WO2018176732A1 - 与cd56分子特异性结合的多肽及其应用 - Google Patents

与cd56分子特异性结合的多肽及其应用 Download PDF

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WO2018176732A1
WO2018176732A1 PCT/CN2017/099487 CN2017099487W WO2018176732A1 WO 2018176732 A1 WO2018176732 A1 WO 2018176732A1 CN 2017099487 W CN2017099487 W CN 2017099487W WO 2018176732 A1 WO2018176732 A1 WO 2018176732A1
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polypeptide
cells
tumor
cell
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张雁
黄鸿兴
赵龙
汪华
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中山大学
广州一代医药科技有限公司
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0646—Natural killers cells [NK], NKT cells
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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    • C12N5/0602—Vertebrate cells
    • C12N5/0693—Tumour cells; Cancer cells

Definitions

  • the invention relates to the field of biomedicine, in particular to a polypeptide capable of specifically binding to a CD56 molecule and an application thereof.
  • the CD56 molecule also known as the neural cell adhesion molecule (NCAM), is a member of the immunoglobulin superfamily.
  • CD56 is a glycoprotein containing a polypeptide chain, and the extracellular portion of the CD56 molecule includes five Ig-like domains and two type III fibronectin-like domains. Due to the different splicing methods of mRNA, three different molecular subtypes are finally formed: 1.120kDa subtype, which is anchored on the cell membrane by GPI (glycosyl-phosphatidylinositol), mainly expressed in normal tissues and well-differentiated tissues, such as NK cells. , nerve tissue, nerve-muscle junction, neuroendocrine tissue, endocrine tissue, etc.
  • GPI glycoprotein containing a polypeptide chain
  • fibronectin-like domains Due to the different splicing methods of mRNA, three different molecular subtypes are finally formed: 1.120kDa subtype, which is anchored on the
  • 2.140kDa and 180kDa subtypes comprising a transmembrane domain, mainly expressed on undifferentiated tissues or malignant tumor cells, such as tumors derived from neuroectodermal (eg retinoblastoma, astrocytoma, neuroblastoma, etc.) ), endocrine tumors, small cell lung cancer, NK/T cell lymphoma/leukemia, multiple myeloma, and acute myeloid leukemia. Therefore, CD56 has been used as a marker for NK cells and the aforementioned tumor cells, and can be used for cell labeling, sorting, and tumor molecular diagnosis.
  • a transmembrane domain mainly expressed on undifferentiated tissues or malignant tumor cells, such as tumors derived from neuroectodermal (eg retinoblastoma, astrocytoma, neuroblastoma, etc.) ), endocrine tumors, small cell lung cancer, NK/T cell lympho
  • CD56 molecules not only play a mechanical anchoring role in cell adhesion, but also an important receptor on the cell surface, regulating the downstream signaling pathways within the cell.
  • Ligands that have been found in the literature include: CD56 molecules (same binding), L1-CAM, FGFR, GNDF, GFR ⁇ , extracellular matrix components such as CSPG, SHPG, collagen, heparin, and the like.
  • CD56 plays an important role in the differentiation process of the nervous system and synaptic plasticity, and the downstream signaling can inhibit the proliferation of astrocytes and neurite outgrowth precursor cells, and more importantly, The downstream signal of CD56 can affect the biological activity of malignant tumor cells, and the loss of CD56 is closely related to the metastatic potential and poor prognosis of some malignant tumors. Therefore, ligands for CD56 molecules (such as antibodies) can act as a potential inhibitor to inhibit tumor growth.
  • CD56 antibodies are involved in the study of tumor therapy targeting CD56 molecules.
  • Previous murine antibodies produced human anti-mouse antibodies (HAMA) in clinical treatment, resulting in therapeutic The time in which the antibody acts in vivo is shortened, its anti-tumor effect is attenuated, and other side effects may be caused.
  • Some researchers have used genetic engineering methods to engineer mouse-derived antibodies to attenuate the HAMA response.
  • anti-CD56 antibody as a targeted transport molecule, combined with radioisotopes, cytotoxic drugs, cytokines, and double titer antibodies (which can simultaneously bind tumor cells).
  • Surface tumor-associated antigens and activation molecules on the surface of immune cells enhance the killing effect on tumor cells. Therefore, the key to the clinical application of tumor immunotherapy is to find highly specific antibodies that bind to the CD56 molecule.
  • the present invention provides a polypeptide which can specifically bind to a CD56 molecule, which has the characteristics of easy preparation, small molecular weight and high application safety.
  • the present invention adopts the following technical scheme: providing a polypeptide which specifically binds to a CD56 molecule, and the polypeptide includes at least one of a polypeptide No. 7 and a polypeptide No. 8, the polypeptide No. 7 and the polypeptide No. 8
  • the amino acid sequences are shown in Sequence 1 and Sequence 2 in the Sequence Listing, respectively.
  • the polypeptide provided by the invention can specifically bind to the CD56 molecule and can be produced by artificial synthesis or genetic engineering.
  • the polypeptide may also be referred to as a non-antibody binding protein.
  • the non-antibody binding protein is a feature of the antibody/antigen-specific binding, and is also referred to as a scaffolds binding protein. ).
  • Such a protein can bind to a specific molecule through a scaffold formed by a surface amino acid in a three-dimensional structure, and has a small molecular weight, a strong permeability, a heat resistance, a good stability, an easy operation, and is suitable for mass production, compared with an antibody.
  • the invention also provides a marker for CD56 positive expressing cells, the marker comprising a polypeptide as described above.
  • the polypeptides provided herein can be used to label, identify, enrich, sort, or purify CD56 positive cells, or to prepare formulations for labeling, identifying, enriching, sorting, or purifying CD56 positive cells.
  • the cells include neurons, astrocytes, Schwann cells, NK cells, and NKT cells, and also include tumors derived from neuroectodermal (eg, retinoblastoma, astrocytoma, neuroblastoma, etc.), endocrine Tumor cells of tumor, NK/T cell lymphoma and small cell lung cancer.
  • Another aspect of the invention provides a formulation for sorting CD56-positive cells, comprising a polypeptide as described above, or a polypeptide magnetic bead complex comprising the polypeptide coupled to a magnetic bead (or polypeptide coating) a magnetic bead complex formed by magnetic beads).
