WO2019104860A1 - 抗PD-1的纳米抗体PD-1/Nb52及其制备方法与应用 - Google Patents

抗PD-1的纳米抗体PD-1/Nb52及其制备方法与应用 Download PDF

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WO2019104860A1
WO2019104860A1 PCT/CN2018/074474 CN2018074474W WO2019104860A1 WO 2019104860 A1 WO2019104860 A1 WO 2019104860A1 CN 2018074474 W CN2018074474 W CN 2018074474W WO 2019104860 A1 WO2019104860 A1 WO 2019104860A1
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nanobody
seq
preparation
nucleic acid
antibody
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卢小玲
杨晓梅
段斯亮
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广西医科大学
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/74—Inducing cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the invention relates to a nano-antibody PD-1/Nb52 against PD-1 in the field of biomedicine and a preparation method and application thereof.
  • PD-1 Programmed death 1
  • PD-1 is a transmembrane glycoprotein molecule expressed on the surface of activated T cells. It is a member of the CD28 superfamily and plays a negative regulatory role in T cell proliferation in the immune response. Play an important regulatory role.
  • PD-1 is mainly expressed in activated T cells and B cells, and is a surface receptor of activated T cells.
  • PD-1 has two ligands, which are cell death-ligand 1 (PD-L1, Also known as B7-H1) and cell death-ligand 1 (PD-L2, aka B7-DC).
  • the tumor microenvironment in the body induces high expression of PD-1 molecules in infiltrating T cells, and tumor cells highly express PD-1 ligands PD-L1 and PD-L2, resulting in PD-1 pathway continuing to activate PD in the tumor microenvironment.
  • PD-1 inhibitors block the binding of PD-1 to PD-L1, block negative regulatory signals, and restore T cells to activity, thereby enhancing the immune response.
  • the highly specific, low-reactive anti-PD-1 monoclonal antibody has been recognized in several clinical trials, such as the drug produced by Merck, named Keytruda (Pembrolizumab), and the name produced by Bristol-Myers Squibb, Opdivo (Nivolumab) nivolumab. medicine.
  • Nano-antibody technology is the latest and smallest antibody molecule developed by biomedical scientists based on traditional antibodies and using molecular biology technology combined with the concept of nanoparticle science to carry out the antibody engineering revolution.
  • Hamers et al. reported that there is a heavy chain antibody in the camel body that naturally lacks the light chain and heavy chain constant region 1 (CH1), and clones its variable region to obtain a single domain antibody consisting of only one heavy chain variable region.
  • VHH variable domain of heavy chain of heavy-chain antibody
  • Nb nanobody
  • the Nanobody has the smallest antigen-binding fragment with complete function, and its crystal structure is elliptical, 2.5 nm in diameter and 4 nm in length.
  • Nanobodies have many unique properties, is very suitable for genetic modification, and has broad application prospects in the accurate diagnosis and targeted treatment of diseases.
  • Nanobodies are much simpler in chemical composition and shape than antibodies, they are not chemically hydrophobic, they are more resistant to heat and acid and alkali, and they are easier to bind to each other or to other compounds, can be encoded by a single gene, and are easily synthesized by microorganisms. .
  • Nano-antibodies are well tolerated by the environment, have high conformational stability, and have lower molecular mass, better clinical therapeutic effects, and these small protein molecules are easier to synthesize and lower in price.
  • the unique properties of Nanobodies show a broader application prospect in the accurate diagnosis of diseases and immunotargeting therapy.
  • the technical problem to be solved by the present invention is how to prepare a medicament for effectively treating tumors.
  • the present invention first provides a nanobody.
  • a Nanobody designated PD-1/Nb52, comprising an antigenic determinant complementary region CDR and a framework region FR;
  • the Nanoparticle antigenic determinant complementary region CDR is CDR1 , CDR2 and CDR3;
  • the amino acid sequence of CDR1 is amino acids 23-32 of SEQ ID No. 8 in the sequence listing;
  • the amino acid sequence of said CDR2 is positions 49-56 of SEQ ID No. 8 in the sequence listing Amino acid;
  • the amino acid sequence of the CDR3 is amino acids 94-112 of SEQ ID No. 8 in the Sequence Listing.
  • the framework region FR of the Nanobody consists of FR1, FR2, FR3 and FR4; wherein the amino acid sequence of FR1 is amino acid 1-22 of SEQ ID No. 8 in the Sequence Listing; amino acid sequence of FR2 Is the amino acids 33-48 of SEQ ID No. 8 in the Sequence Listing; the amino acid sequence of FR3 is amino acids 57-93 of SEQ ID No. 8 in the Sequence Listing; the amino acid sequence of FR4 is in the Sequence Listing Amino acids 113-123 of SEQ ID No. 8.
  • the amino acid sequence of the Nanobody PD-1/Nb52 is as shown in SEQ ID No. 8 of the Sequence Listing.
  • the invention also provides a VHH chain of PD-1 Nanobody PD-1/Nb52, comprising a framework region FR and an antigenic determinant complementary region CDR, wherein the framework region FR is selected from the group consisting of the FR amino acid sequence: SEQ FR1 shown by ID NO: 1, FR2 shown by SEQ ID NO: 2, FR3 shown by SEQ ID NO: 3, FR4 shown by SEQ ID NO: 4;
  • the amino acid sequences of the CDRs of the group are: CDR1 shown in SEQ ID NO: 5, CDR2 shown in SEQ ID NO: 6, and CDR3 shown in SEQ ID NO: 7.
