技术领域Technical Field
本发明属于生物技术领域，涉及一种TPO模拟肽及其应用。The invention belongs to the field of biotechnology and relates to a TPO mimetic peptide and application thereof.
背景技术Background technique
血小板减少症与多种疾病有关，其特征在于通常因血小板产生不足、血小板隔离、缺陷型血小板产生或血小板破坏增加而引起的血液血小板计数异常低下。血小板减少症的症状包括例如不适、瘀伤(例如紫癜)和出血(例如鼻出血或牙龈出血)。Thrombocytopenia is associated with a variety of diseases and is characterized by an abnormally low platelet count in the blood, usually due to insufficient platelet production, platelet sequestration, defective platelet production, or increased platelet destruction. Symptoms of thrombocytopenia include, for example, discomfort, bruising (e.g., purpura), and bleeding (e.g., nosebleeds or bleeding gums).
血小板生成素(TPO)是一种糖基化生长因子，它刺激巨核细胞这种产生大量血小板的骨髓细胞的产生和分化。(参见例如 Kaushansky(2006)N.Engl.J. Med. 354(19)：2034-45)。TPO与在巨核细胞祖细胞上表达的c-Mpl受体结合，从而刺激细胞增殖并分化成血小板。事与愿违的是，患者用重组人TPO治疗导致抗TPO中和抗体的产生，该抗TPO中和抗体与患者的天然存在TPO结合并干扰其活性。(参见例如Kuter和Begley(2002)Blood 100：3457-3469；Li 等(2001)Blood 98：3241-3248；以及Vadhan-Raj等(2000)Ann Intern Med132：364-368)。因此，需要用于血小板减少症相关疾病的患者的新的更好的治疗方法。Thrombopoietin (TPO) is a glycosylated growth factor that stimulates the production and differentiation of megakaryocytes, bone marrow cells that produce large quantities of platelets. (See, e.g., Kaushansky (2006) N. Engl. J. Med. 354(19):2034-45). TPO binds to the c-Mpl receptor expressed on megakaryocyte progenitor cells, thereby stimulating cell proliferation and differentiation into platelets. Conversely, treatment of patients with recombinant human TPO results in the production of anti-TPO neutralizing antibodies that bind to the patient's naturally occurring TPO and interfere with its activity. (See, e.g., Kuter and Begley (2002) Blood 100:3457-3469; Li et al. (2001) Blood 98:3241-3248; and Vadhan-Raj et al. (2000) Ann Intern Med 132:364-368). Therefore, new and better treatments are needed for patients with thrombocytopenia-related disorders.
发明内容Summary of the invention
为了解决现有技术中存在的技术问题，本发明提供了以下的技术方案：In order to solve the technical problems existing in the prior art, the present invention provides the following technical solutions:
本发明提供了以TPOR为靶点的TPO模拟肽，所述TPO模拟肽的氨基酸序列如下：GGCPVGPTLHEWLVSCGG（SEQ ID NO:1）。The present invention provides a TPO mimetic peptide targeting TPOR, wherein the amino acid sequence of the TPO mimetic peptide is as follows: GGCPVGPTLHEWLVSCGG (SEQ ID NO: 1).
本发明提供了一种核苷酸序列，所述核苷酸序列编码前面所述的TPO模拟肽。The present invention provides a nucleotide sequence, which encodes the TPO mimetic peptide mentioned above.
进一步，所述核苷酸序列编码与SEQ ID NO:5所示的序列具有80%序列一致性的序列。Furthermore, the nucleotide sequence encodes a sequence having 80% sequence identity with the sequence shown in SEQ ID NO:5.
在一些实施方案中，所述核苷酸序列包括DNA、RNA或其杂交体，所述核苷酸序列可以是单链的，也可以是双链的。In some embodiments, the nucleotide sequence includes DNA, RNA or a hybrid thereof, and the nucleotide sequence may be single-stranded or double-stranded.
本文中使用的术语“序列一致性”是指两个多核苷酸序列在比较窗口是相同的(即在核苷酸与核苷酸的基础上)。术语“序列一致性百分比”是通过以下来计算 ：在比较窗口比较两个最优对齐的序列、确定在两个序列中出现相同核酸碱基(例如 A、T、C、G、U 或 I)的位置的数量以获得匹配位置的数量、将匹配位置的数量除以在比较窗口(即窗口大小)中的位置的总数量、并将结果乘以 100 以产生序列一致性百分比。As used herein, the term "sequence identity" means that two polynucleotide sequences are identical (i.e., on a nucleotide-by-nucleotide basis) over a comparison window. The term "sequence identity percentage" is calculated by comparing two optimally aligned sequences over a comparison window, determining the number of positions where the same nucleic acid base (e.g., A, T, C, G, U, or I) occurs in the two sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to produce a sequence identity percentage.
在一些实施方案中，所述核苷酸序列包括编码至少一种(例如至少 2、3、4、5、6 或8种)TPO模拟肽的多肽的核酸，其中所述多肽的氨基酸序列与SEQ ID NO:5所述氨基酸序列有至少80%(例如至少81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%)同一性。将百分比(%)氨基酸序列或核酸序列同一性定义为在比对序列和必要时引入空位以达到最大百分比序列同一性之后，候选序列中与参比序列的氨基酸或核酸相同的氨基酸或核酸的百分比。可按本领域技术人员掌握的不同方法实现用于确定百分比序列同一性目的的比对，例如，应用可公开获取的计算机软件例如 BLAST、BLAST-2、ALIGN、ALIGN-2 或Megalign(DNASTAR)软件。测定比对的合适参数，包括对在比较中的全长序列内获得最大比对所需的任何算法，可通过已知方法确定。In some embodiments, the nucleotide sequence includes a nucleic acid encoding at least one (e.g., at least 2, 3, 4, 5, 6, or 8) TPO mimetic peptide polypeptide, wherein the amino acid sequence of the polypeptide is at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 5. Percent (%) amino acid sequence or nucleic acid sequence identity is defined as the percentage of amino acids or nucleic acids in the candidate sequence that are identical to the amino acids or nucleic acids of the reference sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percentage sequence identity. Alignment for the purpose of determining percentage sequence identity can be achieved in different ways known to those skilled in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalign (DNASTAR) software. Appropriate parameters for determining alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared, can be determined by known methods.