  • the polypeptides provided by the present invention are also useful for the diagnosis or typing or treatment of diseases, such as tumors, viral infectious diseases, parasitic diseases, leukemias, etc., and also for the preparation of preparations for the diagnosis or typing or treatment of diseases.
  • the tumor includes a neuroectodermal-derived tumor, an endocrine tumor, an NK/T-cell lymphoma, and a small-cell lung cancer, etc.
  • the neuroectodermal-derived tumor includes, but is not limited to, retinoblastoma, astrocytoma, and neuromuscular Cell tumors, etc.
  • polypeptides of the invention are also useful in the preparation of pharmaceutical targeting formulations.
  • the invention also provides a pharmaceutical formulation comprising the polypeptide described above.
  • Further pharmaceuticals may also include pharmaceutically acceptable excipients or carriers. These carriers or excipients can be specifically selected by those skilled in the art according to the requirements of the pharmaceutical dosage form to be prepared, for example, the carrier can be a diluent, an excipient, a binder, a wetting agent, a disintegrant, an absorption enhancer, and a surface.
  • the carrier can be a diluent, an excipient, a binder, a wetting agent, a disintegrant, an absorption enhancer, and a surface.
  • the auxiliary material may include, for example, a flavoring agent, sweetness Agents, etc.
  • the pharmaceutical dosage form may be, for example, a capsule, a soft capsule, a tablet, an oral solution, a dispersible tablet, a lyophilized powder, an injection or a dropping pill.
  • These carriers or adjuvants may be selected from the corresponding reagents available in the art.
  • the inventors of the present invention screened a plurality of proteins or polypeptides capable of specifically binding to CD56 molecules from a human liver cancer cDNA library by T7 phage display technology, and combined with ELISA technology and PCR, finally selected two strong binding groups to CD56 molecules.
  • the polypeptides are peptides No. 7 and No. 8, respectively, and the polypeptides are synthetically synthesized for subsequent experimental studies.
  • the inventors ligated FITC fluorescein at the end of the polypeptide, and by cellular immunofluorescence experiments, it was found that the polypeptide of the present invention can bind to the surface of CD56+ cells together with the CD56 antibody, and does not bind to the cells of CD56-, demonstrating that the polypeptide of the present invention is in the cell.
  • the surface is capable of binding to CD56 and has a strong specificity.
  • the inventors have found that the polypeptide of the present invention is not toxic to cells, and does not affect the proliferation of cells, nor does it affect the expression rate of CD56, indicating that the polypeptide of the present invention has no stimulating effect on cells, and does not affect the survival state and function of cells.
  • the inventors coated the polypeptide onto the nanomagnetic beads and used the magnetic field environment for cell sorting. As a result, it was found that the positive rate of the polypeptide sorting cells was close to that of the commercial reagent, indicating that the polypeptide of the present invention is feasible in cell sorting.
  • the polypeptide can replace the commercial mouse anti-CD56 antibody and has important clinical application value in sorting natural killer cells (NK cells) and NKT cells.
  • the coding sequence of the polypeptide of the present invention is derived from a human library, can avoid the HAMA reaction in clinical treatment, prolong the action time of the polypeptide in vivo, enhance the anti-tumor effect of the drug, and attenuate other side effects that may cause the body. .
  • the polypeptides screened by the invention have many advantages such as small molecular weight, convenient synthesis, easy storage, and the like, and have high application value in clinical practice.
  • the polypeptide which specifically binds to the CD56 molecule hereinafter may be expressed as CD56-nABP or nABP-CD56.
  • a polypeptide capable of specifically binding to CD56 was screened from a human liver cancer cDNA library, and a polypeptide having strong binding ability was selected by ELISA, and the DNA sequence inserted into the phage was sequenced and analyzed, and the polypeptide was artificially synthesized.
  • Bacto typtone 10g Yeast extract 5g NaCl 5g Agarose 6g
  • the CD56 recombinant protein was diluted to 5 ⁇ g/ml with deionized water or TBS (10 mM Tris-HCl, pH 8.0, 150 mM NaCl).
  • the eluted phage was added to 6 ml of the 5403 host bacteria prepared at the beginning, and shaken at 37 ° C until lysis.
  • the amount of phage added and the amount of phage eluted in each round are shown in Table 1. After three rounds of phage screening, phages capable of binding to CD56 recombinant protein were gradually enriched, and phage with low binding ability were removed.
  • T7Tail-Fiber Monoclonal Antibody (1:2500) (Merck Millipore) diluted with blocking solution was added, and incubated for 1 hour at room temperature.
  • plaques were randomly selected from the phage after the third round of screening, and amplified by 5403 bacteria and detected by ELISA. The results showed that No. 1, No. 7, No. 11, No. 14, No. 15, No. 17, No. 18 No. 22, No. 24, No. 25, No. 27, No. 29, No. 32, No. 35, No. 37, No. 38, No. 40, No. 44, No. 45, the OD value is higher, as shown in Fig. 1.
  • CD56-positive phage gene sequencing CD56-nABP sequence determination and synthesis
  • Phages with higher OD values were selected from the ELISA results for PCR detection and sequencing.
  • DNASTAR software was used to compare the DNA fragments inserted by each phage with T7 phage self-sequences, and BLAST was performed on the NCBI website to determine CD56-
  • the amino acid sequence of nABP is combined with synthetic peptides (Shanghai Qiang Yao Biotechnology Co., Ltd.).
  • a CD56-nABP fluorescent reagent was prepared by adding a fluorescent tag protein such as FITC (green fluorescent) at a N-terminus of a part of CD56-nABP with a purity of >95%.
  • Amino acid sequence of polypeptide No. 7 Ser Ser Val Leu Thr Lys Trp Tyr Leu Val Cys Trp Ser Leu Gly Glu Ser Ile Ser Asn Glu Arg Arg Lys Asn Phe His Leu Val Gly His Trp Gly Cys Leu Arg Lys Leu Thr Phe Val Val Glu (SSVLTKWYLVCWSLGESISNERRKNFHLVGHWGCLRKLTFVVE).