  • the VHH chain of the PD-1 Nanobody PD-1/Nb52 has the amino acid sequence set forth in SEQ ID NO: 8.
  • a label as shown in Table 1 may be attached to the amino terminus or the carboxy terminus of the protein represented by amino acids 1-123 of SEQ ID No. 8 in the Sequence Listing. .
  • the Nanobody PD-1/Nb52 can be artificially synthesized, or the coding gene can be synthesized first, and then biologically expressed.
  • the gene encoding the Nanobody PD-1/Nb52 can be obtained by deleting a codon of one or several amino acid residues in the DNA sequence shown by SEQ ID No. 9 in the Sequence Listing, and/or performing one or several bases. A missense mutation of the base pair, and/or a coding sequence for the tag shown in Table 1 attached at its 5' end and/or 3' end.
  • the present invention also provides a biological material related to the nanobody PD-1/Nb52.
  • the biomaterial related to the nanobody PD-1/Nb52 provided by the present invention is any one of B1) to B12):
  • B2 an expression cassette comprising the nucleic acid molecule of B1);
  • B3 a recombinant vector comprising the nucleic acid molecule of B1);
  • B4 a recombinant vector comprising the expression cassette of B2)
  • B5 a recombinant microorganism comprising the nucleic acid molecule of B1);
  • B7 a recombinant microorganism comprising the recombinant vector of B3);
  • B9 a transgenic animal cell line comprising the nucleic acid molecule of B1);
  • B11 a transgenic animal cell line comprising the recombinant vector of B3);
  • B12 A transgenic animal cell line comprising the recombinant vector of B4).
  • the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA.
  • the expression cassette containing the nucleic acid molecule encoding the Nanobody PD-1/Nb52 described in B2) which is also referred to as the PD-1/Nb52 gene expression cassette, is capable of expressing the Nanobody in the host cell.
  • the DNA of PD-1/Nb52 which may include not only a promoter that initiates transcription of the Nanobody PD-1/Nb52 gene, but also a terminator that terminates transcription of the Nanobody PD-1/Nb52 gene.
  • the expression cassette may further comprise an enhancer sequence.
  • a recombinant vector containing the Nanobody PD-1/Nb52 gene expression cassette can be constructed using an existing expression vector.
  • the vector may be a plasmid, a cosmid, a phage or a viral vector.
  • the recombinant vector may be a recombinant vector obtained by introducing the nucleic acid molecule of B1) into pComb3.
  • the recombinant vector of B3) is a gene encoding the Nanobody PD-1/Nb52 (the nucleotide sequence is nucleus 1-369 of SEQ ID No. 9 in the Sequence Listing) The glycoside was introduced into the recombinant vector pComb3-PD-1/Nb52 obtained in pComb3, and the recombinant vector pComb3-PD-1/Nb52 expressed the Nanobody PD-1/Nb52 shown in SEQ ID No. 8.
  • the microorganism may be yeast, bacteria, algae or fungi.
  • the transgenic animal cell line does not include a propagation material; and the recombinant microorganism may be a recombinant microorganism obtained by introducing the nucleic acid molecule of B1) into Escherichia coli WK6.
  • One of ordinary skill in the art can readily mutate the nucleotide sequence of the Nanobody PD-1/Nb52 of B1) of the present invention using known methods, such as directed evolution and point mutation.
  • Those artificially modified, having nucleotides which are 75% or more identical to the nucleotide sequence of the Nanobody PD-1/Nb52 of the B1) of the present invention, as long as the Nanobody PD-1/ Nb52 and having Nanobody PD-1/Nb52 activity both are nucleotide sequences derived from the present invention and are equivalent to the sequences of the present invention.
  • identity refers to sequence similarity to a native nucleic acid sequence. “Identity” includes 75% or more, or 85% or more, or 90% or more, or 95% or more with the nucleotide sequence of the protein of SEQ ID No. 8 of the present invention. Highly identical nucleotide sequence. Identity can be evaluated using the naked eye or computer software. Using computer software, the identity between two or more sequences can be expressed in percentage (%), which can be used to evaluate the identity between related sequences.
  • the above 75% or more of the identity may be 75%, 80%, 85%, 90% or 95% or more.
  • nucleic acid molecule of B1) is 1) or 2) or 3) below:
  • nucleotide sequence is the cDNA molecule or DNA molecule of SEQ ID No. 9 in the Sequence Listing;
  • the present invention also provides a derivative antibody of the Nanobody PD-1/Nb52.
  • the derivative antibody of the Nanobody PD-1/Nb52 provided by the present invention is as follows a) or b) or c) or d) or e):
  • a fusion antibody comprising a) the single chain antibody
  • the present invention also provides a preparation method of the nano-antibody PD-1/Nb52.
  • the preparation method of the nano-antibody PD-1/Nb52 provided by the invention comprises introducing a nucleic acid molecule encoding the nano-antibody PD-1/Nb52 into a recipient cell to obtain a transgene expressing the nano-antibody PD-1/Nb52.
  • the cells were cultured to obtain the Nanobody PD-1/Nb52.
