技术领域technical field
本发明属于生物医药领域，涉及一种C端-C端(羧基端-羧基端)连接的二价或双特异性，以及多价或多特异性抗体复合物的制备方法，特别是纳米抗体复合物的制备方法。The invention belongs to the field of biomedicine, and relates to a C-terminal-C-terminal (carboxyl-carboxyl-terminal) connection bivalent or bispecific, and a method for preparing a multivalent or multispecific antibody complex, especially a nanobody complex method of preparation.
背景技术Background technique
到目前为止，美国FDA已经批准超过50个单克隆抗体药物进入市场，这些药物在癌症，感染，自身免疫性疾病等病症的治疗上发挥了巨大的作用。据统计，2013年全球抗体药物的销售额达到750亿美元，占据全部生物药物销售额的一半。除了已经上市的单克隆抗体外，还有超过300个抗体处于研发当中。在这些抗体中，以抗体片段合成多价，多特异性的工程抗体由于其更优的特性正在占据越来越高的比重(Holliger,P.and P.J.Hudson(2005)."Engineered antibody fragments and the rise of single domains."NatureBiotechnology 23(9):1126-1136)。多价或多特异性抗体相比抗体单体而言具有以下几方面的优势。第一，因为具有多抗原结合部位，多价或多特异性抗体具有更高的亲和力，所以以在体内或体外结合靶标分子更加快速和稳定。第二，由于分子量增加，从而延长在体内循环的的半衰期，使得药物作用时间变长。第三，多特异性抗体能够识别不同的抗原，这赋予了常规抗体所没有的新功能。例如应用于特异性T细胞募集(bispecific T cell engager,BiTE)以杀伤肿瘤细胞，其中以单链可变区抗体(Single chain fragment variable,scFv)作为单元构筑的双特异性抗体(Blinatumomab)能够有效的治疗白血病和非霍奇金淋巴瘤，已于2014年底由美国FDA批准上市。除此之外多特异性还被广泛应用于治疗体内多种毒素或病原体以及体外诊断等领域，具有广阔的应用前景和市场。So far, the US FDA has approved more than 50 monoclonal antibody drugs to enter the market, and these drugs have played a huge role in the treatment of cancer, infection, autoimmune diseases and other diseases. According to statistics, the global sales of antibody drugs reached 75 billion US dollars in 2013, accounting for half of all biological drug sales. In addition to the monoclonal antibodies already on the market, more than 300 antibodies are under development. Among these antibodies, antibody fragments are used to synthesize multivalent and multispecific engineered antibodies due to their better characteristics (Holliger, P. and P.J. Hudson (2005). "Engineered antibody fragments and the rise of single domains."Nature Biotechnology 23(9):1126-1136). Compared with antibody monomers, multivalent or multispecific antibodies have the following advantages. First, because of having multiple antigen-binding sites, multivalent or multispecific antibodies have higher affinity, so they can bind target molecules more quickly and stably in vivo or in vitro. Second, due to the increase in molecular weight, the half-life of circulation in the body is prolonged, making the drug's action time longer. Third, multispecific antibodies can recognize different antigens, which confers new functions that conventional antibodies do not have. For example, it is applied to the recruitment of specific T cells (bispecific T cell engager, BiTE) to kill tumor cells, in which the bispecific antibody (Blinatumomab) constructed with a single chain variable region antibody (Single chain fragment variable, scFv) as a unit can effectively The treatment of leukemia and non-Hodgkin's lymphoma has been approved by the US FDA for marketing at the end of 2014. In addition, multi-specificity is also widely used in the treatment of various toxins or pathogens in the body and in vitro diagnosis and other fields, and has broad application prospects and markets.