  • Amino acid sequence of polypeptide No. 8 Ser Ser Val Tyr Cys Lys Gln Thr Asn Lys Lys Lys Arg Lys Glu Arg Lys Arg Asp Thr Gly Met Lys Gly Asp Ile Ile Gly Thr Gly Val Asn Asp Leu Ala Pro Leu Arg Gly Pro Ile Lys Ala Ser Thr Met Val Ala Val Ala Gln Asp Met Ala Gly Thr Gly Ala Asp Phe Leu Phe Leu (SSVYCKQTNKKKRKERKRDTGMKGDIIGTGVNDLAPLRGPIKASTMVAVAQDMAGTGADFLFL).
  • the coverslips were sterilized with alcohol and placed in a 6-well plate.
  • the coverslips were coated with 0.1% rat tail type I collagen for 1 h at room temperature.
  • DAPI was added to protect the nuclei from light for 5 min, washed once with PBS, and then sealed with anti-fluorescence quenching tablets.
  • Anti-Mouse IgG fluorescent secondary antibody (1:1000) (Abeam, Cambridge, UK) was added, and incubated for 1 hour at room temperature in the dark.
  • CTSC-2 cells are CD56-cells with no CD56 antibody on the cell surface and no peptide fluorescence, as shown in Figure 4, indicating that CD56-nABP polypeptide does not bind to CTSC-2 cells, indicating that CD56-nABP peptide binding is more High specificity.
  • U2OS cells were resuspended and resuspended, and 3000 cells were seeded per well on a 96-well plate.
  • Cells were cultured in 100 ⁇ l (DF+10% FBS) medium and cultured in a CO 2 incubator for 24 h. After the cells were attached, different concentrations were added.
  • the polypeptide (0.1 ⁇ g/ml, 0.5 ⁇ g/ml, 1 ⁇ g/ml, 5 ⁇ g/ml, 10 ⁇ g/ml, 25 ⁇ g/ml, 50 ⁇ g/ml, 100 ⁇ g/ml) was assayed for cell viability after 72 h with CCK8.
  • Figures A and B show the toxic effects of peptides No. 7 and No. 8 on U2OS cells, respectively.
  • the results of statistical analysis showed that there was no significant difference in cell viability between U2OS cells cultured in medium with different concentration gradients and in medium without peptide addition, indicating that No.7 and No.8 peptides had no toxic effects on U2OS cells.
  • U2OS and CTSC-2 cells were cultured in 24-well plates, cultured in 500 ⁇ l (DF+10% FBS) medium, and cultured in CO 2 incubator for 24 h. After cell attachment, add peptide No. 7 and peptide No. 8, respectively. The final concentration was 10 ⁇ g/mL, and a negative control group without peptide was established. The polypeptide was added once every 48 hours, and the cells were counted every day for 7 days to draw a cell proliferation curve.
  • the female mice weighed 18.4 g and injected 400 ⁇ l of CD56-nABP peptide No. 8 from the tail vein of the mouse at a concentration of 1 mg/ml. Imaging was started after anesthetized mice by intraperitoneal injection of 60 ul of sodium pentobarbital (concentration: 20 mg/ml). Fluorescence imaging taken every 15 minutes (Fig. 7) showed that the blood circulation of CD56-nABP polypeptide was distributed to the whole body of mice, and the lip of the mouse also showed fluorescence; after 1 h, the fluorescence of the polypeptide gradually weakened and gradually enriched into the liver of the mouse. In the kidney and intestine, the polypeptide remained only in the liver and intestine after 3.5 h, indicating that it was mainly metabolized by the liver.
  • mice The liver, spleen, lung and kidney of the mice were dissected, and frozen sections were observed under a confocal microscope. The results are shown in Fig. 8.
  • Each organ has a small amount of peptide residue, indicating that the polypeptide enters the various organs after blood circulation in the mouse. However, after 3.5 hours of fluorescence photography, there are fewer peptides remaining in each organ, and most of them may have been catabolized in the body.
  • the magnetic beads were washed 3 times with 1 ml of a preservation solution (PBS containing 0.1% BSA, 0.01% Tween, 0.05% NaN 3 , pH 7.4), and the magnetic beads were resuspended in 500 ⁇ l of the preservation solution and stored at 4 ° C.
  • a preservation solution PBS containing 0.1% BSA, 0.01% Tween, 0.05% NaN 3 , pH 7.4
  • the cells were separated by magnetic fluid, and the positive and negative cell populations were collected, and the CD56 flow antibody was added to the machine for detection.
  • the sorting results are shown in Fig. 9.
  • the curves 1, 2, and 3 in the figure represent the experimental results of the negative control, the experimental group, and the positive control group, respectively.
  • the positive rate of U2OS cells sorted by the No. 8 polypeptide-magnetic bead complex reached 84.6%
  • the positive rate of U2OS cells sorted by the commercial Meitianjing sorting reagent was 97.9%. Sorting U2OS cells with a peptide-magnetic bead complex is feasible and is close to the sorting effect of Mirin.
  • the single magnetic beads sorted U2OS cells reached 47.9%, indicating that the magnetic beads have a certain non-specific adsorption effect on the cells, but also far lower than the sorting effect of the peptide-magnetic beads, which needs to be studied in the follow-up study.
  • the sorting rates of the negative control group, the experimental group and the positive control group were 2.74%, 1.6%, and 54.9%, respectively, when the CD56-type CTSC-2 cells were sorted by the peptide-magnetic bead complex No.8.
  • the polypeptide of the invention does not bind to CTSC-2 cells, and there is less non-specific adsorption.
  • the results of the cell sorting experiment are similar to those of the No. 8 polypeptide, and will not be described again.
  • the present invention screens a plurality of proteins or polypeptides capable of specifically binding to CD56 molecules from a human liver cancer cDNA library by T7 phage display technology, and combines ELISA technology and PCR to finally select two strong binding forces with CD56 molecules.
  • the polypeptides are peptides No. 7 and No. 8, respectively, and the polypeptides are synthetically synthesized for subsequent experimental studies.
  • the inventors ligated FITC fluorescein at the end of the polypeptide, and by cellular immunofluorescence experiments, it was found that the polypeptide of the present invention can bind to the surface of CD56+ cells together with the CD56 antibody, and does not bind to the cells of CD56-, demonstrating that the polypeptide of the present invention is on the cell surface. It is able to bind to CD56 and has strong specificity.