  • the nucleotide sequence of the nucleic acid molecule encoding the Nanobody PD-1/Nb52 is shown in SEQ ID No. 9 of the Sequence Listing.
  • the recipient cell may be a microbial cell, such as Escherichia coli, specifically Escherichia coli WK6.
  • the present invention also provides the use of any of the following A1-A8:
  • A1 The use of the nanobody PD-1/Nb52 in the preparation of a tumor suppressor or a tumor cell inhibitor;
  • the preparation method of the nano-antibody PD-1/Nb52 is used for preparing a tumor suppressor or a tumor cell inhibitor
  • Nanobody PD-1/Nb52 in the preparation of a product for inhibiting PD-1 activity or promoting T cell proliferation
  • A6 The use of the biological material in the preparation of a product for inhibiting PD-1 activity or promoting T cell proliferation;
  • the preparation method of the nanobody PD-1/Nb52 is used for preparing a product for inhibiting PD-1 activity or promoting T cell proliferation.
  • the above products may be drugs.
  • the present invention provides a PD-1-resistant Nanobody PD-1/Nb52, a nucleotide sequence encoding the Nanobody PD-1/Nb52, and a host cell, and a preparation method and application thereof.
  • the Nanobody PD-1/Nb52 can be highly expressed in E. coli, applied to the development of PD-1 molecular detection reagents, preparation of tumor inhibitors or tumor cell inhibitors, and preparation of drugs for inhibiting PD-1 activity and promoting T cell proliferation. .
  • Figure 1 is a DNA electrophoresis map of a nanobody.
  • the DNA bands from left to right are: 2000 bp molecular marker, the second is PCR product, and the PCR product band is about 400 bp;
  • Lane M represents protein molecular weight Marker in KDa
  • Figure 3A shows the results of binding experiments of Nanobody PD-1/Nb52 with 293T cells not transfected with PD-1
  • Figure 3B shows the results of binding experiments of Nanobody PD-1/Nb52 with 293T cells transfected with PD-1.
  • the present invention provides a nanobody derived from camel, the name of which is PD-1/Nb52, and the amino acid sequence of the Nanobody PD-1/Nb52 is shown in SEQ ID No. 8 of the Sequence Listing, and is represented by SEQ ID No. The nucleotide sequence of 9 is encoded.
  • the nucleotide electrophoresis pattern of the Nanobody PD-1/Nb52 is shown in Figure 1.
  • the first channel is a 2000 bp molecular marker, and the remaining channels are PCR products.
  • the PCR product band is about 400 bp.
  • the DNA fragment between the PstI and NotI recognition sequences of the vector pComb3 (Biovector product) was replaced with the DNA molecule shown in SEQ ID No. 9, and the other sequences were unchanged, and the recombinant vector pComb3-PD-1/Nb52, pComb3-PD was obtained.
  • -1/Nb52 differs from pComb3 only in that the DNA fragment between the PstI and NotI recognition sequences of pComb3 is replaced with the DNA molecule shown by SEQ ID No. 9.
  • the recombinant vector pComb3-PD-1/Nb52 expressed the Nanobody PD-1/Nb52 shown in SEQ ID No. 8.
  • pComb3-PD-1/Nb52 was introduced into Escherichia coli WK6 to obtain recombinant strain WK6-pComb3-PD-1/Nb52.
  • step (2) Take 1 mL of the step (2) The culture medium cultured overnight is inoculated into 300-350 mL TB culture solution, and cultured at 25-37 ° C on a shaker until the OD value reaches 0.6-1, and IPTG is added to obtain WK6-pComb3-PD- 1/Nb52 medium, the concentration of IPTG in WK6-pComb3-PD-1/Nb52 medium was 1 mM, and the WK6-pComb3-PD-1/Nb52 medium was shaken at 20-30 °C (shaker) Incubate overnight (10-14 hours) at 220-250 rpm to obtain WK6-pComb3-PD-1/Nb52 induction solution;
  • step (3) The WK6-pComb3-PD-1/Nb52 induction solution of step (3) is centrifuged at 4 ° C to collect the cells;
  • Nanoscale Res Lett 2014, 9:528 Nanoparticle PD-1/Nb52 was prepared by nickel column ion affinity chromatography in .
  • the SDA-PAGE electropherograms of the Nanobody PD-1/Nb52 are shown in Figure 2.
  • the size of the Nanobody PD-1/Nb52 is about 15KDa. The results show that the purity of the nano-antibody PD-1/Nb52 obtained by the above method can reach more than 90%.
  • Nanobody PD-1/Nb52 The binding rate of Nanobody PD-1/Nb52 to PD-1 was detected by 293T cells stably transfected with PD-1, and the Nanobody PD-1/Nb52 (1 ⁇ g) of Example 1 was added to 1-6 ⁇ 10 6 293T cells. Incubate in the dark at 4 °C for 20-40 min, wash twice with PBS, add 5 ⁇ l of PE anti-HA tag antibody (abcam, Clone: 20B12) for 20-40 min at 4 °C, wash twice with PBS, and transfer the sample to BACKMAN flow. The cytometer, the results are shown in Fig. 3B, and 293T cells not transfected with PD-1 were used as controls as shown in Fig. 3A.