现有制备二价或双特异性，以及多价或多特异性抗体大部分采用的是重组基因表达技术，具体表现为将抗体片段的基因序列顺序连接起来，抗体基因序列之间插入柔性肽段的基因如甘氨酸丝氨酸重复序列或天然抗体铰链区的序列，以提高抗体单元的自由度(Cuesta,A.M.,et al.(2010).，Trends in Biotechnology 28(7):355-362)。这种常规方法虽然已经被广泛的应用，但是其技术属性决定其只能合成C端-N端(羧基端-氨基端)连接的多抗体复合物。由于抗体活性区域大多在N端附近，所以C端-N端连接的方式容易造成N端的空间位阻影响抗体的活性(van Lith,S.A.M.,et al.(2017)，Bioconjug Chem 28(2):539-548)。因此，发展C端-C端连接的技术是最为理想的方式。现有C端-C端连接的技术多采用二硫键桥接和点击化学法，二硫键桥接法多不稳定而且易受本地二硫键影响，而点击化学法过程较多而且复杂(Witte,M.D.,et al.(2012)，Proceedings of the NationalAcademy of Sciences of the United States of America 109(30):11993-11998)，因此急需发展一种简便的稳定的C端-C端连接技术。Most of the current preparations of bivalent or bispecific, as well as multivalent or multispecific antibodies, use recombinant gene expression technology, which is specifically represented by sequentially linking the gene sequences of antibody fragments and inserting flexible peptides between antibody gene sequences Genes such as the glycine serine repeat sequence or the sequence of the natural antibody hinge region to increase the degree of freedom of the antibody unit (Cuesta, A.M., et al. (2010)., Trends in Biotechnology 28(7):355-362). Although this conventional method has been widely used, its technical attributes determine that it can only synthesize C-terminal-N-terminal (carboxyl-amino-terminal) linked multi-antibody complexes. Since the active region of the antibody is mostly near the N-terminal, the way of C-terminal-N-terminal connection is likely to cause steric hindrance at the N-terminal to affect the activity of the antibody (van Lith, S.A.M., et al. (2017), Bioconjug Chem 28(2): 539-548). Therefore, developing the technology of C-terminal-C-terminal connection is the most ideal way. Existing C-terminal-C-terminal connection technologies mostly use disulfide bridge bridging and click chemistry. Disulfide bridging methods are mostly unstable and easily affected by local disulfide bonds, while click chemistry processes are more complex and complicated (Witte, M.D., et al. (2012), Proceedings of the National Academy of Sciences of the United States of America 109(30):11993-11998), so it is urgent to develop a simple and stable C-terminal-C-terminal connection technology.
现有多价或多特异性抗体的构筑单元主要有scFv和纳米抗体(nanobody,singledomain antibody,sdAb)两种。纳米抗体也称单域抗体，是发现于骆驼科体内的一种抗体分子。其大小只有常规单克隆抗体的十分之一，却具备与其相当的抗原结合能力；在稳定性，原核表达，结合隐藏抗原表位等方面更胜一筹。由于相比scFv具有更小的结构，所以纳米抗体更加适合做为多价或多特异性抗体的构筑单元，具有广阔的应用前景和市场(Muyldermans,S.(2013).Vol 82 82:775-797)。The building blocks of existing multivalent or multispecific antibodies mainly include scFv and nanobody (single domain antibody, sdAb). Nanobodies, also known as single-domain antibodies, are antibody molecules found in camelids. Its size is only one-tenth of that of conventional monoclonal antibodies, but it has equivalent antigen-binding ability; it is superior in stability, prokaryotic expression, and binding to hidden epitopes. Due to its smaller structure than scFv, nanobodies are more suitable as building blocks for multivalent or multispecific antibodies, and have broad application prospects and markets (Muyldermans, S. (2013). Vol 82 82:775- 797).
由于二价或双特异性，多价或多特异性抗体的重要作用，已有C端-N端连接合成方式需要改良，发展简单的稳定的C端-C端连接是一种最理想的合成方式。Due to the important role of bivalent or bispecific, multivalent or multispecific antibodies, the existing C-terminal-N-terminal linkage synthesis method needs to be improved, and the development of a simple and stable C-terminal-C-terminal linkage is an ideal synthesis Way.
发明内容Contents of the invention
为了解决上述问题，本发明提供了用于以单域抗体或纳米抗体为构筑单元合成共价的C端-C端连接的二价抗体复合物的方法。In order to solve the above problems, the present invention provides a method for synthesizing a covalent C-terminal-C-terminal linked bivalent antibody complex using a single domain antibody or nanobody as a building block.