进一步，所述核苷酸序列还编码抗体的Fc区，所述抗体的Fc区与TPO模拟肽之间有连接子连接。Furthermore, the nucleotide sequence also encodes the Fc region of the antibody, and the Fc region of the antibody is connected to the TPO mimetic peptide via a linker.
进一步，所述连接子的核苷酸序列如SEQ ID NO:6所示。Furthermore, the nucleotide sequence of the linker is shown in SEQ ID NO:6.
进一步，所述编码抗体的Fc区的核苷酸序列如SEQ ID NO:7所示。Furthermore, the nucleotide sequence encoding the Fc region of the antibody is shown in SEQ ID NO:7.
在一些实施方案中，所述抗体的Fc区连接至少一个TPO模拟肽。In some embodiments, the Fc region of the antibody is linked to at least one TPO mimetic peptide.
本文使用的“多肽”、“肽”和“蛋白质”可互换使用，是指任何氨基酸的肽键链，不论长度或翻译后修饰。在一些实施方案中，治疗性肽(例如TPO模拟肽)可在羧基端和氨基端的一个或两个上侧接间隔基氨基酸序列(或间隔基序列)。可使用间隔基序列例如降低对模拟物的结构约束、允许模拟物更容易地采取生物活性构象和/或使肽更有效地存在于抗体支架的环境下。As used herein, "polypeptide", "peptide" and "protein" are used interchangeably and refer to any peptide bond chain of amino acids, regardless of length or post-translational modification. In some embodiments, the therapeutic peptide (e.g., TPO mimetic peptide) can be flanked by a spacer amino acid sequence (or spacer sequence) at one or both of the carboxyl terminus and the amino terminus. The spacer sequence can be used, for example, to reduce structural constraints on the mimetic, allow the mimetic to more easily adopt a biologically active conformation, and/or allow the peptide to be more effectively present in the context of an antibody scaffold.
本发明提供了一种融合蛋白，所述融合蛋白包括一个或多个前面所述的TPO模拟肽。The present invention provides a fusion protein, wherein the fusion protein comprises one or more of the TPO mimetic peptides described above.
进一步，前面所述的TPO模拟肽连接到抗体的Fc区。Furthermore, the aforementioned TPO mimetic peptide is linked to the Fc region of an antibody.
进一步，所述抗体的Fc区的氨基酸序列如SEQ ID NO:3所示。Furthermore, the amino acid sequence of the Fc region of the antibody is shown in SEQ ID NO:3.
进一步，所述TPO模拟肽与抗体的Fc区中间有连接氨基酸序列连接。Furthermore, the TPO mimetic peptide is connected to the Fc region of the antibody via a connecting amino acid sequence.
进一步，所述连接氨基酸序列如SEQ ID NO:2所示。Furthermore, the connecting amino acid sequence is shown in SEQ ID NO:2.
在一些实施方案中，所述一个或多个前面所述的TPO模拟肽在结构上可存在于抗体中心裂口的位置上。所述一个或多个前面所述的TPO模拟肽整合到轻链多肽的恒定区。在一些实施方案中，所述抗体还包括至少一个TPO模拟肽的重链多肽。更具体的实施方案中，所述至少一个TPO模拟肽整合到抗体的重链多肽的铰链区、铰链区的氨基端与铰链区上游的重链多肽区之间的连接处、铰链区的羧基端与铰链区下游的重链多肽区之间的连接处、或始于重链多肽铰链区的氨基酸上游少于20个氨基酸或重链多肽铰链区的羧基端下游至少20个氨基酸内的位置。In some embodiments, the one or more aforementioned TPO mimetic peptides may be structurally present at the position of the central cleft of the antibody. The one or more aforementioned TPO mimetic peptides are integrated into the constant region of the light chain polypeptide. In some embodiments, the antibody further comprises a heavy chain polypeptide of at least one TPO mimetic peptide. In a more specific embodiment, the at least one TPO mimetic peptide is integrated into the hinge region of the heavy chain polypeptide of the antibody, the junction between the amino terminus of the hinge region and the heavy chain polypeptide region upstream of the hinge region, the junction between the carboxyl terminus of the hinge region and the heavy chain polypeptide region downstream of the hinge region, or a position less than 20 amino acids upstream of the amino acids of the hinge region of the heavy chain polypeptide or at least 20 amino acids downstream of the carboxyl terminus of the hinge region of the heavy chain polypeptide.
进一步，所述抗体还可包括单链抗体、单链 Fv 片段 (scFv)、Fd 片段、Fab 片段、Fab’片段或 F(ab’)2 片段。Furthermore, the antibody may also include a single-chain antibody, a single-chain Fv fragment (scFv), a Fd fragment, a Fab fragment, a Fab' fragment or a F(ab')2 fragment.
进一步，所述抗体来自于人或非人哺乳动物。Furthermore, the antibody is from a human or a non-human mammal.
进一步，所述抗体来自于人。Furthermore, the antibody is from human.
进一步，所述抗体的中还包括异源部分。Furthermore, the antibody further comprises a heterologous portion.