  • Figure 3A is the percentage of binding of the blank control and PD-1 Nanobody PD-1/Nb52 to 293T cells not transfected with PD-1, respectively;
  • Figure 3B is the blank control and PD-1 Nanobody PD-1/Nb52, respectively. Percentage of binding of PD-1 to 293T cells; in Figures 3A and 3B, the horizontal axis is the fluorescence intensity (PE), the vertical axis is the number percent (% of Max), S2 is the blank control, and S1 is the PD-1 Nanobody PD-1. /Nb52. The results shown in the figure show that PD-1 Nanobody PD-1/Nb52 binds well to 293T cells stably transfected with PD-1.
  • PE fluorescence intensity
  • S2 is the blank control
  • S1 is the PD-1 Nanobody PD-1. /Nb52.

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Abstract

公开了抗PD-1的纳米抗体PD-1/Nb52及其制备方法与应用。该纳米抗体PD-1/Nb52能够在大肠杆菌内高效表达，可用于制备肿瘤抑制剂或肿瘤细胞抑制剂、抑制PD-1活性或促进T细胞增殖的药物。

Description

抗PD-1的纳米抗体PD-1/Nb52及其制备方法与应用 技术领域
本发明涉及生物医学领域中抗PD-1的纳米抗体PD-1/Nb52及其制备方法与应用。
背景技术
程序性死亡受体1(programmed death 1，PD-1)是活化T细胞表面表达的一种跨膜糖蛋白分子，属CD28超家族成员，对T细胞增殖起负性调节作用，在免疫应答中起重要调节作用。PD-1主要在激活的T细胞和B细胞中表达，是激活型T细胞的一种表面受体，PD-1有两个配体，分别是细胞程式死亡-配体1(PD-L1，又名B7-H1)和细胞程式死亡-配体1(PD-L2，又名B7-DC)。机体内的肿瘤微环境会诱导浸润的T细胞高表达PD-1分子，肿瘤细胞会高表达PD-1的配体PD-L1和PD-L2，导致肿瘤微环境中PD-1通路持续激活PD-L1与PD-1联接后，T细胞功能被抑制，不能向免疫系统发出攻击肿瘤的信号。PD-1抑制剂可以阻断PD-1与PD-L1的结合，阻断负向调控信号，使T细胞恢复活性，从而增强免疫应答。高特异性、低反应性的抗PD-1单抗已在多项临床试验中获得肯定，例如默沙东公司生产的名称为Keytruda(Pembrolizumab)的药物，以及施贵宝公司生产的名称为Opdivo(Nivolumab)nivolumab的药物。
但是，抗体药物应用存在很多问题，比如研发周期长，生产成本过高；难以大规模生产；稳定性差易降解，贮存成本高；容易被污染，维护成本费用高昂；并具有免疫原性等，限制了其在临床上的应用范围。
纳米抗体技术，是生物医学科学家在传统抗体的基础上，运用分子生物学技术结合纳米粒子科学的概念进行的抗体工程革命，从而研 发出的最新和最小的抗体分子。1993年Hamers等报道，骆驼体内存在着天然缺失轻链和重链恒定区1(CH1)的重链抗体，克隆其可变区得到只由一个重链可变区组成的单域抗体，称为VHH(variable domain of heavy chain of heavy-chain antibody)，现已被重新命名为“纳米抗体”(nanobody，Nb)。纳米抗体具有完整功能的最小的抗原结合片段，其晶体结构呈椭圆形，直径2.5nm，长4nm。Nb具有许多独特的性质，很适合进行基因改造，在疾病的精确诊断和靶向治疗等方面展现了广阔的应用前景。纳米抗体在化学组成和形状上比抗体简单许多，不具有化学疏水性，其抗热性和抗酸碱性更强，更容易相互结合或与其他化合物结合，能被单基因编码，容易用微生物合成。纳米抗体对环境具有良好的耐受性，具备高度的构象稳定性，而且分子质量更小，临床的治疗效果更好，同时这些小蛋白分子更容易合成，价格也更低。纳米抗体的独特的性质，使其在疾病的精确诊断和免疫靶向治疗等方面展现了更为广阔的应用前景。
发明内容
本发明所要解决的技术问题是，如何制备有效治疗肿瘤的药物。
为解决上述技术问题，本发明首先提供了纳米抗体。
本发明的第一方面，提供了一种纳米抗体，称为PD-1/Nb52，该纳米抗体包含抗原决定簇互补区CDR和骨架区FR；所述纳米抗体的抗原决定簇互补区CDR由CDR1、CDR2和CDR3组成；所述CDR1的氨基酸序列为序列表中SEQ ID No.8的第23-32位氨基酸；所述CDR2的氨基酸序列为序列表中SEQ ID No.8的第49-56位氨基酸；所述CDR3的氨基酸序列为序列表中SEQ ID No.8的第94-112位氨基酸。