一种C端-C端连接的二价抗体或双特异性抗体复合物的制备方法，包括如下步骤：A method for preparing a C-terminal-C-terminal-connected bivalent antibody or bispecific antibody complex, comprising the following steps:
(1)通过基因重组在抗体的C末端融合FGE识别氨基酸序列，通过体外加入FGE进行催化，得C末端定点醛基修饰的抗体；(1) FGE recognition amino acid sequence is fused to the C-terminus of the antibody by genetic recombination, and FGE is added in vitro for catalysis to obtain an antibody modified by the C-terminal site-specific aldehyde group;
(2)将C末端定点醛基修饰的抗体与醛基反应性同双官能接头，在冰冻条件下反应，得二价抗体复合物，其中所述C末端定点醛基修饰的抗体与醛基反应性同双官能接头的摩尔比为1:0.4～1.2；(2) Reacting the C-terminally-directed aldehyde-modified antibody with the aldehyde-reactive bifunctional linker under frozen conditions to obtain a bivalent antibody complex, wherein the C-terminally-directed aldehyde-modified antibody reacts with the aldehyde group The molar ratio of the same bifunctional linker is 1:0.4～1.2;
(3)将C末端定点醛基修饰的抗体与醛基反应性同双官能接头，在冰冻条件下反应，得单一同双官能接头连接的抗体，其中所述C末端定点醛基修饰的抗体与醛基反应性同双官能接头的摩尔比为1:5～15；(3) Reacting the C-terminally-directed aldehyde group-modified antibody with the aldehyde-reactive bifunctional linker under frozen conditions to obtain an antibody linked to a single homobifunctional linker, wherein the C-terminus-directed aldehyde-group-modified antibody is combined with the bifunctional linker The molar ratio of aldehyde group reactivity to bifunctional linker is 1:5-15;
(4)将步骤(3)得到的抗体和C末端定点醛基修饰的抗体，在冰冻条件下反应，得双特异性抗体复合物，其中步骤(3)得到的抗体和C末端定点醛基修饰的抗体的可变区不同。(4) React the antibody obtained in step (3) with the antibody modified by the C-terminal site-directed aldehyde group under frozen conditions to obtain a bispecific antibody complex, wherein the antibody obtained in step (3) and the C-terminal site-directed aldehyde group modified The variable regions of the antibodies are different.
进一步，在上述技术方案中，在步骤(2)、步骤(3)以及步骤(4)中，所述的冰冻条件为-5℃～30℃，优选为-10℃～25℃，更优选为-20℃。Further, in the above technical solution, in step (2), step (3) and step (4), the freezing condition is -5°C to 30°C, preferably -10°C to 25°C, more preferably -20°C.
进一步，在上述技术方案中，在步骤(2)、步骤(3)以及步骤(4)中，所述的在冰冻条件下反应是指在-5℃～-30℃的温度下反应2～48h，所述温度优选-10℃～-25℃，更优选为-20℃，所述反应时间优选为10-30h，更优选为24h。Further, in the above technical solution, in step (2), step (3) and step (4), the reaction under freezing conditions refers to the reaction at a temperature of -5°C to -30°C for 2 to 48 hours , the temperature is preferably -10°C to -25°C, more preferably -20°C, and the reaction time is preferably 10-30h, more preferably 24h.
进一步，在上述技术方案中，在步骤(2)、步骤(3)以及步骤(4)中，所述的在冰冻条件下反应的反应体系的pH值为4.0～7.5，pH值优选为4-5，更优选为4。Further, in the above technical scheme, in step (2), step (3) and step (4), the pH value of the reaction system reacting under freezing conditions is 4.0-7.5, and the pH value is preferably 4- 5, more preferably 4.
进一步，在上述技术方案中，在步骤(1)中，所述抗体为纳米抗体、单链抗体scFV或可变区抗体Fab。Further, in the above technical solution, in step (1), the antibody is a nanobody, a single-chain antibody scFv or a variable region antibody Fab.
进一步，在上述技术方案中，在步骤(1)中，所述的FGE识别氨基酸序列为含有半胱氨酸-X-脯氨酸-X-精氨酸，所述X为任意天然氨基酸，优选FGE识别氨基酸序列为LCTPSR。进一步，在上述技术方案中，在步骤(2)、步骤(3)中，所述的醛基反应性同双官能接头为R-L-R，其中R为包含氨基、酰肼基、氧氨基、苯肼基或吡啶肼基的醛基反应性基团，L为具有-(CH2CH2-O)n和/或-(O-CH2CH2)n作为构成单元的聚合物，其中，n为1至100的整数，n优选为1到50，更优选为10到30。Further, in the above technical solution, in step (1), the FGE recognition amino acid sequence contains cysteine-X-proline-X-arginine, and the X is any natural amino acid, preferably The amino acid sequence recognized by FGE is LCTPSR. Further, in the above technical scheme, in step (2) and step (3), the aldehyde reactive homobifunctional linker is RLR, wherein R is a or an aldehyde reactive group of a pyridinehydrazine group, L is a polymer having -(CH2 CH2 -O)n and/or -(O-CH2 CH2 )n as a constituent unit, wherein n is 1 n is an integer from 1 to 100, and n is preferably 1 to 50, more preferably 10 to 30.