本文公开内容涉及含有TPO模拟肽的有治疗活性的融合蛋白(这些融合蛋白在下文中称为“重组抗体”)。在一些实施方案中，重组抗体含有本公开内容的血小板生成素(TPO)模拟肽(这些抗体在下文中称为“TPO 模拟抗体”)。本公开内容还涉及重组抗体的治疗活性片段(例如TPO模拟抗体的治疗活性片段)。例如，可以在各种诊断和/或治疗应用中使用重组抗体及其片段。The disclosure herein relates to therapeutically active fusion proteins containing TPO mimetic peptides (these fusion proteins are hereinafter referred to as "recombinant antibodies"). In some embodiments, the recombinant antibodies contain the thrombopoietin (TPO) mimetic peptides of the disclosure (these antibodies are hereinafter referred to as "TPO mimetic antibodies"). The disclosure also relates to therapeutically active fragments of recombinant antibodies (e.g., therapeutically active fragments of TPO mimetic antibodies). For example, recombinant antibodies and fragments thereof can be used in various diagnostic and/or therapeutic applications.
本公开内容全文中使用的术语“重组抗体”和“TPO 模拟抗体”是指含有至少一个(例如至少1、2、3 或4个)本公开内容的肽(例如TPO模拟肽)的整个或完整抗体(例如IgM、IgG(包括IgG1、IgG2、IgG3和IgG4)、IgA、IgD 或 IgE)分子。重组抗体(例如TPO模拟抗体)还包括完整抗体，其具有至少一个TPO模拟肽和杂合恒定区或其部分，例如G2/G4杂合恒定区(参见例如Burton等 (1992)Adv.Immun.51：1-18；Canfield等(1991) J.Exp.Med.173：1483-1491；以及 Mueller等(1997)Mol.Immunol.34(6)：441-452)。例如(并按照Kabat 编号)，IgG1和IgG4恒定区含有G249G250残基，而IgG2恒定区不含残基249，但却含有 G250。在其中249-250区域源自G2序列的G2/G4杂合恒定区中，可对该恒定区进行进一步修饰以便在249位引入甘氨酸残基，产生具有G249/G250的G2/G4融合物。含有G249/G250的其它恒定结构域杂合体还可用作本公开内容的TPO模拟抗体的支架。The terms "recombinant antibody" and "TPO mimetic antibody" as used throughout the present disclosure refer to a whole or intact antibody (e.g., IgM, IgG (including IgG1, IgG2, IgG3 and IgG4), IgA, IgD or IgE) molecule containing at least one (e.g., at least 1, 2, 3 or 4) peptides of the present disclosure (e.g., TPO mimetic peptides). Recombinant antibodies (e.g., TPO mimetic antibodies) also include intact antibodies having at least one TPO mimetic peptide and a hybrid constant region or a portion thereof, such as a G2/G4 hybrid constant region (see, e.g., Burton et al. (1992) Adv. Immun. 51: 1-18; Canfield et al. (1991) J. Exp. Med. 173: 1483-1491; and Mueller et al. (1997) Mol. Immunol. 34(6): 441-452). For example (and according to Kabat numbering), IgG1 and IgG4 constant regions contain G249G250 residues, while IgG2 constant regions do not contain residue 249, but do contain G250. In a G2/G4 hybrid constant region in which the 249-250 region is derived from a G2 sequence, the constant region can be further modified to introduce a glycine residue at position 249, resulting in a G2/G4 fusion with G249/G250. Other constant domain hybrids containing G249/G250 can also be used as scaffolds for the TPO mimetic antibodies of the present disclosure.
在一些实施方案中，可使融合蛋白与异源部分缀合。异源部分可为例如异源多肽、治疗剂(例如毒素或药物)或可检测标记，例如但不限于放射性标记、酶标记、荧光标记或发光标记。合适的异源多肽包括例如用于纯化治疗性抗体或片段的抗原标签(例如FLAG、聚组氨酸、血凝素(HA)、谷胱甘肽-S-转移酶(GST)或麦芽糖结合蛋白(MBP))。异源多肽还包括用作诊断标记或检测标记的多肽，例如萤光素酶、绿色荧光蛋白(GFP)或氯霉素乙酰基转移酶(CAT)。合适的放射性标记包括例如32P、33P、14C、125I、131I、35S和3H。合适的荧光标记包括而不限于荧光素、异硫氰酸荧光素(FITC)、GFP、DyLight 488、藻红蛋白(PE)、碘化丙锭(PI)、PerCP、PE-Alexa Fluor 700、Cy5、别藻蓝蛋白和Cy7。发光标记包括例如各种发光镧系元素(例如铕或铽)螯合物的任一种。例如，合适的铕螯合物包括二亚乙基三胺五乙酸(DTPA)的铕螯合物。酶标记包括例如碱性磷酸酶、CAT、萤光素酶和辣根过氧化物酶。In some embodiments, the fusion protein can be conjugated to a heterologous portion. The heterologous portion can be, for example, a heterologous polypeptide, a therapeutic agent (e.g., a toxin or a drug) or a detectable label, such as, but not limited to, a radiolabel, an enzyme label, a fluorescent label, or a luminescent label. Suitable heterologous polypeptides include, for example, antigen tags (e.g., FLAG, polyhistidine, hemagglutinin (HA), glutathione-S-transferase (GST), or maltose binding protein (MBP)) for purifying therapeutic antibodies or fragments. Heterologous polypeptides also include polypeptides used as diagnostic markers or detection markers, such as luciferase, green fluorescent protein (GFP), or chloramphenicol acetyltransferase (CAT). Suitable radiolabels include, for example,32 P,33 P,14 C,125 I,131 I,35 S, and3 H. Suitable fluorescent labels include, but are not limited to, fluorescein, fluorescein isothiocyanate (FITC), GFP, DyLight 488, phycoerythrin (PE), propidium iodide (PI), PerCP, PE-Alexa Fluor 700, Cy5, allophycocyanin and Cy7. Luminescent labels include, for example, any of various luminescent lanthanide (e.g., europium or terbium) chelates. For example, suitable europium chelates include europium chelates of diethylenetriaminepentaacetic acid (DTPA). Enzyme labels include, for example, alkaline phosphatase, CAT, luciferase and horseradish peroxidase.