优选地，该纳米抗体的骨架区FR由FR1、FR2、FR3和FR4组成；其中所述FR1的氨基酸序列为序列表中SEQ ID No.8的第1-22位氨基酸；所述FR2的氨基酸序列为序列表中SEQ ID No.8的第33-48位氨基酸；所述FR3的氨基酸序列为序列表中SEQ ID No.8的第57-93位 氨基酸；所述FR4的氨基酸序列为序列表中SEQ ID No.8的第113-123位氨基酸。
优选地，所述纳米抗体PD-1/Nb52的氨基酸序列如序列表中SEQ ID No.8所示。
本发明也相应提供了一种PD-1纳米抗体PD-1/Nb52的VHH链，包括骨架区FR和抗原决定簇互补区CDR，所述骨架区FR选自下组的FR的氨基酸序列：SEQ ID NO：1所示的FR1，SEQ ID NO：2所示的FR2，SEQ ID NO：3所示的FR3，SEQ ID NO：4所示的FR4；所述抗原决定簇互补区CDR选自下组的CDR的氨基酸序列：SEQ ID NO：5所示的CDR1，SEQ ID NO：6所示的CDR2，SEQ ID NO：7所示的CDR3。
优选地，所述的PD-1纳米抗体PD-1/Nb52的VHH链，它具有SEQ ID NO：8所示的氨基酸序列。
为了使所述纳米抗体PD-1/Nb52便于纯化，可在序列表中SEQ ID No.8的第1-123位氨基酸所示的蛋白质的氨基末端或羧基末端连接上如表1所示的标签。
表1、标签的序列
标签 残基 序列
Poly-Arg 5-6(通常为5个) RRRRR
Poly-His 2-10(通常为6个) HHHHHH
FLAG 8 DYKDDDDK
Strep-tag II 8 WSHPQFEK
c-myc 10 EQKLISEEDL
HA 9 YPYDVPDYA
本发明另外一方面，所述纳米抗体PD-1/Nb52可人工合成，也可 先合成其编码基因，再进行生物表达得到。所述纳米抗体PD-1/Nb52的编码基因可通过将序列表中SEQ ID No.9所示的DNA序列中缺失一个或几个氨基酸残基的密码子，和/或进行一个或几个碱基对的错义突变，和/或在其5′端和/或3′端连上表1所示的标签的编码序列得到。
为解决上述技术问题，本发明还提供了与所述纳米抗体PD-1/Nb52相关的生物材料。
本发明所提供的与所述纳米抗体PD-1/Nb52相关的生物材料，为B1)至B12)中的任一种：
B1)编码所述纳米抗体PD-1/Nb52的核酸分子；
B2)含有B1)所述核酸分子的表达盒；
B3)含有B1)所述核酸分子的重组载体；
B4)含有B2)所述表达盒的重组载体；
B5)含有B1)所述核酸分子的重组微生物；
B6)含有B2)所述表达盒的重组微生物；
B7)含有B3)所述重组载体的重组微生物；
B8)含有B4)所述重组载体的重组微生物；
B9)含有B1)所述核酸分子的转基因动物细胞系；
B10)含有B2)所述表达盒的转基因动物细胞系；
B11)含有B3)所述重组载体的转基因动物细胞系；
B12)含有B4)所述重组载体的转基因动物细胞系。
上述生物材料中，所述核酸分子可以是DNA，如cDNA、基因组DNA或重组DNA；所述核酸分子也可以是RNA，如mRNA或hnRNA等。
上述生物材料中，B2)所述的含有编码所述纳米抗体PD-1/Nb52的核酸分子的表达盒，又称为PD-1/Nb52基因表达盒，是指能够在宿主细胞中表达纳米抗体PD-1/Nb52的DNA，该DNA不但可包括启动纳米抗体PD-1/Nb52基因转录的启动子，还可包括终止纳米抗体PD-1/Nb52基因转录的终止子。进一步，所述表达盒还可包括增强子序 列。
可用现有的表达载体构建含有所述纳米抗体PD-1/Nb52基因表达盒的重组载体。
上述生物材料中，所述载体可为质粒、黏粒、噬菌体或病毒载体。
上述生物材料中，所述重组载体可为将B1)所述核酸分子导入到pComb3中得到的重组载体。在本发明的一个实施例中，B3)所述重组载体为将所述纳米抗体PD-1/Nb52的编码基因(核苷酸序列为序列表中SEQ ID No.9的第1-369位核苷酸)导入pComb3中得到的重组载体pComb3-PD-1/Nb52，重组载体pComb3-PD-1/Nb52表达SEQ ID No.8所示的纳米抗体PD-1/Nb52。
上述生物材料中，所述微生物可为酵母、细菌、藻或真菌。
上述生物材料中，所述转基因动物细胞系不包括繁殖材料；所述重组微生物可为将B1)所述核酸分子导入到大肠杆菌WK6中得到的重组微生物。
本领域普通技术人员可以很容易地采用已知的方法，例如定向进化和点突变的方法，对本发明的B1)所述纳米抗体PD-1/Nb52的核苷酸序列进行突变。那些经过人工修饰的，具有与本发明的B1)所述纳米抗体PD-1/Nb52的核苷酸序列75％或75％以上同一性的核苷酸，只要编码所述纳米抗体PD-1/Nb52且具有纳米抗体PD-1/Nb52活性，均是衍生于本发明的核苷酸序列并且等同于本发明的序列。
这里使用的术语“同一性”指与天然核酸序列的序列相似性。“同一性”包括与本发明的编码SEQ ID No.8所示的蛋白质的核苷酸序列具有75％或更高，或85％或更高，或90％或更高，或95％或更高同一性的核苷酸序列。同一性可以用肉眼或计算机软件进行评价。使用计算机软件，两个或多个序列之间的同一性可以用百分比(％)表示，其可以用来评价相关序列之间的同一性。