  • the polypeptide is not toxic to the cells, and does not affect the proliferation of the cells, nor does it affect the expression rate of the cells CD56, indicating that the polypeptide of the present invention has no stimulating effect on the cells, and does not affect the survival state and function of the cells, which gives Subsequent cell sorting provides the feasibility of ensuring the safety of the peptide to the desired sorted cells.
  • the polypeptide of the present invention can be retained in mice for up to 3.5 hours, indicating that the catabolism of the polypeptide is slow in mice, and the polypeptide of the present invention has the potential as a targeted drug carrier, which can greatly prolong the drug.
  • the retention time in the body does not rapidly catabolize with blood circulation, thereby improving the therapeutic effect of the drug and prolonging the administration interval.
  • the inventors also coated the polypeptide onto the nanomagnetic beads by EDC and NHSS cross-linking agent, and used the magnetic field environment for cell sorting. As a result, it was found that the positive rate of the sorted cells of the polypeptide of the present invention was close to the commercial reagent, indicating that the polypeptide was in the cell fraction. The selection is feasible.
  • the polypeptide can replace the commercial mouse anti-CD56 antibody and has important clinical application value in sorting natural killer cells (NK cells) and NKT cells. NK cells are an important immune cell of the human body and participate in the body. Anti-tumor, anti-viral infection, also involved in type II hypersensitivity and graft versus host response.
  • NK cells are mainly found in peripheral blood, accounting for 5-10% of PBMC, and NK cells in lymph nodes and bone marrow.
  • NKT cells are a subpopulation of T cells.
  • the cell surface has both T cell receptor TCR and NK cell receptor, which plays a role in the innate immunity of the human body, mainly regulating immune regulation and cytotoxicity.
  • the NK and NKT cell populations which can be isolated from human peripheral blood can be expanded and cultured in vitro, and returned to the patient, thereby enhancing the immunity of the body and increasing the tumor cells. , the lethality of viruses and parasites.
  • the coding sequence of the polypeptide is derived from a human library, the HAMA response can be avoided in clinical treatment, prolonging the action time of the polypeptide in vivo, enhancing the anti-tumor effect of the drug, and attenuating other side effects that may cause the body, and we
  • the selected polypeptide has many advantages such as small molecular weight, convenient synthesis, easy storage, and the like, and has high application value in clinical practice.