进一步，在上述技术方案中，所述FGE的基因编码序列来源于结核分枝杆菌或人类。Further, in the above technical solution, the gene coding sequence of FGE is derived from Mycobacterium tuberculosis or human.
进一步，在上述技术方案中，在步骤(3)中，将在冰冻条件下反应后的反应产物，通过体积排阻层析分离，得单一同双官能接头连接的抗体。Further, in the above technical scheme, in step (3), the reaction product reacted under frozen conditions is separated by size exclusion chromatography to obtain a single antibody linked to a bifunctional linker.
进一步，在上述技术方案中，在步骤(4)中，所述的步骤(3)得到的抗体与C末端定点醛基修饰的抗体的摩尔比为1:1～3。Further, in the above technical solution, in step (4), the molar ratio of the antibody obtained in step (3) to the antibody modified by the C-terminal site-directed aldehyde group is 1:1-3.
在上述技术方案中，在步骤(2)、步骤(3)和步骤(4)中所述的反应体系中还加入还原剂，将生成的C＝N双键还原成C-N单键可增加腙键或者肟键的稳定性，对于所述还原剂以及其加入量不做特别限定，本领域技术人员可以根据常规技术选择适当的还原剂以及加入量，优选地，还原剂可使用硼氢化钠、氰基硼氢化钠等，加入量为需还原物质摩尔量的10倍以上。In the above technical scheme, a reducing agent is also added to the reaction system described in step (2), step (3) and step (4), and the generated C=N double bond is reduced to a C-N single bond to increase the hydrazone bond Or the stability of the oxime bond, there is no special limitation on the reducing agent and its addition amount, those skilled in the art can select the appropriate reducing agent and the addition amount according to conventional techniques, preferably, the reducing agent can use sodium borohydride, cyanide Base sodium borohydride, etc., the amount added is more than 10 times the molar amount of the substance to be reduced.
本发明中，所述的FGE为甲酰甘氨酸生成酶，所述的C端-C端连接的二价抗体复合物是指两个相同的抗体各自通过羧基端与同双官能接头连接而得到的二聚体，所述C端-C端连接的双特异性抗体复合物是指两个具有不同可变区的抗体各自通过羧基端与同双官能接头连接而得到的具有双特异性的抗体复合物。所述单一同双官能接头连接的抗体是指同双官能接头两端中只有一端连接了抗体而形成的复合物。In the present invention, the FGE is a formylglycine-generating enzyme, and the C-terminal-C-terminal-linked bivalent antibody complex refers to two identical antibodies that are each connected to a homobifunctional linker through a carboxyl terminal. Dimer, the C-terminal-C-terminal linked bispecific antibody complex refers to a bispecific antibody complex obtained by linking two antibodies with different variable regions to the same bifunctional linker through the carboxyl terminal thing. The single antibody linked to the bifunctional linker refers to a complex formed by linking the antibody to only one of the two ends of the bifunctional linker.
本发明优选的技术方案中，所述的C末端定点醛基修饰的抗体，可以按照如下方法制备得到：In the preferred technical solution of the present invention, the antibody modified by the C-terminus-directed aldehyde group can be prepared according to the following method:
①在抗体的基因序列3’端加入甲酰甘氨酸生成酶(FGE)的识别肽段的基因序列，将该重组基因连接于载体，转入大肠杆菌表达宿主菌或毕赤酵母或哺乳动物细胞等宿主细胞中进行重组表达，表达的抗体经提取、纯化得3’端(C-端)融合有FGE识别氨基酸序列的重组抗体，所述提取以及纯化均可以采用本领域常规的方法进行，如纯化可采用金属螯合亲和层析初步纯化，再经体积排阻层析精细纯化等；其中所述FGE识别肽段优选LCTPSR，所述质粒优选pET21a或pET23a；① Add the gene sequence of the recognition peptide of formylglycine-generating enzyme (FGE) to the 3' end of the gene sequence of the antibody, connect the recombinant gene to the vector, and transfer it into E. coli expression host bacteria or Pichia pastoris or mammalian cells, etc. Recombinant expression is carried out in host cells, and the expressed antibody is extracted and purified to obtain a recombinant antibody fused with an FGE recognition amino acid sequence at the 3' end (C-terminus). The extraction and purification can be performed by conventional methods in the art, such as purification Metal chelate affinity chromatography can be used for preliminary purification, and then finely purified by size exclusion chromatography; wherein the FGE recognition peptide is preferably LCTPSR, and the plasmid is preferably pET21a or pET23a;
②在体外，将该重组抗体和一定量的FGE混合，在适当的缓冲液条件和温度下FGE催化重组抗体的C-端的标签转化为侧链带有醛基基团，即得到本发明所述的C末端定点醛基修饰的抗体，其中，所述重组抗体与FGE的加入量优选为按摩尔比1:5～10，缓冲液优选三乙醇胺-盐酸缓冲液，pH 7.0-9.0，缓冲液中加入1-5mM巯基乙醇，离子强度50-150mM氯化钠，催化温度优选18℃至30℃。② In vitro, the recombinant antibody is mixed with a certain amount of FGE, and under appropriate buffer conditions and temperature, FGE catalyzes the conversion of the C-terminal label of the recombinant antibody into a side chain with an aldehyde group, that is, the present invention can be obtained. The C-terminal site-directed aldehyde group-modified antibody, wherein, the added amount of the recombinant antibody and FGE is preferably 1:5-10 by molar ratio, and the buffer is preferably triethanolamine-hydrochloric acid buffer, pH 7.0-9.0, in the buffer 1-5mM mercaptoethanol is added, the ionic strength is 50-150mM sodium chloride, and the catalytic temperature is preferably 18°C to 30°C.