上述75％或75％以上同一性，可为75％、80％、85％、90％或95％以上的同一性。
上述生物材料中，B1)所述核酸分子为下述1)或2)或3)：
1)核苷酸序列是序列表中SEQ ID No.9的cDNA分子或DNA分子；
2)与1)限定的核苷酸序列具有75％或75％以上同一性，且编码所述纳米抗体PD-1/Nb52的cDNA分子或基因组DNA分子；
3)在严格条件下与1)限定的核苷酸序列杂交，且编码所述纳米抗体PD-1/Nb52的cDNA分子或基因组DNA分子。
为解决上述技术问题，本发明还提供了所述纳米抗体PD-1/Nb52的衍生抗体。
本发明所提供的所述纳米抗体PD-1/Nb52的衍生抗体，为下述a)或b)或c)或d)或e)：
a)含有所述纳米抗体PD-1/Nb52的单链抗体；
b)含有a)所述单链抗体的融合抗体；
c)含有所述纳米抗体PD-1/Nb52的融合抗体；
d)含有所述纳米抗体PD-1/Nb52的Fab；
e)含有所述纳米抗体PD-1/Nb52的完整抗体。
为解决上述技术问题，本发明还提供了所述纳米抗体PD-1/Nb52的制备方法。
本发明所提供的所述纳米抗体PD-1/Nb52的制备方法，包括将编码所述纳米抗体PD-1/Nb52的核酸分子导入受体细胞得到表达所述纳米抗体PD-1/Nb52的转基因细胞，培养所述转基因细胞，得到所述纳米抗体PD-1/Nb52。
上述纳米抗体PD-1/Nb52的制备方法中，所述编码所述纳米抗体PD-1/Nb52的核酸分子的核苷酸序列如序列表中SEQ ID No.9所示。
上述纳米抗体PD-1/Nb52的制备方法中，所述受体细胞可为微生物细胞，如大肠杆菌，具体可为大肠杆菌WK6。
为解决上述技术问题，本发明还提供了下述A1-A8中的任一种用途：
A1、所述纳米抗体PD-1/Nb52在制备肿瘤抑制剂或肿瘤细胞抑制剂中的应用；
A2、所述生物材料在制备肿瘤抑制剂或肿瘤细胞抑制剂中的应用；
A3、所述纳米抗体PD-1/Nb52的衍生抗体在制备肿瘤抑制剂或肿瘤细胞抑制剂中的应用；
A4、所述纳米抗体PD-1/Nb52的制备方法在制备肿瘤抑制剂或肿瘤细胞抑制剂中的应用；
A5、所述纳米抗体PD-1/Nb52在制备抑制PD-1活性或促进T细胞增殖产品中的应用；
A6、所述生物材料在制备抑制PD-1活性或促进T细胞增殖产品中的应用；
A7、所述衍生抗体在制备抑制PD-1活性或促进T细胞增殖产品中的应用；
A8、所述纳米抗体PD-1/Nb52的制备方法在制备抑制PD-1活性或促进T细胞增殖产品中的应用。
上述产品可为药物。
扩增编码序列表中SEQ ID No.8所示的氨基酸序列或其任一片段氨基酸序列的核酸分子的引物对，也属于本发明的保护范围。
本发明提供了一种抗PD-1的纳米抗体PD-1/Nb52、编码该纳米抗体PD-1/Nb52的核苷酸序列及宿主细胞，以及其制备方法和应用。该纳米抗体PD-1/Nb52能够在大肠杆菌内高效表达，应用于PD-1分子检测试剂的研发，制备肿瘤抑制剂或肿瘤细胞抑制剂以及制备抑制PD-1活性和促进T细胞增殖的药物。
附图说明
图1是纳米抗体的DNA电泳图，从左到右凝胶孔的DNA条带分别是：第一道为2000bp的分子标记，第二道为PCR产物，PCR产物带约为400bp；
图2是PD-1纳米抗体PD-1/Nb52经镍柱树脂凝胶亲和层析纯化后的SDS-PAGE的电泳图；泳道M表示蛋白分子量Marker，单位为KDa；
图3A为纳米抗体PD-1/Nb52与未转染PD-1的293T细胞的结合实验结果；图3B为纳米抗体PD-1/Nb52与转染PD-1的293T细胞的结合实验结果。
具体实施方式
下面结合具体实施方式对本发明进行进一步的详细描述，给出的实施例仅为了阐明本发明，而不是为了限制本发明的范围。
下述实施例中的实验方法，如无特殊说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
下述实施例中的大肠杆菌WK6经东南大学生命科学研究院万亚坤实验室同意后，公众可从广西医科大学获得该生物材料，该生物材料只为重复本发明的相关实验所用，不可作为其它用途使用。
实施例1、纳米抗体的制备
本发明提供了来源于骆驼的1种纳米抗体，其名称为PD-1/Nb52，该纳米抗体PD-1/Nb52的氨基酸序列如序列表中SEQ ID No.8所示，由SEQ ID No.9的核苷酸序列编码。
纳米抗体PD-1/Nb52的核苷酸电泳图如图1所示，其中第一道为2000bp的分子标记，其余孔道为PCR产物，PCR产物带约为400bp。
将载体pComb3(Biovector产品)的PstI和NotI识别序列间的DNA片段替换为SEQ ID No.9所示的DNA分子，其他序列均不变，得到重组载体pComb3-PD-1/Nb52，pComb3-PD-1/Nb52与pComb3的差别仅在于将pComb3的PstI和NotI识别序列间的DNA片段替换为SEQ ID No.9所示的DNA分子。重组载体pComb3-PD-1/Nb52表达SEQ ID No.8所示的纳米抗体PD-1/Nb52。将pComb3-PD-1/Nb52导入大肠杆菌WK6中，得到重组菌WK6-pComb3-PD-1/Nb52。
纳米抗体的具体制备步骤如下：