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Abstract

一种与CD56分子特异性结合的多肽及其应用，所述多肽选自7号多肽、8号多肽中的至少一种，所述7号多肽、8号多肽的氨基酸序列分别如序列表中序列1、序列2所示。上述多肽能与CD56分子特异性结合，可以用人工合成或基因工程的方法生产。与抗体比较，该多肽具有易于制备，分子量小，不易产生免疫排斥反应等特点；且毒副作用小，与CD56+细胞的结合，对目标细胞没有明显的杀伤和抑制增殖的效果。所述多肽可用于作为CD56阳性表达细胞的标记物，也可作为免疫荧光CD56抗体的代替物等。

Description

与CD56分子特异性结合的多肽及其应用 技术领域
本发明涉及生物医药领域，特别涉及能与CD56分子特异性结合的多肽及其应用。
背景技术
CD56分子，又叫神经细胞粘附分子(neural cell adhesion molecule,NCAM)，是免疫球蛋白超家族的一个成员。CD56是一个含有多肽链的糖蛋白，CD56分子的胞外部分包括5个Ig样的结构域和2个Ⅲ型纤粘蛋白样结构域。由于其mRNA的不同剪接方式，最后生成3种不同的分子亚型：1.120kDa亚型，由GPI(glycosyl-phosphatidylinositol)锚定在细胞膜上，主要表达于正常组织和分化良好的组织，如NK细胞，神经组织，神经-肌肉连接处，神经内分泌组织，内分泌组织等。2.140kDa和180kDa亚型，包含一个跨膜结构域，主要表达于未分化组织或恶性肿瘤细胞上，如神经外胚层来源的肿瘤(如视网膜母细胞瘤、星形细胞瘤、神经母细胞瘤等)、内分泌肿瘤、小细胞肺癌，NK/T细胞淋巴瘤/白血病、多发性骨髓瘤和急性髓系白血病等。因此，CD56已经被用于NK细胞和上述肿瘤细胞的标志物，可用于细胞标记、分选和肿瘤分子诊断。
CD56分子不仅在细胞粘附中起机械的锚定作用，还是细胞表面一个重要的受体，调节细胞内的下游信号通路。文献中报道已发现的配体包括：CD56分子(同种结合)，L1-CAM，FGFR，GNDF，GFRα，细胞外基质成分如CSPG，SHPG，胶原蛋白，肝素等。研究发现，CD56在神经系统的分化过程和突触的可塑性中具有重要作用，并且利用下游的信号传导可以抑制星形胶质细胞和神经凸起伸长前体细胞的增殖，更为重要的是，CD56下游信号可以影响恶性肿瘤细胞的生物活性，并且CD56失表达与一些恶性肿瘤转移潜能和预后差密切相关。因此，CD56分子的配体(如抗体等)可以作为一种潜在的抑制剂来抑制肿瘤生长。
目前，在以CD56分子为靶标的肿瘤治疗的研究中都涉及到CD56抗体，以往的鼠源性抗体在临床治疗中会产生人抗鼠抗体反应(human anti-mouse antibodies,HAMA)，导致治疗性抗体在体内作用时间缩短，减弱其抗肿瘤的作用，以及可能引起身体上的其他副作用。有研究者采用基因工程的方法来改造鼠源性抗体，来减弱HAMA反应。但是单纯的抗CD56抗体的治疗作用并不十分理想，进而有研究利用抗CD56抗体作为一个靶向运输的分子，联合放射性同位素、细胞毒性药物、细胞因子、双效价抗体(能同时结合肿瘤细胞表面的肿瘤相关抗原和免疫细胞表面的激活分子)等，增强对肿瘤细胞的杀伤作用。因此，在肿瘤免疫治疗的临床应用中的关键是找到与CD56分子结合的高度特异性抗体。
发明内容
本发明为弥补现有技术的不足，提供一种能与CD56分子特异性结合的多肽，其具有易于制备，分子量小，应用安全性高等特点。
本发明为达到其目的，采用的技术方案如下：提供与CD56分子特异性结合的多肽，所述多肽包括7号多肽、8号多肽中的至少一种，所述7号多肽、8号多肽的氨基酸序列分别如序列表中序列1、序列2所示。
本发明提供的多肽能与CD56分子特异性结合，可以用人工合成或基因工程的方法生产。该多肽亦可称之为一种非抗体结合蛋白(non-antibody binding protein)，非抗体结合蛋白是相对于抗体/抗原特异性结合的特征而言，又称之为支架结合蛋白(scaffolds binding protein)。这类蛋白在三维结构上通过其表面氨基酸形成的支架能结合特定的分子，与抗体比较，具有分子量小，渗透力强，耐热，稳定性好，容易操作和适合大规模生产等特点。
本发明还提供一种CD56阳性表达细胞的标记物，该标记物包括如上文所述的多肽。
本发明提供的多肽可应用于标记、鉴定、富集、分选或纯化CD56阳性细胞，或应用于制备用于标记、鉴定、富集、分选或纯化CD56阳性细胞的制剂。所述细胞包括神经元、星形细胞、雪旺氏细胞、NK细胞和NKT细胞，也包含神经外胚层来源的肿瘤(如视网膜母细胞瘤、星形细胞瘤、神经母细胞瘤等)、内分泌肿瘤、NK/T细胞淋巴瘤和小细胞肺癌的肿瘤细胞。
本发明另一方面提供一种用于分选CD56阳性细胞的制剂，包括如上文所述的多肽、或包括所述多肽与磁珠偶联形成的多肽磁珠复合物(或称为多肽包被磁珠形成的多肽磁珠复合物)。
本发明提供的多肽还可用于疾病的诊断或分型或治疗，例如肿瘤、病毒感染性疾病、寄生虫病、白血病等，也可应用于制备用于疾病的诊断或分型或治疗的制剂。所述肿瘤包括神经外胚层来源的肿瘤、内分泌肿瘤、NK/T细胞淋巴瘤和小细胞肺癌等，所述神经外胚层来源的肿瘤包括但不限于视网膜母细胞瘤、星形细胞瘤、神经母细胞瘤等。
本发明的多肽还可应用于药物靶向制剂的制备。
本发明还提供一种药物制剂，其中包括上文所述的多肽。进一步的该药物还可包括药学上允许的辅料或载体。这些载体或辅料，本领域技术人员可根据所需制备的药物剂型等要求而具体选择，例如载体可以为稀释剂、赋形剂、粘合剂、湿润剂、崩解剂、吸收促进剂、表面活性剂、吸附载体、润滑剂中的一种或多种；辅料例如可以包括有香味剂、甜味 剂等。药物剂型例如可以是胶囊、软胶囊、片剂、口服液、分散片、冻干粉针、注射液或滴丸等。这些载体或辅料可选用本领域现有的相应试剂。
本申请发明人通过T7噬菌体展示技术从人的肝癌cDNA文库中筛选出来多条能够特异性结合CD56分子的蛋白或多肽，结合ELISA技术和PCR，最终挑选出两条与CD56分子结合力较强的多肽，分别为7号和8号多肽，并且人工合成了多肽进行后续的实验研究。发明人在多肽的末端连接了FITC荧光素，通过细胞免疫荧光实验，发现本发明的多肽能够与CD56抗体共同结合在CD56+细胞表面，并且不与CD56-的细胞结合，证明本发明的多肽在细胞表面能够与CD56结合，并且有较强的特异性。进一步地，发明人发现本发明的多肽对细胞没有毒性，并且不影响细胞的增殖，也不影响细胞CD56的表达率，说明本发明的多肽对细胞没有刺激作用，不影响细胞的生存状态和功能，这就给后续的细胞分选提供了可行性，保证了多肽对所需分选细胞的安全性。多肽注射入小鼠体内后可以在小鼠体内滞留长达3.5h，说明小鼠对多肽的分解代谢过程较为缓慢，具备作为靶向药物载体的潜力，可以大大延长药物在体内的存留时间，不会快速的随着血液循环而分解代谢，由此提高药物的治疗效果，延长给药间隔。