在一些实施方案中，与相应的分离的TPO模拟肽相比，本文所述融合蛋白具有多个优势。首先，融合蛋白的血清半寿期延长。其次，可稳定融合蛋白支架内治疗性肽的活性区域的构象，就其与靶标结合而言，赋予它比分离的治疗性肽更大的活性和特异性。另外，与其分离的天然TPO肽对应物相比，本文描述的融合蛋白有多个额外优势。例如，给予哺乳动物受试者(例如人受试者)时，融合蛋白显著降低对天然TPO产生有害免疫应答的可能性。这与采用天然TPO蛋白的重组形式的治疗相反，后者通常导致在患者中产生TPO中和抗体，该抗体干扰患者的天然存在的TPO的活性。In some embodiments, the fusion proteins described herein have multiple advantages compared to the corresponding isolated TPO mimetic peptides. First, the serum half-life of the fusion protein is extended. Secondly, the conformation of the active region of the therapeutic peptide within the fusion protein scaffold can be stabilized, giving it greater activity and specificity than the isolated therapeutic peptide in terms of its binding to the target. In addition, the fusion proteins described herein have multiple additional advantages compared to their isolated natural TPO peptide counterparts. For example, when administered to a mammalian subject (e.g., a human subject), the fusion protein significantly reduces the likelihood of producing a harmful immune response to natural TPO. This is in contrast to treatments using recombinant forms of natural TPO proteins, which typically result in the production of TPO neutralizing antibodies in patients that interfere with the activity of the patient's naturally occurring TPO.
本文中使用的术语“抗体”包括例如嵌合化抗体或嵌合抗体、人源化抗体、去免疫化人抗体和完全人抗体。抗体支架可以在各种物种的任一种中制备或可来源于各种物种的任一种，例如哺乳动物，例如人、非人类灵长类动物 ( 例如猴、狒狒或黑猩猩 )、马、牛、猪、绵羊、山羊、狗、猫、兔、豚鼠、沙鼠、仓鼠、大鼠和小鼠。The term "antibody" as used herein includes, for example, chimeric antibodies or chimeric antibodies, humanized antibodies, deimmunized human antibodies, and fully human antibodies. Antibody scaffolds can be prepared in or derived from any of a variety of species, such as mammals, such as humans, non-human primates (e.g., monkeys, baboons, or chimpanzees), horses, cows, pigs, sheep, goats, dogs, cats, rabbits, guinea pigs, gerbils, hamsters, rats, and mice.
本文使用的术语“融合蛋白”，也可称为“抗体片段”或“治疗活性抗体片段”是指以下(例如TPO模拟肽的片段)：其(i)在结构上保持至少一个(例如至少1、2、3或4个；见上)治疗性肽(例如TPO模拟肽)存在于本公开内容的完整融合蛋白上；和(ii)功能上保持至少10%(例如至少12%、至少15%、至少20%、至少25%、至少30%、至少35%、至少40%、至少45%、至少50%、至少55%、至少60%、至少65%、至少70%、至少75%、至少80%、至少85%、至少90%、至少95%、至少98%、至少99%、至少99.5%或100%或更多)的完整治疗性抗体的治疗活性。The term "fusion protein", also referred to as "antibody fragment" or "therapeutically active antibody fragment" as used herein, refers to the following (e.g., a fragment of a TPO mimetic peptide): it (i) structurally retains at least one (e.g., at least 1, 2, 3 or 4; see above) therapeutic peptide (e.g., TPO mimetic peptide) present on the intact fusion protein of the present disclosure; and (ii) functionally retains at least 10% (e.g., at least 12%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5% or 100% or more) of the therapeutic activity of the intact therapeutic antibody.
本发明提供了如下任一项所述的方法，包括：The present invention provides any of the following methods, comprising:
（i）一种检测TPOR的方法，所述方法包括使用前面所述的TPO模拟肽、前面所述的融合蛋白，其中前面所述的TPO模拟肽、前面所述的融合蛋白可连接不影响其功能的可检测标记物。(i) A method for detecting TPOR, said method comprising using the aforementioned TPO mimetic peptide or the aforementioned fusion protein, wherein the aforementioned TPO mimetic peptide or the aforementioned fusion protein may be linked to a detectable marker that does not affect its function.
（ii）一种体外非治疗目的的恢复或提升血小板、红细胞、白细胞、血红蛋白的方法，所述方法包括向体外细胞或组织施用前面所述的TPO模拟肽、前面所述的融合蛋白的步骤。(ii) An in vitro method for restoring or increasing platelets, red blood cells, white blood cells, and hemoglobin for non-therapeutic purposes, the method comprising the step of administering the aforementioned TPO mimetic peptide or the aforementioned fusion protein to in vitro cells or tissues.
进一步，所述恢复或提升血小板、红细胞、白细胞、血红蛋白包括恢复或提升其活性、数量及存活时间。Furthermore, the restoration or enhancement of platelets, red blood cells, white blood cells, and hemoglobin includes restoration or enhancement of their activity, quantity, and survival time.
（iii）一种制备前面所述的融合蛋白的方法，所述方法包括使用前面所述的核苷酸序列通过载体转化到细胞中，通过培养细胞，从细胞培养液中分离纯化获得前面所述的融合蛋白。(iii) A method for preparing the aforementioned fusion protein, the method comprising using the aforementioned nucleotide sequence to transform into cells via a vector, culturing the cells, and isolating and purifying the aforementioned fusion protein from the cell culture fluid.
本发明提供了一种产品，所述产品包括前面所述的TPO模拟肽、前面所述的核苷酸序列、和/或前面所述的融合蛋白及其衍生物。The present invention provides a product, which comprises the aforementioned TPO mimetic peptide, the aforementioned nucleotide sequence, and/or the aforementioned fusion protein and derivatives thereof.