本发明优选的技术方案中，所述C端-C端连接的二价抗体复合物，可按如下方法制备得到：将上述得到的C末端定点醛基修饰的抗体与醛基反应性同双官能接头，按照摩尔比1:0.5～1.0混合，在-5℃～-30℃、pH 4-5的条件下反应2-24h，可完成醛基反应性同双官能接头与抗体C末端醛基的连接，从而形成共价稳定的C端-C端连接的二价抗体复合物，最终收率为50％左右。In the preferred technical solution of the present invention, the C-terminus-C-terminus-linked bivalent antibody complex can be prepared as follows: the C-terminus-specific aldehyde group-modified antibody obtained above and the aldehyde group-reactive homobifunctional The linker is mixed according to the molar ratio of 1:0.5~1.0, and reacted for 2-24h under the conditions of -5°C~-30°C, pH 4-5, which can complete the aldehyde group reactivity with the bifunctional linker and the C-terminal aldehyde group of the antibody. Linked to form a covalently stable C-terminal-C-terminal linked bivalent antibody complex with a final yield of about 50%.
本发明优选的技术方案中，所述C端-C端连接的双特异性抗体复合物，可按如下方法制备得到：In the preferred technical solution of the present invention, the C-terminal-C-terminal linked bispecific antibody complex can be prepared as follows:
(1)将上述得到的C末端定点醛基修饰的抗体与醛基反应性同双官能接头，按照摩尔比1:5-15混合，在-5℃～-30℃、pH 4-5的条件下反应2-24h，可完成醛基反应性同双官能接头与抗体C末端醛基的连接，获得单一同双官能接头连接的抗体；其中，优选地，反应产物可经体积排阻色谱分离，得纯化的单一同双官能接头连接的抗体，用于下一步反应。(1) Mix the C-terminus-specific aldehyde-modified antibody obtained above with the aldehyde-reactive bifunctional linker at a molar ratio of 1:5-15, at -5°C to -30°C, pH 4-5 After reacting for 2-24 hours, the aldehyde group-reactive homobifunctional linker can be connected to the C-terminal aldehyde group of the antibody to obtain a single homobifunctional linker-linked antibody; wherein, preferably, the reaction product can be separated by size exclusion chromatography, The purified monofunctional linker-linked antibody was obtained for the next reaction.
(2)将上述步骤(1)得到的单一同双官能接头连接的抗体与C末端定点醛基修饰的抗体按照摩尔比1:1～3混合，在-5℃～-30℃、pH 4-5的条件下反应2-24h，其中，单一同双官能接头连接的抗体与C末端定点醛基修饰的抗体的可变区不同，从而可得到C端-C端连接的双特异性抗体复合物，最终收率为30％左右。(2) Mix the monofunctional linker-linked antibody obtained in the above step (1) with the C-terminal site-directed aldehyde-modified antibody at a molar ratio of 1:1 to 3. React for 2-24 hours under the conditions of 5, wherein the variable region of the antibody linked to the single homobifunctional linker is different from that of the antibody modified by the C-terminal site-directed aldehyde group, so that a C-terminal-C-terminal linked bispecific antibody complex can be obtained , and the final yield is about 30%.