发明人将多肽包被到纳米磁珠上，利用磁场环境进行细胞分选，结果发现多肽分选细胞的阳性率接近商品化的试剂，表明本发明多肽在细胞分选中具有可行性。该多肽可替代商品化的鼠源性抗CD56抗体，在分选自然杀伤细胞(NK细胞)、NKT细胞中具有重要的临床应用价值。
本发明的多肽的编码序列来源于人的文库，在临床治疗中可以避免HAMA反应，延长多肽在体内的作用时间，增强其运载药物的抗肿瘤的作用，以及减弱可能引起身体上的其他副作。而且，本发明筛选的多肽具有分子量小，合成方便，容易保存等诸多优点，在临床有较高的应用价值。
附图说明
图1.CD56噬菌体与CD56重组蛋白结合的OD值
图2CD56非抗体结合蛋白噬菌体PCR结果
图3CD56-nABP与CD56抗体免疫荧光共成像
图4CD56-nABP与CD56抗体的非特异性结合研究
图5CD56-nABP的细胞毒性研究
图6CD56-nABP细胞增殖作用
图7CD56-nABP体内代谢荧光成像分析
图8CD56-nABP在小鼠脏器代谢荧光观察
图9CD56-nABP磁性细胞分选研究
具体实施方式
下面结合附图对本发明的技术方案做进一步说明：
文中的百分比若未特别说明，均为质量百分比；文中使用的试剂若未特别说明，均为可从市面购买获得的试剂。下文实施例中所进行的实验操作若未特别说明，均为本领域技术人员所掌握的常规技术操作。
下文中关于CD56分子特异性结合的多肽可表述为CD56-nABP或nABP-CD56。
实施例一 T7噬菌体肝癌cDNA文库筛选CD56非抗体结合蛋白
利用T7噬菌体展示技术，从人的肝癌cDNA文库中筛选能够与CD56特异性结合的多肽，通过ELISA挑选出结合力较强的多肽，并测序分析插入噬菌体的DNA序列，人工合成多肽。
1.1培养基的制备
(1)LB液体培养基的配制(1L体积)：
成分 重量
Bacto typtone 10g
Yeast extract 5g
NaCl 10g
用二级水溶解，并用1N NaOH调节pH到7.5，高压蒸汽灭菌20min。
(2)LB固体培养基的配制(1L体积)：
成分 重量
Bacto typtone 10g
Yeast extract 5g
NaCl 10g
Agar 15g
用二级水溶解，并用1N NaOH调节pH到7.5，高压蒸汽灭菌20min。
(3)Top agarose配制(1L体积)：
成分 重量
Bacto typtone 10g
Yeast extract 5g
NaCl 5g
Agarose 6g
用二级水溶解，并用1N NaOH调节pH到7.5，高压蒸汽灭菌20min。
(4)M9LB培养基的配制：
成分 体积
20×M9salts 5ml
20％glucose 2ml
1M MgSO4 0.1ml
LB 100ml
(5)20×M9salts的配制(1L体积)：
成分 重量
NH4Cl 20g
KH2PO4 60g
Na2HPO4·7H2O 120g
用二级水溶解，高压蒸汽灭菌20min。
1.2包被96孔板：
(1)用去离子水洗板几次，在纸上拍干。
(2)用去离子水或TBS(10mM Tris-HCl,pH8.0,150mM NaCl)稀释CD56重组蛋白至5μg/ml。
(3)加入100μl稀释好的蛋白，4℃孵育过夜，用封口膜包好。
(4)用300μl 1×TBS洗板3次。
(5)用水配好5％的脱脂牛奶，每孔加入200μl。
(6)室温孵育60min或者4℃过夜。
(7)用去离子水洗板5次，然后每孔加入200μl水，封好板保存在4℃。
1.3噬菌体筛选步骤(参照说明书进行)：
(1)接种适量的5403宿主菌(50μl)和抗生素到50ml的M9LB培养基，摇菌到OD为0.5-0.6。
(2)配制1×TBST(10mM Tris-HCl,pH 8.0,150mM NaCl,0.1％
Figure PCTCN2017099487-appb-000001
)。
(3)加入所需的噬菌体，如果体积小于100μl，用1×TBST补足。
(4)室温孵育30min。
(5)用1×TBST洗板5次，每孔大于200μl。
(6)加入200μl T7洗脱液，室温孵育10-20min。
(7)转移洗脱的噬菌体到无菌EP管，取10μl计算噬菌体滴度。
(8)将洗脱的噬菌体加入到一开始准备好的6ml的5403宿主菌，37℃摇菌直到裂解。
(9)再次进行第二轮，第三轮噬菌体筛选过程，步骤与第一轮筛选基本相同。
每一轮加入的噬菌体量和洗脱的噬菌体量如表1所示，经过三轮的噬菌体筛选，能够与CD56重组蛋白结合的噬菌体逐渐富集起来，结合力低下的噬菌体被去除。
表1 CD56噬菌体四轮筛选富集的结果
Figure PCTCN2017099487-appb-000002
1.4ELISA法检测噬菌体单克隆与CD56重组蛋白的结合力：
(1)96孔板上每孔加入30μl浓度为5μg/ml的CD56重组蛋白，4℃包被过夜。
(2)洗去未结合的重组蛋白，再用3％的脱脂奶粉室温封闭1h。
(3)将第三轮筛选后的噬菌体培养在LB固体培养基，随机挑取45个噬菌体单克隆并扩增，将噬菌体溶菌产物加入到孔中，37℃孵育1h。
(4)洗板后加入100μl用封闭液稀释的T7Tail-Fiber Monoclonal Antibody(1:2500)(Merck Millipore)，室温孵育1h。
(5)洗板后加入100μl用封闭液稀释的Anti-Mouse IgG-HRP(1:5000)(Abcam，Cambridge，UK)，室温孵育1h。
(6)洗板后加入100μl TMB显色底物，室温避光孵育15-60min。
(7)加入100μl的250mM HCl终止反应。
(8)在450nm波长处检测各孔的吸光度(OD)。
从第三轮筛选后的噬菌体中随机挑选45个噬菌斑，在5403菌中扩增后进行ELISA检测，结果发现1号，7号，11号，14号，15号，17号，18号，22号，24号，25号，27号，29号，32号，35号，37号，38号，40号，44号，45号OD值较高，如图1所示。
1.5CD56阳性噬菌体基因测序，CD56-nABP序列确定和合成