进一步，所述产品还包括其他治疗性肽。Furthermore, the product also includes other therapeutic peptides.
进一步，所述其他治疗性肽包括拮抗剂肽、激动剂肽、肽模拟物。Furthermore, the other therapeutic peptides include antagonist peptides, agonist peptides, and peptide mimetics.
进一步，所述产品还包括药学上可接受的载体。Furthermore, the product also includes a pharmaceutically acceptable carrier.
进一步，所述药学上可接受的载体包括助流剂、甜味剂、稀释剂、防腐剂、染料/着色剂、增味剂、表面活性剂、润湿剂、分散剂、悬浮剂、稳定剂、等渗剂、pH 调节和/或缓冲剂、溶剂、表面活性剂或乳化剂。Furthermore, the pharmaceutically acceptable carrier includes a glidant, a sweetener, a diluent, a preservative, a dye/colorant, a flavor enhancer, a surfactant, a wetting agent, a dispersant, a suspending agent, a stabilizer, an isotonic agent, a pH adjusting and/or buffering agent, a solvent, a surfactant or an emulsifier.
在一些实施方案中，所述产品包括载体、细胞、试剂、疫苗、试剂盒、芯片、试纸。In some embodiments, the product includes a vector, a cell, a reagent, a vaccine, a kit, a chip, or a test paper.
在一些实施方案中，可将前面所述的TPO模拟肽的核苷酸序列插入含有转录和翻译调节序列的表达载体中，所述转录和翻译调节序列包括例如启动子序列、核糖体结合部位、转录起始序列和终止序列、翻译起始序列和终止序列、转录终止子信号、聚腺苷酸化信号和增强子或激活因子 (activator) 序列。调节序列包括启动子及转录起始序列和终止序列。另外，表达载体可包括不止一个复制系统，从而可保持在两种不同的生物中，例如在哺乳动物或昆虫细胞中用于表达，在原核宿主中用于克隆和扩增。在一些实施方案中，所述载体整合到宿主细胞基因组中。可通过同时引入抗药基因例如大肠杆菌gpt(Mulligan和Berg(1981)Proc.Natl.Acad.Sci.USA，78：2072)或Tn5 neo(Southern和 Berg(1982)Mol.Appl.Genet.1：327)，来选择具有稳定整合的DNA的细胞。可将选择标记基因与待表达的DNA基因序列连接，或者通过共转染导入相同细胞中 (Wigler等 (1979)Cell 16：77)。In some embodiments, the nucleotide sequence of the TPO mimetic peptide described above can be inserted into an expression vector containing transcriptional and translational regulatory sequences, including, for example, promoter sequences, ribosome binding sites, transcriptional start and stop sequences, translational start and stop sequences, transcriptional terminator signals, polyadenylation signals, and enhancers or activator sequences. Regulatory sequences include promoters and transcriptional start and stop sequences. In addition, the expression vector can include more than one replication system, so that it can be maintained in two different organisms, such as in mammalian or insect cells for expression, and in prokaryotic hosts for cloning and amplification. In some embodiments, the vector is integrated into the host cell genome. Cells with stably integrated DNA can be selected by simultaneously introducing drug resistance genes such as Escherichia coli gpt (Mulligan and Berg (1981) Proc. Natl. Acad. Sci. USA, 78: 2072) or Tn5 neo (Southern and Berg (1982) Mol. Appl. Genet. 1: 327). The selectable marker gene can be linked to the DNA gene sequence to be expressed or introduced into the same cell by co-transfection (Wigler et al. (1979) Cell 16:77).
本发明提供了治疗有效量的前面所述的TPO模拟肽、和/或前面所述的融合蛋白在制备用于增加受试者的血小板生成的药物组合物中的应用。The present invention provides the use of a therapeutically effective amount of the aforementioned TPO mimetic peptide and/or the aforementioned fusion protein in the preparation of a pharmaceutical composition for increasing platelet production in a subject.
进一步，前面所述的应用，其中所述受试者患有血小板减少症相关病症和/或血小板不足相关的癌症。Furthermore, the aforementioned use, wherein the subject suffers from a thrombocytopenia-related disorder and/or a platelet deficiency-related cancer.
进一步，前面所述的应用，其中所述病症包括伯-索综合征、特发性血小板减少性紫癜、威-奥综合征、脾功能亢进、血栓性微血管病、弥散性血管内凝血、肝素诱发的血小板减少症、冯维勒布兰德病、变型冯维勒布兰德病、因 HIV 感染所致的血小板减少症、因慢性肝病所致的血小板减少症、药物诱导性血小板机能不全和/或格兰兹曼血小板机能不全、急性放射病、各种射线照射引起的骨髓抑制和/或胃肠道损伤、肿瘤放射治疗所引起的骨髓抑制和/或胃肠道损伤、化学治疗或外科手术结合进行的放射治疗所引起的骨髓抑制和/或胃肠道损伤、核与辐射损伤、肿瘤放射治疗引起的正常组织细胞的辐射损伤、慢性辐射综合症。Further, the aforementioned application, wherein the diseases include Berkeley-Sauer syndrome, idiopathic thrombocytopenic purpura, Willebrand-Oberleutz syndrome, hypersplenism, thrombotic microangiopathy, disseminated intravascular coagulation, heparin-induced thrombocytopenia, von Willebrand disease, variant von Willebrand disease, thrombocytopenia caused by HIV infection, thrombocytopenia caused by chronic liver disease, drug-induced thrombocytopenia and/or Glanzmann's thrombocytopenia, acute radiation sickness, bone marrow suppression and/or gastrointestinal damage caused by various radiation irradiations, bone marrow suppression and/or gastrointestinal damage caused by tumor radiotherapy, bone marrow suppression and/or gastrointestinal damage caused by radiotherapy combined with chemotherapy or surgery, nuclear and radiation damage, radiation damage to normal tissue cells caused by tumor radiotherapy, and chronic radiation syndrome.