本发明相对于现有技术具有如下优点和效果：Compared with the prior art, the present invention has the following advantages and effects:
本发明通过在冰冻条件下实现C端-C端连接的二价或双特异性抗体复合物，条件温和，反应速度快，收率较高。相比常规方法所生产的C端-N端连接的二价或双特异性抗体复合物本发明能够得到更高的亲和力(如图1)，所述亲和力是基于表面等离子体共振所测得平衡解离常数。相比现有制备C端-C端连接的二价或双特异性抗体的二硫桥接法或点击化学法，本发明具有更好的特异性，同时步骤较少，表现在只在C端生成的醛基进行连接，生成的腙键或肟键在生理条件下稳定并且用还原剂还原后不可逆，同时由于添加了聚乙二醇的接头，其血液半衰期能够延长。The invention realizes the C-terminal-C-terminal linked bivalent or bispecific antibody complex under freezing conditions, with mild conditions, fast reaction speed and high yield. Compared with the C-terminal-N-terminal linked bivalent or bispecific antibody complexes produced by conventional methods, the present invention can obtain higher affinity (as shown in Figure 1), the affinity is based on the equilibrium measured by surface plasmon resonance dissociation constant. Compared with the existing disulfide bridging method or click chemistry method for preparing C-terminal-C-terminal-linked bivalent or bispecific antibodies, the present invention has better specificity and fewer steps, and is only generated at the C-terminal The hydrazone bond or oxime bond generated is stable under physiological conditions and irreversible after being reduced with a reducing agent. At the same time, due to the addition of polyethylene glycol linkers, its blood half-life can be extended.
附图说明Description of drawings
图1为一纳米抗体的结构、通过传统方法制备C-N连接的二价或双特异性纳米抗体结构以及通过本发明制备C-C连接的二价或双特异性纳米抗体结构的示意图。Figure 1 is a schematic diagram of the structure of a Nanobody, the structure of a C-N linked bivalent or bispecific Nanobody prepared by a traditional method, and the structure of a C-C linked bivalent or bispecific Nanobody prepared by the present invention.
图2A为使用荧光分子对醛基化修饰纳米抗体进行标记，在SDS-PAGE上对其分离的结果，图2B为在高效液相色谱法联用点喷雾电离轨道离子阱质谱对醛基化修饰纳米抗体精确定量，所示图谱为去卷积后分子量-丰度图。Figure 2A is the result of labeling the aldehyde-modified nanobody with fluorescent molecules and separating it on SDS-PAGE, and Figure 2B is the result of the aldehyde-modified nanobody using high-performance liquid chromatography coupled with point spray ionization orbital ion trap mass spectrometry Nanobodies are accurately quantified, and the spectra shown are deconvoluted molecular weight-abundance maps.
图3为加入双酰肼功能化PEG400连接头，在不同pH值和温度下反应后24小时后的产物在SDS-PAGE上的分离结果。Fig. 3 is the separation result of the product on SDS-PAGE after adding the bishydrazide functionalized PEG400 linker and reacting at different pH values and temperatures for 24 hours.
图4A为高效液相色谱法测定二价抗体产率在-20℃冰冻温度下随时间的变化，图4B为在不同温度下二价纳米抗体产率随时间的变化。Fig. 4A is the change with time of the yield of the bivalent antibody determined by high performance liquid chromatography at a freezing temperature of -20° C., and Fig. 4B is the change of the yield of the bivalent nanobody with time at different temperatures.
图5A为分别加入不同长度连接头的二价纳米抗体在SDS-PAGE上的分离结果，图5B为体积排阻层析对含有不同长度接头二价纳米抗体的分离纯化。Figure 5A shows the separation results on SDS-PAGE of bivalent Nanobodies with linkers of different lengths, and Figure 5B shows the separation and purification of bivalent Nanobodies with linkers of different lengths by size exclusion chromatography.
图6为利用体积排阻层析对连接了同双官能接头的纳米抗体B单体进行分离Figure 6 is the separation of Nanobody B monomer linked with the same bifunctional linker by size exclusion chromatography
具体实施方式Detailed ways
下述非限制性实施例可以使本领域的普通技术人员更全面地理解本发明，但不以任何方式限制本发明。下述实施例中，如无特殊说明，所使用的实验方法均为常规方法，所用材料、试剂等均可从生物或化学公司购买。The following non-limiting examples can enable those skilled in the art to understand the present invention more fully, but do not limit the present invention in any way. In the following examples, unless otherwise specified, the experimental methods used are conventional methods, and the materials and reagents used can be purchased from biological or chemical companies.