从ELISA结果中选取OD值较高的噬菌体进行PCR检测并测序，采用DNASTAR软件经过与T7噬菌体自身序列比对，分析每个噬菌体插入的DNA片段，并在NCBI网站上进行BLAST，最后确定CD56-nABP的氨基酸序列并，并人工合成多肽(上海强耀生物科技有限公司)。根据需要在一部分CD56-nABP的N末端添加FITC(绿色荧光)等荧光标签蛋白，纯度＞95％，制备CD56-nABP荧光试剂。
经过PCR检测发现大多数片段的长度在200bp-700bp之间，图2中示出了部分片段的PCR结果。将噬菌体测序后，测序结果用DNASTAR软件比对拼接出每个插入噬菌体的序列，并在NCBI上进行BLSAT，最后人工合成两条非抗体结合蛋白SEQ7和SEQ8，氨基酸序列分别如下所示：
7号多肽的氨基酸序列：Ser Ser Val Leu Thr Lys Trp Tyr Leu Val Cys Trp Ser Leu Gly Glu Ser Ile Ser Asn Glu Arg Arg Lys Asn Phe His Leu Val Gly His Trp Gly Cys Leu Arg Lys Leu Thr Phe Val Val Glu(SSVLTKWYLVCWSLGESISNERRKNFHLVGHWGCLRKLTFVVE)。
8号多肽的氨基酸序列：Ser Ser Val Tyr Cys Lys Gln Thr Asn Lys Lys Lys Arg Lys Glu Arg Lys Arg Asp Thr Gly Met Lys Gly Asp Ile Ile Gly Thr Gly Val Asn Asp Leu Ala Pro Leu Arg Gly Pro Ile Lys Ala Ser Thr Met Val Ala Val Ala Gln Asp Met Ala Gly Thr Gly Ala Asp Phe Leu Phe Leu(SSVYCKQTNKKKRKERKRDTGMKGDIIGTGVNDLAPLRGPIKASTMVAVAQDMAGTGADFLFL)。
实施例二、CD56-nABP多肽与细胞结合的特异性研究
2.1 CD56-nABP多肽与U2OS细胞、CTSC-2细胞结合的特异性研究
(1)盖玻片用酒精消毒后放置在6孔板中，室温下用0.1％的鼠尾Ⅰ型胶原包被盖玻片1h。
(2)用PBS洗一次后，将细胞接种到盖玻片上，在37℃培养箱培养24h待细胞贴壁后，加入7号和8号多肽孵育结合30min。
(3)再用PBS洗一次，加入4％PFA固定15min。
(4)用PBS洗3次后用封闭液(5％BSA+10％山羊血清)孵育1h。
(5)加入稀释过后的CD56抗体(1:100)在4℃孵育过夜。
(6)然后用PBS洗3次，加入Anti-Mouse IgG荧光二抗(1:1000)，室温下避光孵育1h。
(7)用PBS洗3次后加入DAPI避光染核5min，PBS洗1次后，用防荧光淬灭封片剂封片。
(8)激光扫描共聚焦显微镜下观察和拍照。
2.2CD56-nABP与NK92、Jurkat、PBMC等细胞结合的特异性研究：
(1)细胞重悬后加入10μg/ml的多肽(7号或8号多肽)，混匀并在37℃培养箱孵育30min。
(2)再用PBS洗一次，加入4％PFA固定15min。
(3)用PBS洗3次后用封闭液(5％BSA+10％山羊血清)孵育1h。
(4)加入稀释过后的CD56抗体(1:100)在4℃孵育过夜。
(5)用PBS洗3次后，加入Anti-Mouse IgG荧光二抗(1:1000)(Abcam，Cambridge，UK)，室温下避光孵育1h。
(6)用PBS洗3次后加入DAPI试剂避光染核5min，PBS洗1次后，用防荧光淬灭封片剂封片。
(7)激光扫描共聚焦显微镜下观察和拍照。
实验结果：应用U2OS，NK92和Jurkat细胞珠的荧光标记实验显示带有FITC荧光的CD56-nABP和CD56抗体均可共同标记这些CD56+细胞，图3显示CD56-nABP和CD56抗体荧光标记的共聚焦显微镜观察图像，带有FITC荧光的CD56-nABP与CD56抗体重叠在一起，说明CD56-nABP能与细胞表面的CD56分子结合。而CTSC-2细胞是CD56-的细胞，其细胞表面无CD56抗体，也无多肽荧光，如图4所示，表明CD56-nABP多肽不与CTSC-2细胞结合，说明CD56-nABP多肽结合具有较高的特异性。
实施例三、CD56-nABP多肽的细胞毒性实验
U2OS细胞消化重悬后细胞计数，96孔板上每孔接种3000个细胞，用100μl(DF+10％FBS)培养基培养细胞，在CO2培养箱培养24h，待细胞贴壁后加入不同浓度的多肽(0.1μg/ml，0.5μg/ml，1μg/ml，5μg/ml，10μg/ml，25μg/ml，50μg/ml，100μg/ml)，在72h后用CCK8检测细胞活性。
结果如图5所示，图A、B分别为7号、8号多肽对U2OS细胞的毒性作用。统计学分析结果显示，在加入不同浓度梯度的多肽的培养基和未加入多肽的培养基中培养U2OS细胞，其细胞活力没有显著性差别，说明7号和8号多肽对U2OS细胞没有毒性作用。
实施例四、CD56-nABP多肽对细胞增殖的影响
U2OS和CTSC-2细胞培养在24孔板，用500μl(DF+10％FBS)培养基培养细胞，在CO2培养箱培养24h待细胞贴壁后，分别加入7号多肽和8号多肽，使其终浓度为10μg/mL，并设立一个不加多肽的阴性对照组，每隔48h添加一次多肽，并每天计数细胞，连续计数7天，绘制细胞增殖曲线。
实验结果：见图6，图A、B分别是U2OS、CTSC-2细胞的实验结果，与阴性对照组比较，各组细胞的增殖能力无显著性差异(图6)，CD56-nABP对细胞增殖能力的无显著影响。
实施例五、CD56-nABP在小鼠体内代谢实验
雌性小鼠体重18.4g，从小鼠尾静脉注射入400ul CD56-nABP的8号多肽，浓度1mg/ml。腹腔注射60ul的戊巴比妥钠(浓度为20mg/ml)麻醉小鼠后开始摄像。每隔15min拍摄的荧光摄像(图7)显示，CD56-nABP多肽血液循环分布到小鼠全身，以及小鼠唇部也有荧光；1h后，多肽荧光逐渐衰弱，并逐渐富集到小鼠肝、肾和肠，3.5h后多肽只留存于肝脏和肠腔，表明主要经过肝脏代谢。
解剖小鼠肝、脾、肺和肾脏，冰冻切片共聚焦显微镜下观察，结果如图8所示，各个脏器均有少量的多肽残留，说明多肽在小鼠体内经过血液循环后进入到各个脏器，但是在经过3.5小时的荧光拍照后，各个脏器残留的多肽较少，可能大部分已经在体内分解代谢。
实施例六、CD56-nABP多肽磁珠细胞分选研究