进一步，所述癌症包括实体肿瘤癌症和血液肿瘤癌症。Furthermore, the cancer includes solid tumor cancer and blood tumor cancer.
进一步，所述实体肿瘤癌症包括来自肠、胰腺、脑、膀胱、乳腺、前列腺、肺、乳腺、卵巢、子宫、肝脏、肾脏、脾脏、胸腺、甲状腺、神经组织、上皮组织、淋巴结、骨、肌肉和皮肤的癌症。Further, the solid tumor cancer includes cancers from the intestine, pancreas, brain, bladder, breast, prostate, lung, breast, ovary, uterus, liver, kidney, spleen, thymus, thyroid, neural tissue, epithelial tissue, lymph node, bone, muscle and skin.
进一步，所述血液肿瘤癌症在血液形成组织，或在免疫系统的细胞中开始的癌症。Further, the hematologic malignancy cancer is a cancer that begins in blood-forming tissue, or in cells of the immune system.
在一些实施方案中，所述血液肿瘤癌症包括白血病、淋巴瘤、和多发性骨髓瘤，所述白血病包括自急性骨髓性白血病(AML)、骨髓增生异常综合征(MDS)以及急性淋巴母细胞性白血病(ALL)。In some embodiments, the hematological cancer includes leukemia, lymphoma, and multiple myeloma, including acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), and acute lymphoblastic leukemia (ALL).
在一些实施方案中，所述血小板减少症相关病症由化学治疗药剂引起。在一些实施方案中，所述血小板减少症相关病症由给予受试者的化学治疗方案或放射治疗方案。在一些具体的实施方案中，所述化学治疗方案包括给予受试者细胞毒性剂：环磷酰胺、泰素、甲氨蝶呤、氮芥、硫唑嘌呤、苯丁酸氮芥、氟尿嘧啶、顺铂、诺考达唑、羟基脲、长春新碱、长春碱、依托泊苷、多柔比星、博来霉素、卡铂、吉西他滨、紫杉醇、托泊替康和硫鸟嘌呤。在一些具体的实施方案中，所述放射治疗方案包括X射线或γ辐射。In some embodiments, the thrombocytopenia-related condition is caused by a chemotherapeutic agent. In some embodiments, the thrombocytopenia-related condition is caused by a chemotherapeutic regimen or a radiotherapy regimen given to a subject. In some specific embodiments, the chemotherapeutic regimen includes giving a subject a cytotoxic agent: cyclophosphamide, taxol, methotrexate, nitrogen mustard, azathioprine, chlorambucil, fluorouracil, cisplatin, nocodazole, hydroxyurea, vincristine, vinblastine, etoposide, doxorubicin, bleomycin, carboplatin, gemcitabine, paclitaxel, topotecan and thioguanine. In some specific embodiments, the radiotherapy regimen includes X-rays or gamma radiation.
在一些实施方案中，受试者是哺乳动物，包括牛、羊、马、狗、鼠、兔、鸡等哺乳动物。In some embodiments, the subject is a mammal, including cows, sheep, horses, dogs, mice, rabbits, chickens and the like.
进一步，所述药物组合物还包括药学上可接受的载体。Furthermore, the pharmaceutical composition also includes a pharmaceutically acceptable carrier.
在本文中使用的术语“药学上可接受的”描述了生物学上或其他方面不期望的材料，即，不会引起不可接受水平的生物学效果或以有害的方法互相作用的材料。As used herein, the term "pharmaceutically acceptable" describes a material that is biologically or otherwise undesirable, ie, a material that does not cause unacceptable levels of biological effects or interact in a deleterious manner.
进一步，所述药物组合物包括鼻喷剂、口服制剂、栓剂或肠胃外制剂的形式。Furthermore, the pharmaceutical composition includes the form of nasal spray, oral preparation, suppository or parenteral preparation.
术语“肠胃外”可包括皮下、静脉内、肌内、关节内、滑膜内、胸骨内、鞘内、肝内、病灶内和颅内的注射或输注。The term "parenteral" may include subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional, and intracranial injection or infusion.
进一步，所述药物组合物还包括降低辐射暴露的副作用或用于降低化学治疗的副作用的活性剂。Further, the pharmaceutical composition also includes an active agent for reducing the side effects of radiation exposure or for reducing the side effects of chemotherapy.
进一步，所述降低化学治疗的副作用的活性剂包括抗生素、麻醉剂、止吐药、类固醇、螯合剂和/或利尿药。Furthermore, the active agent for reducing the side effects of chemotherapy includes antibiotics, anesthetics, antiemetics, steroids, chelating agents and/or diuretics.
本发明提供了前面所述的TPO模拟肽、和/或前面所述的融合蛋白在制备检测受试者TPOR水平的产品中的应用。The present invention provides the use of the aforementioned TPO mimetic peptide and/or the aforementioned fusion protein in the preparation of a product for detecting the TPOR level of a subject.
有益效果Beneficial Effects
本发明提供了一种TPO模拟肽及其衍生品，TPO模拟肽具有刺激血小板生成的功能，在预防和治疗血小板缺乏症中具有重要的应用价值，同时在预防或治疗射线或化学药剂导致的血小板缺乏具有明显的效果。The present invention provides a TPO mimetic peptide and its derivatives. The TPO mimetic peptide has the function of stimulating platelet production and has important application value in preventing and treating thrombocytopenia. It also has obvious effects in preventing or treating thrombocytopenia caused by radiation or chemical agents.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
图1是SDS-PAGE电泳检测抗体纯度结果图；Figure 1 is a graph showing the results of antibody purity detection by SDS-PAGE electrophoresis;
图2是ELISA实验结果图；Fig. 2 is an ELISA experimental result diagram;
图3是小鼠外周血象检测结果图。FIG. 3 is a graph showing the results of peripheral blood test of mice.