(1)将WK6-pComb3-PD-1/Nb52涂布在含有氨苄青霉素和葡萄糖的LB平板(LB平板中，氨苄青霉素和葡萄糖的浓度分别为100μg/mL和20mg/mL)上，25-37℃培养过夜(10-14小时)；
(2)挑选单个菌落接种在5mL含有氨苄青霉素的LB培养液(LB培养液中，氨苄青霉素的浓度为100μg/mL)中，25-37℃摇床培养过夜(10-14小时)；
(3)取1mL步骤(2)培养过夜的培养液接种至300-350mL TB培养液中，25-37℃摇床培养至OD值达到0.6-1时，加入IPTG，得到WK6-pComb3-PD-1/Nb52培养液，使WK6-pComb3-PD-1/Nb52培养液中IPTG的浓度为1mM，将WK6-pComb3-PD-1/Nb52培养液在20-30℃下于摇床(摇床的转速为220-250rpm)上培养过夜(10-14小时)，得到WK6-pComb3-PD-1/Nb52诱导液；
(4)将步骤(3)的WK6-pComb3-PD-1/Nb52诱导液于4℃下离心，收集菌体；
(5)利用文献(Zhu M,Hu Y,Li G,Ou W,Mao P,Xin S,Wan Y:Combining magnetic nanoparticle with biotinylated nanobodies for rapid and sensitive detection of influenza H3N2.Nanoscale Res Lett 2014,9:528.)中的渗透法，获得抗体粗提液；
(6)利用文献(Zhu M,Hu Y,Li G,Ou W,Mao P,Xin S,Wan Y:Combining magnetic nanoparticle with biotinylated nanobodies for rapid and sensitive detection of influenza H3N2.Nanoscale Res Lett 2014,9:528.)中的镍柱离子亲和层析法制备纳米抗体PD-1/Nb52。纳米抗体PD-1/Nb52的SDA-PAGE电泳图分别如图2，纳米抗体PD-1/Nb52的大小约为15KDa。结果显示，上述方法得到的纳米抗体PD-1/Nb52的纯度可以达到90％以上。
实施例2、纳米抗体与PD-1结合率的测定
纳米抗体PD-1/Nb52与PD-1结合率测定(直接法)
用稳转PD-1的293T细胞检测纳米抗体PD-1/Nb52与PD-1结合率，将实施例1的纳米抗体PD-1/Nb52(1μg)加入1-6×10 6个上述293T细胞中4℃避光孵育20-40min，PBS洗涤2次后，加入5μl PE anti-HA tag antibody(abcam，Clone:20B12)4℃孵育20-40min，PBS洗涤2次后，将样品上BACKMAN流式细胞仪，结果如图3B所示，未转染PD-1的293T细胞作为对照如图3A所示。图3A是空白对照和PD-1纳米抗体PD-1/Nb52分别与未转染PD-1的293T细胞的结合百分率；图3B是空白对照和PD-1纳米抗体PD-1/Nb52分别稳转PD-1的293T细胞的结合百分率；图3A和3B中横轴为荧光强度(PE)，纵轴为数量百分比(％of Max)，S2代表空白对照，S1代表PD-1纳米抗体PD-1/Nb52。图示结果可以看出PD-1纳米抗体PD-1/Nb52能够很好的与稳转PD-1的293T细胞结合。
最后应说明的是：以上实施例仅用以说明本发明的技术方案，而非对其限制；尽管参照前述实施例对本发明进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本发明各实施例技术方案的精神和范围。
Figure PCTCN2018074474-appb-000001
Figure PCTCN2018074474-appb-000002
Figure PCTCN2018074474-appb-000003
Figure PCTCN2018074474-appb-000004
Figure PCTCN2018074474-appb-000005
Figure PCTCN2018074474-appb-000006

Claims (10)

  1. 纳米抗体，包含抗原决定簇互补区；其特征在于：
    所述纳米抗体的抗原决定簇互补区由CDR1、CDR2和CDR3组成；
    所述CDR1的氨基酸序列为序列表中SEQ ID No.8的第23-32位氨基酸；
    所述CDR2的氨基酸序列为序列表中SEQ ID No.8的第49-56位氨基酸；
    所述CDR3的氨基酸序列为序列表中SEQ ID No.8的第94-112位氨基酸。
  2. 根据权利要求1所述的纳米抗体，其特征在于：所述纳米抗体由所述抗原决定簇互补区和所述骨架区组成。
  3. 根据权利要求1或2所述的纳米抗体，其特征在于：所述纳米抗体的氨基酸序列如序列表中SEQ ID No.8所示。
  4. 与权利要求1或2或3所述纳米抗体相关的生物材料，为B1)至B12)中的任一种：
    B1)编码权利要求1或2或3所述纳米抗体的核酸分子；
    B2)含有B1)所述核酸分子的表达盒；
    B3)含有B1)所述核酸分子的重组载体；
    B4)含有B2)所述表达盒的重组载体；
    B5)含有B1)所述核酸分子的重组微生物；
