的ELISA鉴定的抗体为：SouthernBiotech(1170-01)和SouthernBiotech(1170-05，以健康成年人和健康小鼠血清作为对照。
6)杂交组合获得人源化抗体转基因小鼠
将第二步1)，2)，3)，4)，5)步获得的小鼠进行杂交繁育，经过PCR和ELISA 检测，最终获得高表达人免疫球蛋白，而不表达(或低表达)鼠免疫球蛋白的人源化抗体转基因小鼠用于下一步的研究。
3.全人源化抗体的获得
1)OVA免疫第2步6)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(SigmaA7641)抗原，终浓度为5mg/ml，加入50ug 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MIXPACTM注射器使之乳化，每只小鼠，注射剂量为200ul(0.5mg)，进行皮下注射免疫。
二免：
①初免后第16天进行二免，用PBS稀释抗原，终浓度为2.5mg/ml，加入25ug 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MIXPACTM注射器使之乳化，每只小鼠注射剂量为200ul(0.25mg)，进行腹腔注射免疫。
三免：
①二免第16天后进行三免，用PBS稀释抗原，终浓度为1.25mg/ml，加入12.5ug 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(0.25mg)进行腹腔注射免疫。
2)小鼠抗体检测
第3次免疫后10天，分别取编号为3554，3555，3556号小鼠取血进行ELISA检测，以健康成年人和野生型小鼠作为对照，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：
①小鼠IgG的含量检测
使用试剂盒为小鼠IgG ELISA试剂盒(Abcam，ab157719)
3554：检测限度以下
3555：检测限度以下
3556：检测限度以下
健康成年人：检测限度以下
野生型小鼠：0.7mg/ml
结果显示：免疫后的人源化抗体小鼠的鼠源IgG表达量极低。
②小鼠血清中人IgG的检测
使用试剂盒为人IgG ELISA试剂盒(Abcam，ab100547)
3554：10.3mg/ml
3555：8.1mg/ml
3556：8.2mg/ml
健康成年人：10.2mg/ml
野生型小鼠：＜0.1ng/ml
结果显示：免疫后的人源化抗体小鼠的人源IgG表达量较高。
③OVA抗体亲和力测定
选取3556号小鼠，将其脾细胞和杂交瘤细胞融合，筛选单克隆抗体，ELISA筛选表达量最高的3个细胞克隆，利用竞争性ELISA检测法(CEB459Ge，Cloud-Clone Corp)进行抗OVA抗体亲和力检测，结果显示最高亲和力的抗体为230pM。
本发明对人免疫球蛋白基因的改造方案，在其它哺乳动物中同样适用，例如，猪等哺乳动物作为宿主动物同样可实现本发明所述全人源化抗体表达的有益效果。

Claims (17)

  1. 一种核酸分子，包括了人免疫球蛋白基因或其片段，其特征在于：还包括宿主动物IgM恒定区基因片段。
  2. 如权利要求1所述的核酸分子，所述宿主动物IgM恒定区位于原人免疫球蛋白IgM恒定区的相应区域。
  3. 如权利要求1或2所述的核酸分子，所述宿主IgM恒定区基因片段为宿主IgHCμ的CH2部分序列、CH3、CH4、TM1和TM2序列以及其之间的序列和相关PolyA信号序列。
  4. 如权利要求1-3任一所述的核酸分子，所述人免疫球蛋白基因或其片段与宿主动物IgM恒定区基因片段的连接结构如图1-1所示。
  5. 如权利要求1-4任一所述的核酸分子，IgM恒定区基因片段如SEQ ID NO.1<1>～<4425>或SEQ ID NO.3<1>～<4386>所示。
  6. 如权利要求1-5任一所述的核酸分子，还包括FRT序列、人Ig内含子序列(如hIgM、hIgD)。
  7. 如权利要求6所述的核酸分子，所述FRT序列如SEQ ID NO.1<4426>～<4459>，人Ig内含子序列包括SEQ ID NO.1<4460>～<4717>；或者FRT序列如SEQ ID NO.3<4387>～<4420>，人Ig内含子序列包括SEQ ID NO.3<4421>～<4678>。
  8. 如权利要求1-5任一所述的核酸分子，所述核酸分子中宿主动物IgM恒定区的基因片段与人免疫球蛋白IgM基因或其片段，以及与FRT、人Ig内含子连接结构如图1-2所示。
  9. 如权利要求1-8任一所述的核酸分子，所述人免疫球蛋白基因或其片段敲除了人IgHCμ的CH2部分序列、CH3、CH4、TM1和TM2序列以及其之间的序列和相关PolyA信号序列。
  10. 如权利要求1-9任一所述的核酸分子，所述人免疫球蛋白基因或其片段包括IgH的V区基因、D区基因、J区基因。
  11. 如权利要求10所述的核酸分子，所述人免疫球蛋白重链基因还包括hCγ、hCα、hCδ和/或hCξ重链基因和/或hLCR。
  12. 12.一种载体，包含如权利要求1-11任意所述核酸分子。
  13. 13.一种细胞，包含如权利要求1-11任意所述核酸分子或权利要求12所述载体。
  14. 14.一种人源化抗体，由权利要求1-11任一核酸分子重排、编码制得。
  15. 权利要求1-11任一所述核酸分子或权利要求12所述载体或权利要求13所述细胞在制备人源化抗体中的应用。
  16. 权利要求1-11任一所述核酸分子或权利要求12所述载体或权利要求13所述细胞在制备 转基因动物中的应用。
  17. 采用权利要求1-11任一所述核酸分子或权利要求12所述载体或权利要求13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)向宿主细胞或胚胎导入含有所述核酸分子的载体；
    (4)将含有人Ig的细胞植入宿主动物的胚胎内或体细胞克隆；
    (5)繁育杂合、纯合的转人Ig基因的动物1
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EP2820947A1 (en) * 2013-07-05 2015-01-07 B Cell Design Transgenic non-human mammal for producing chimeric human immunoglobulin E antibodies

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