具体实施方式Detailed ways
除非另有说明，否则本文使用的全部科技术语都具有本公开内容所属领域普通技术人员通常理解的相同含义。万一发生抵触，则以本文件(包括定义)为准。下面描述了优选的方法与材料，然而类似或等同于本文描述的方法与材料也可用于实施或检验本发明公开的方法和组合物。所有出版物、专利申请、专利和本文提及的其它参考文献均通过引用以其整体结合到本文中。Unless otherwise indicated, all scientific and technological terms used herein have the same meanings as those of ordinary skill in the art to which the present disclosure belongs. In the event of a conflict, this document (including definitions) shall prevail. Preferred methods and materials are described below, but methods and materials similar or equivalent to those described herein may also be used to implement or test the methods and compositions disclosed in the present invention. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety.
实施例1质粒构建Example 1 Plasmid Construction
1、实验方法1. Experimental methods
将2C1多肽（TPO模拟肽）的核苷酸序列与人IgG1Fc连接，然后连接入表达载体pCDNA3.1，构建重组表达质粒pCDNA3.1-2C1。pCDNA3.1-2C1经酶切鉴定以及测序确证。The nucleotide sequence of 2C1 polypeptide (TPO mimetic peptide) was connected to human IgG1Fc, and then connected to the expression vector pCDNA3.1 to construct the recombinant expression plasmid pCDNA3.1-2C1. pCDNA3.1-2C1 was identified by enzyme digestion and confirmed by sequencing.
2、实验结果2. Experimental results
经测序后得到pCDNA3.1-2C1中目的基因的核苷酸序列和氨基酸序列，如表1所示。After sequencing, the nucleotide sequence and amino acid sequence of the target gene in pCDNA3.1-2C1 were obtained, as shown in Table 1.
表1Table 1
实施例2 2C1-Fc融合蛋白表达纯化及检测Example 2 Expression, purification and detection of 2C1-Fc fusion protein
1、实验方法1. Experimental methods
（1）表达纯化(1) Expression and purification
根据试剂盒说明书，利用ExpiCHO转染试剂盒将pCDNA3.1-2C1转染进哺乳动物细胞ExpiCHO中，125 r/min，37℃，5% CO2细胞摇床培养，8-10 d后收集细胞培养上清，ProteinAFF蛋白柱进行纯化。用pH3.0的柠檬酸缓冲液洗脱，收集流出液，并用1 mol/LpH8.5 TRIS-HCL缓冲液中和，用0.01 mol/LpH7.2PBS透析72 h，0.22 μm滤膜过滤除菌。BCA方法检测纯化抗体浓度，SDS-PAGE电泳检测抗体纯度。According to the kit instructions, pCDNA3.1-2C1 was transfected into mammalian cells ExpiCHO using the ExpiCHO transfection kit, and cultured in a cell shaker at 125 r/min, 37°C, and 5% CO2. After 8-10 days, the cell culture supernatant was collected and purified using a ProteinAFF protein column. The protein was eluted with pH 3.0 citric acid buffer, and the effluent was collected and neutralized with 1 mol/L pH 8.5 TRIS-HCL buffer, dialyzed with 0.01 mol/L pH 7.2 PBS for 72 h, and sterilized by 0.22 μm filter membrane. The concentration of the purified antibody was detected by the BCA method, and the purity of the antibody was detected by SDS-PAGE electrophoresis.
（2）ELISA检测(2) ELISA test
包被液稀释人或鼠源C-MPL（血小板生成素受体）至1 μg/mL，100 μL每孔加入酶联板中，4℃过夜；PBST清洗3次后，5%牛奶封闭，37℃孵育1 h；吸弃封闭液，加入封闭液梯度稀释的2C1-Fc融合蛋白，起始浓度为1 μg/mL，37℃孵育1 h；PBST清洗3次后，加入羊抗人(Fab')2-HRP二抗室温反应45 min；PBST清洗3次后，TMB显色。1 mol/L的硫酸终止后检测450 nm处吸光度（A450）值。Human or mouse C-MPL (thrombopoietin receptor) was diluted to 1 μg/mL in the coating solution, and 100 μL was added to each well of the enzyme-linked plate at 4°C overnight; after washing with PBST for 3 times, the plate was blocked with 5% milk and incubated at 37°C for 1 h; the blocking solution was discarded, and 2C1-Fc fusion protein diluted in the blocking solution was added with a starting concentration of 1 μg/mL, and incubated at 37°C for 1 h; after washing with PBST for 3 times, goat anti-human (Fab')2-HRP secondary antibody was added and reacted at room temperature for 45 min; after washing with PBST for 3 times, TMB was used for color development. After terminating with 1 mol/L sulfuric acid, the absorbance (A450) value at 450 nm was detected.
2、实验结果2. Experimental results
使用SDS-PAGE电泳检测抗体纯度的结果如图1所示，结果显示，2C1-Fc融合蛋白纯化结果优良，能够用于后续实验。同时也进行了ELISA实验检测450 nm处吸光度值，结果如图2所示，结果显示，ELISA检测结果优异。The results of using SDS-PAGE electrophoresis to detect the purity of the antibody are shown in Figure 1. The results show that the purification results of 2C1-Fc fusion protein are excellent and can be used for subsequent experiments. ELISA experiments were also performed to detect the absorbance value at 450 nm. The results are shown in Figure 2. The results show that the ELISA test results are excellent.