下述实施例中所用的材料：Materials used in the following examples:
纳米抗体A：编码基因序列如SEQ ID NO.1，其中第511-528位的序列ctgtgcaccccgtctcgt为FGE识别序列；Nanobody A: coding gene sequence such as SEQ ID NO.1, wherein the sequence ctgtgcaccccgtctcgt at positions 511-528 is the FGE recognition sequence;
纳米抗体B：编码基因序列如SEQ ID NO.2，其中第499-516位的序列ctgtgcaccccgtctcgt为FGE识别序列。Nanobody B: coding gene sequence such as SEQ ID NO.2, wherein the sequence ctgtgcaccccgtctcgt at positions 499-516 is the FGE recognition sequence.
实施例1纳米抗体的C末端定点修饰Example 1 C-terminal site-specific modification of Nanobodies
将纳米抗体A的编码基因序列克隆至表达载体质粒pET23a中，导入大肠杆菌T7Shuffle(DE3)中进行表达，收集菌体，并细胞破碎，从细胞破碎上清中利用金属螯合亲和层析HisTrap HP 5mL(GE Healthcare)纯化纳米抗体，最终产量在100毫克每升发酵液左右。将纯化的纳米抗体经超滤浓缩换液至三乙醇胺缓冲液(25mM TEAM，pH9.0，150mM NaCl，1mM巯基乙醇)，纳米抗体浓度为5mg/mL，加入最终浓度为纳米抗体摩尔量约十分之一即1mg/mL的纯度为>90％FGE酶(FGE酶分子量约为33kDa，纳米抗体分子量约为18kDa)，在18℃条件下温和震荡催化反应20小时，反应后离心去除蛋白沉淀，得到羧基端定点醛基化修饰的纳米抗体A，命名为纳米抗体A0。The coding gene sequence of Nanobody A was cloned into the expression vector plasmid pET23a, introduced into Escherichia coli T7Shuffle (DE3) for expression, the bacteria were collected, and the cells were broken, and the supernatant from the broken cells was analyzed by metal chelation affinity chromatography HisTrap HP 5mL (GE Healthcare) was used to purify Nanobodies, and the final yield was about 100 mg per liter of fermentation broth. The purified nanobody was concentrated by ultrafiltration and replaced with triethanolamine buffer solution (25mM TEAM, pH9.0, 150mM NaCl, 1mM mercaptoethanol), the nanobody concentration was 5mg/mL, and the final concentration was about ten One-third of the purity of 1mg/mL is >90% FGE enzyme (the molecular weight of FGE enzyme is about 33kDa, and the molecular weight of nanobody is about 18kDa). The reaction is catalyzed by gentle shaking at 18°C for 20 hours, and the protein precipitate is removed by centrifugation after the reaction. The Nanobody A modified by site-directed aldehydes at the carboxyl terminal was obtained, and named as Nanobody A0.
用高效液相色谱联用高分辨电喷雾电离质谱准确检测醛基修饰效率，图2B所示为蛋白质质谱图去卷积后分子量-丰度图，在图谱中可见修饰前和修饰后的蛋白质(纳米抗体)谱峰，醛基化修饰后，蛋白的分量约减少18Da，通过比较两者的积分峰面积可以确定蛋白的醛基化修饰效率。也可以将反应后的蛋白溶液经超滤浓缩换液至酸性缓冲液(如0.1M醋酸缓冲液pH 4.0含150mM NaCl)，随后加入终浓度为500μmol/L带有酰肼基团的荧光分子Lucifer Yellow CH lithium slat(Thermo Fisher)对醛基进行标记，经SDS-PAGE的分析也可对C末端醛基修饰效率初步定量，如图2A所示，由于标记上Lucifer Yellow荧光分子，蛋白在紫外成像下可见明显荧光，同时蛋白由于标记上一个荧光分子导致分子量增加在SDS-PAGE上表现为迁移率降低，对比没有标记上的蛋白条带向上迁移，可通过灰度扫描粗略对醛基化修饰进行定量。High-performance liquid chromatography coupled with high-resolution electrospray ionization mass spectrometry was used to accurately detect the modification efficiency of aldehyde groups. Figure 2B shows the deconvoluted molecular weight-abundance map of the protein mass spectrogram, and the protein before and after modification can be seen in the map ( Nanobody) spectral peak, after the formylation modification, the protein component is reduced by about 18Da, and the formylation modification efficiency of the protein can be determined by comparing the integrated peak areas of the two. The reacted protein solution can also be concentrated by ultrafiltration and replaced to an acidic buffer (such as 0.1M acetate buffer pH 4.0 containing 150mM NaCl), and then add a fluorescent molecule Lucifer with a hydrazide group at a final concentration of 500μmol/L Yellow CH lithium slat (Thermo Fisher) was used to label the aldehyde group, and the efficiency of the modification of the C-terminal aldehyde group could also be quantified by SDS-PAGE analysis. As shown in Figure 2A, due to the labeling of Lucifer Yellow fluorescent molecules, the protein was imaged in ultraviolet light Significant fluorescence can be seen below, and at the same time, the molecular weight of the protein increases due to the labeling of a fluorescent molecule, which is manifested as a decrease in mobility on SDS-PAGE. Compared with the upward migration of the protein band without labeling, the aldehyde modification can be roughly carried out by grayscale scanning. Quantitative.