6.1CD56-nABP多肽-磁珠复合物的制备
(1)取40μl的300nm直径磁珠，用500μl buffer2(含0.1M MES，0.5M NaCl的PBS溶液，pH5.0)清洗磁珠3次。
(2)用500μl buffer2重悬磁珠后加入100μl的8号多肽，1mg EDC,1.5mg NHSS，摇床孵育2h。
(3)加入5mgBSA和5mg Gly,继续摇床孵育4h。
(4)用1ml PBS清洗磁珠3次。
(5)再用1ml保存液(含0.1％BSA,0.01％Tween,0.05％NaN3的PBS，pH7.4)清洗磁珠3次，500μl保存液重悬磁珠，4℃保存。
6.2CD56-nABP多肽细胞磁性分选研究
(1)将U2OS细胞和CTSC-2细胞消化成单细胞悬液，300g离心弃上清。
(2)分别加入100μl多肽-磁珠复合物(实验组)和100μl磁珠(阴性对照)，另外一组加 入20μlCD56MicroBeads(美天旎，阳性对照)。
(3)混匀后4℃孵育15min。
(4)分别加入1ml buffer1清洗，然后300g离心5min,500μl buffer1重悬。
(5)磁液分离细胞，收集阳性和阴性细胞群，加入CD56流式抗体上机检测。
实验结果：分选结果如图9所示，图中曲线1、2、3分别代表阴性对照、实验组和阳性对照组的实验结果。从图9可见，通过8号多肽-磁珠复合物分选出来的U2OS细胞的阳性率达到84.6％，用商业化的美天旎分选试剂分选出来的U2OS细胞阳性率为97.9％，说明用多肽-磁珠复合物分选U2OS细胞是可行的，并且已经接近美天旎的分选效果。在阴性对照组，即单独的磁珠分选U2OS细胞达到47.9％，说明磁珠对细胞有一定的非特异性吸附作用，但是也远低于多肽-磁珠的分选效果，需要在后续研究中优化实验条件，降低磁珠的非特异性吸附作用。并且用8号多肽-磁珠复合物分选CD56-的CTSC-2细胞过程中，阴性对照组、实验组和阳性对照组的分选率分别是2.74％、1.6％、54.9％，可见，本发明多肽不与CTSC-2细胞结合，非特异性吸附较少。对于7号多肽，其细胞分选实验结果与8号多肽相似，不再赘述。
综上，本发明通过T7噬菌体展示技术从人的肝癌cDNA文库中筛选出来多条能够特异性结合CD56分子的蛋白或多肽，结合ELISA技术和PCR，最终挑选出两条与CD56分子结合力较强的多肽，分别为7号和8号多肽，并且人工合成了多肽进行后续的实验研究。发明人在多肽的末端连接了FITC荧光素，通过细胞免疫荧光实验，发现本发明多肽能够与CD56抗体共同结合在CD56+细胞表面，并且不与CD56-的细胞结合，证明本发明的多肽在细胞表面能够与CD56结合，并且有较强的特异性。进一步地，发明人发现多肽对细胞没有毒性，并且不影响细胞的增殖，也不影响细胞CD56的表达率，说明本发明多肽对细胞没有刺激作用，不影响细胞的生存状态和功能，这就给后续的细胞分选提供了可行性，保证了多肽对所需分选细胞的安全性。将本发明多肽注射入小鼠体内后可以在小鼠体内滞留长达3.5h，说明小鼠对多肽的分解代谢过程较为缓慢，本发明多肽具有作为靶向药物载体的潜力，可以大大延长药物在体内的存留时间，不会快速的随着血液循环而分解代谢，由此提高药物的治疗效果，延长给药间隔。
发明人还通过EDC与NHSS交联剂将多肽包被到纳米磁珠上，利用磁场环境进行细胞分选，结果发现本发明多肽分选细胞的阳性率接近商品化的试剂，表明多肽在细胞分选中具有可行性。该多肽可替代商品化的鼠源性抗CD56抗体，在分选自然杀伤细胞(NK细胞)、NKT细胞中具有重要的临床应用价值。NK细胞是人体的一种重要的免疫细胞，参与机体 的抗肿瘤、抗病毒感染，还参与第Ⅱ型超敏反应和移植物抗宿主反应。NK细胞主要存在于外周血中，占PBMC的5-10％，在淋巴结和骨髓中也有NK细胞。NKT细胞是T细胞的一个亚群，其细胞表面既有T细胞受体TCR，又有NK细胞受体，在人体的固有免疫中发挥作用，主要起免疫调节和细胞毒作用。
因此，利用本发明的CD56分子结合多肽，可以从人体外周血中分离出的NK和NKT细胞群体，在体外进行扩增培养，并回输到病人体内，增强机体的免疫力，提高对肿瘤细胞、病毒和寄生虫的杀伤力。由于多肽的编码序列来源于人的文库，在临床治疗中可以避免HAMA反应，延长多肽在体内的作用时间，增强其运载药物的抗肿瘤的作用，以及减弱可能引起身体上的其他副作用，并且我们筛选的多肽具有分子量小，合成方便，容易保存等诸多优点，在临床有较高的应用价值。
以上所述，仅是本发明的较佳实施例而已，并非对本发明做任何形式上的限制，故凡未脱离本发明技术方案的内容，依据本发明的技术实质对以上实施例所做的任何简单修改、等同变化与修饰，均仍属于本发明技术方案的范围内。

Claims (10)

  1. 与CD56分子特异性结合的多肽，其特征在于，所述多肽包括7号多肽、8号多肽中的至少一种，所述7号多肽、8号多肽的氨基酸序列分别如序列表中序列1、序列2所示。
  2. 一种CD56阳性表达细胞的标记物，其特征在于，包括如权利要求1所述的多肽。
  3. 如权利要求1所述的多肽的应用，其特征在于，包括在标记、鉴定、富集、分选或纯化CD56阳性细胞中的应用，或在制备用于标记、鉴定、富集、分选或纯化CD56阳性细胞的制剂中的应用。
  4. 根据权利要求3所述的应用，其特征在于，所述细胞为CD56表达阳性细胞，所述细胞包括神经元、星形细胞、雪旺氏细胞、NK细胞、NKT细胞；和/或，所述细胞包括神经外胚层来源的肿瘤、内分泌肿瘤、NK/T细胞淋巴瘤和小细胞肺癌的肿瘤细胞，所述神经外胚层来源的肿瘤包括但不限于视网膜母细胞瘤、星形细胞瘤、神经母细胞瘤。
  5. 一种用于分选CD56阳性细胞的制剂，其特征在于，包括如权利要求1所述的多肽、或包括所述多肽包被于磁珠而形成的多肽磁珠复合物。
  6. 如权利要求1所述的多肽在制备用于疾病的诊断或分型或治疗的制剂中的应用，其特征在于，所述疾病包括肿瘤、病毒感染、寄生虫病、或白血病。
  7. 根据权利要求6所述的应用，其特征在于，所述肿瘤包括神经外胚层来源的肿瘤、内分泌肿瘤、NK/T细胞淋巴瘤和小细胞肺癌，所述神经外胚层来源的肿瘤包括但不限于视网膜母细胞瘤、星形细胞瘤、神经母细胞瘤。
  8. 如权利要求1所述的多肽在制备药物靶向制剂中的应用。
  9. 一种药物制剂，其特征在于，其活性成分包括如权利要求1所述的多肽。
  10. 根据权利要求9所述的药物制剂，其特征在于，还包括药学上允许的辅料或载体。
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