实施例3小鼠外周血象检测Example 3 Mouse peripheral blood test
1、实验方法1. Experimental methods
将小鼠装在特制的小鼠照射盒中固定，6.0Gy60Coγ射线全身照射，照射剂量率为46.66 cGy/min，照射当天定义为试验第0天。受照小鼠随机分为溶剂对照组（IR）和2C1-Fc处理组，每组6只。2C1-Fc用生理盐水稀释至0.5 mg/mL。小鼠于照射后2h分别皮下注射生理盐水或者2C1-Fc，给药体积0.2 mL/鼠，即100 μg 2C1-Fc/鼠。自小鼠远端尾静脉采血10 μL/只/次，用全自动血细胞分析仪测定外周血细胞含量。采血时间为射线照射前2天、射线照射后第1、7、10、14、18、30天。The mice were fixed in a special mouse irradiation box and irradiated with 6.0Gy60Coγ-rays throughout the body at a dose rate of 46.66 cGy/min. The day of irradiation was defined as day 0 of the experiment. The irradiated mice were randomly divided into a solvent control group (IR) and a 2C1-Fc treatment group, with 6 mice in each group. 2C1-Fc was diluted to 0.5 mg/mL with normal saline. Mice were subcutaneously injected with normal saline or 2C1-Fc 2h after irradiation, with a dosing volume of 0.2 mL/mouse, i.e., 100 μg 2C1-Fc/mouse. Blood was collected from the distal tail vein of the mice at a volume of 10 μL/mouse/time, and the peripheral blood cell content was determined using an automatic blood cell analyzer. The blood was collected 2 days before irradiation and on days 1, 7, 10, 14, 18, and 30 after irradiation.
2、实验结果2. Experimental results
2C1-Fc治疗给药对6.0Gyγ射线照射小鼠外周血象的影响结果如图3所示。The results of the effect of 2C1-Fc therapeutic administration on the peripheral blood count of mice irradiated with 6.0 Gy γ rays are shown in FIG3 .
其中，经外周血象检测外周血白细胞数，结果如图3A所示，6.0Gyγ射线全身照射后小鼠外周血白细胞急剧下降，7天后才开始缓慢恢复。2C1-Fc给药可加速白细胞的恢复，2C1-Fc给药组于照射后第1天降至最低值后开始迅速恢复。在照射后第7-18天，2C1-Fc给药组与溶剂对照组相比，差异极其显著或非常显著。Among them, the number of peripheral blood leukocytes was detected by peripheral blood image, and the results are shown in Figure 3A. After 6.0Gy γ-ray whole-body irradiation, the number of peripheral blood leukocytes of mice dropped sharply and began to recover slowly after 7 days. 2C1-Fc administration can accelerate the recovery of leukocytes. The 2C1-Fc administration group began to recover rapidly after the number dropped to the lowest value on the first day after irradiation. On the 7th to 18th day after irradiation, the difference between the 2C1-Fc administration group and the solvent control group was extremely significant or very significant.
经外周血象检测外周血红细胞数，结果如图3B所示，6.0Gyγ射线照射后，两组小鼠外周血红细胞数进行性下降，照后7天内各组间无明显差别，溶剂对照组于照后18天达最低值为（3.3±0.8）×1012/L，此后逐渐恢复。2C1-Fc给药组于照射后第10天达最低值为（7.4±0.2）×1012/L。照后10-18天，2C1-Fc给药组的红细胞数与溶剂对照组相比差异极其显著。The peripheral blood red blood cell count was detected by peripheral blood examination. The results are shown in Figure 3B. After 6.0Gy γ-ray irradiation, the peripheral blood red blood cell count of the two groups of mice decreased progressively. There was no significant difference between the groups within 7 days after irradiation. The solvent control group reached the lowest value of (3.3±0.8)×1012 /L on the 18th day after irradiation, and then gradually recovered. The 2C1-Fc administration group reached the lowest value of (7.4±0.2)×1012 /L on the 10th day after irradiation. From 10 to 18 days after irradiation, the red blood cell count of the 2C1-Fc administration group was significantly different from that of the solvent control group.
经外周血象检测外周血血小板数，结果如图3C所示，6.0Gyγ射线全身照射后小鼠外周血血小板数量进行性下降，溶剂对照组于照射后10天达最低值为（56±16）×109/L，此后才逐渐恢复。2C1-Fc给药组小鼠血小板在第7天达最低值（604±98）×109/L，此后开始恢复。在照后第7-18天，2C1-Fc给药组血小板检测值高于同期溶剂对照组，差异显著或极其显著。The number of peripheral blood platelets was detected by peripheral blood image analysis. The results are shown in Figure 3C. After 6.0Gy γ-ray whole-body irradiation, the number of peripheral blood platelets in mice decreased progressively. The solvent control group reached the lowest value of (56±16)×109 /L 10 days after irradiation, and then gradually recovered. The platelet count of mice in the 2C1-Fc administration group reached the lowest value of (604±98)×109 /L on the 7th day, and then began to recover. On the 7th to 18th day after irradiation, the platelet detection value of the 2C1-Fc administration group was higher than that of the solvent control group in the same period, and the difference was significant or extremely significant.
经外周血象检测外周血血红蛋白数，结果如图3D所示，6.0Gyγ射线照射小鼠外周血红蛋白含量在照射后进行性下降，溶剂对照组于照射后18天达最低值，为照射前的34.6%，2C1-Fc给药组在照射后第10天达最低值，为照射前的76.3%。提示2C1-Fc给药可改善照射后小鼠外周血血红蛋白数的降低并促进其恢复。The peripheral blood hemoglobin count was detected by peripheral blood image, and the results are shown in Figure 3D. The peripheral hemoglobin content of mice irradiated with 6.0Gy γ-rays decreased progressively after irradiation. The solvent control group reached the lowest value on the 18th day after irradiation, which was 34.6% of the value before irradiation, and the 2C1-Fc administration group reached the lowest value on the 10th day after irradiation, which was 76.3% of the value before irradiation. This suggests that 2C1-Fc administration can improve the decrease in the peripheral blood hemoglobin count of mice after irradiation and promote its recovery.
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| CN118384261B (en)* | 2024-06-27 | 2024-08-20 | 中国人民解放军军事科学院军事医学研究院 | Delta-T3 HP and medical application of combined rhTPO as radioprotectant |
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