    B6)含有B2)所述表达盒的重组微生物；
    B7)含有B3)所述重组载体的重组微生物；
    B8)含有B4)所述重组载体的重组微生物；
    B9)含有B1)所述核酸分子的转基因动物细胞系；
    B10)含有B2)所述表达盒的转基因动物细胞系；
    B11)含有B3)所述重组载体的转基因动物细胞系；
    B12)含有B4)所述重组载体的转基因动物细胞系。
  5. 根据权利要求4所述的生物材料，其特征在于：B1)所述核酸分子为下述1)或2)或3)：
    1)核苷酸序列是序列表中SEQ ID No.9的cDNA分子或DNA分子；
    2)与1)限定的核苷酸序列具有75％或75％以上同一性，且编码权利要求1或2或3所述纳米抗体的cDNA分子或基因组DNA分子；
    3)在严格条件下与1)限定的核苷酸序列杂交，且编码权利要求1或2或3所述纳米抗体的cDNA分子或基因组DNA分子。
  6. 权利要求1或2或3所述纳米抗体的衍生抗体，为下述a)或b)或c)或d)或e)：
    a)含有权利要求1或2或3所述纳米抗体的单链抗体；
    b)含有a)所述单链抗体的融合抗体；
    c)含有权利要求1或2或3所述纳米抗体的融合抗体；
    d)含有权利要求1或2或3所述纳米抗体的Fab；
    e)含有权利要求1或2或3所述纳米抗体的完整抗体。
  7. 权利要求1或2或3所述纳米抗体的制备方法，包括将编码权利要求1或2或3所述纳米抗体的核酸分子导入受体细胞得到表达所述纳米抗体的转基因细胞，培养所述转基因细胞，得到所述纳米抗体。
  8. 根据权利要求7所述的方法，其特征在于：所述编码权利要求1或2或3所述纳米抗体的核酸分子的核苷酸序列如序列表中SEQ ID No.9所示。
  9. 根据权利要求7所述的方法，其特征在于：所述受体细胞为微生物细胞。
  10. 下述A1-A8中的任一种用途：
    A1、权利要求1或2或3所述纳米抗体在制备肿瘤抑制剂或肿瘤细胞抑制剂中的应用；
    A2、权利要求4-5中任一所述生物材料在制备肿瘤抑制剂或肿瘤细胞抑制剂中的应用；
    A3、权利要求6所述衍生抗体在制备肿瘤抑制剂或肿瘤细胞抑制剂中的应用；
    A4、权利要求7或8所述方法在制备肿瘤抑制剂或肿瘤细胞抑制剂中的应用；
    A5、权利要求1或2或3所述纳米抗体在制备抑制PD-1活性或促进T细胞增殖产品中的应用；
    A6、权利要求4-5中任一所述生物材料在制备抑制PD-1活性或促进T细胞增殖产品中的应用；
    A7、权利要求6所述衍生抗体在制备抑制PD-1活性或促进T细胞增殖产品中的应用；
    A8、权利要求7或8或9所述方法在制备抑制PD-1活性或促进T细胞增殖产品中的应用。
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CN111423510B (zh) * 2019-01-10 2024-02-06 迈威(上海)生物科技股份有限公司 重组抗人pd-1抗体及其应用
CN110423277B (zh) * 2019-03-21 2022-09-06 南京东极医药科技有限公司 Pd-1的纳米抗体及其临床应用
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105175544A (zh) * 2015-10-20 2015-12-23 安徽瀚海博兴生物技术有限公司 一种抗pd-1人源化单克隆抗体及其应用
CN106432494A (zh) * 2015-08-11 2017-02-22 哈尔滨誉衡药业股份有限公司 新型抗‑pd‑1抗体
CN107074947A (zh) * 2014-08-05 2017-08-18 马布奎斯特公司 结合至pd‑1的免疫试剂

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105061597B (zh) * 2015-06-09 2016-04-27 北京东方百泰生物科技有限公司 一种抗pd-1的单克隆抗体及其获得方法
WO2017024515A1 (en) * 2015-08-11 2017-02-16 Wuxi Biologics (Cayman) Inc. Novel anti-pd-1 antibodies
CN106267191A (zh) * 2016-09-03 2017-01-04 山西纳安生物科技有限公司 纳米抗体生物药透皮给药制剂系统及制备方法和应用

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107074947A (zh) * 2014-08-05 2017-08-18 马布奎斯特公司 结合至pd‑1的免疫试剂
CN106432494A (zh) * 2015-08-11 2017-02-22 哈尔滨誉衡药业股份有限公司 新型抗‑pd‑1抗体
CN105175544A (zh) * 2015-10-20 2015-12-23 安徽瀚海博兴生物技术有限公司 一种抗pd-1人源化单克隆抗体及其应用

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