实施例2在冰冻条件下实现制备C-C连接二价纳米抗体Example 2 Realize the preparation of C-C linked bivalent Nanobodies under frozen conditions
将实施例1制备得到的羧基端定点醛基化修饰的纳米抗体A(纳米抗体A0)超滤浓缩换液至酸性缓冲液，0.1M醋酸缓冲液pH 4.0或0.1M MES缓冲液pH5.5，或者中性缓冲液0.2M PBS pH7.4，纳米抗体A0的浓度为2mg/mL即100μmol/L。取一定量配置好的纳米抗体A0，加入双官能接头HZ-PEG-HZ 400(双酰肼聚乙二醇-400)和还原剂氰基硼氢化钠，其中HZ-PEG-HZ 400和氰基硼氢化钠在反应体系中的终浓度分别为50μmol/L和1mmol/L，即，在反应体系中PEG与纳米抗体的摩尔比为1:2，氰基硼氢化钠和纳米抗体的摩尔比为10:1。将加入好的反应混合物，放入低温槽中降温至-30℃，使反应混合物成为冰冻状态，然后调整至不同温度，分别为-30℃，-20℃，-10℃以及-5℃，反应0～25h。其中，将反应混合物成为冰冻状态然后调整至各反应温度，这样能够缩短样品结冰的时间，从而缩短反应时间，本发明中将所述反应混合物直接放入相应温度也可以。另外，还设有37℃和-80℃反应条件，即如上所述，换液至不同酸性缓冲液的纳米抗体中，加入HZ-PEG-HZ 400和还原剂(加入量同上)后，将反应液温度调整至37℃和-80℃后，继续反应0～25h。图3为在不同pH值和温度下反应24小时后的反应产物在SDS-PAGE上的电泳图，可见在-20℃度和pH 4.0的条件下可见明显的二价纳米抗体条带。The Nanobody A (Nanobody A0) modified by site-directed aldehydesylation at the carboxyl terminal prepared in Example 1 was concentrated by ultrafiltration and replaced with an acidic buffer, 0.1M acetate buffer pH 4.0 or 0.1M MES buffer pH 5.5, Or neutral buffer solution 0.2M PBS pH7.4, the concentration of Nanobody A0 is 2 mg/mL, that is, 100 μmol/L. Take a certain amount of configured nanobody A0, add bifunctional linker HZ-PEG-HZ 400 (bishydrazide polyethylene glycol-400) and reducing agent sodium cyanoborohydride, wherein HZ-PEG-HZ 400 and cyano The final concentrations of sodium borohydride in the reaction system were 50 μmol/L and 1 mmol/L, respectively, that is, the molar ratio of PEG to nanobody in the reaction system was 1:2, and the molar ratio of sodium cyanoborohydride to nanobody was 10:1. Put the added reaction mixture into a low-temperature tank to cool down to -30°C, make the reaction mixture into a frozen state, and then adjust to different temperatures, respectively -30°C, -20°C, -10°C and -5°C. 0~25h. Wherein, the reaction mixture is frozen and then adjusted to each reaction temperature, which can shorten the time for the sample to freeze, thereby shortening the reaction time. In the present invention, the reaction mixture can be directly placed at the corresponding temperature. In addition, there are also reaction conditions of 37°C and -80°C, that is, as mentioned above, the liquid is changed to the nanobody of different acidic buffer, and after adding HZ-PEG-HZ 400 and reducing agent (the amount added is the same as above), the reaction After the solution temperature was adjusted to 37°C and -80°C, the reaction was continued for 0-25h. Figure 3 is the electrophoresis of the reaction product on SDS-PAGE after reacting at different pH values and temperatures for 24 hours, it can be seen that obvious bivalent nanobody bands can be seen under the conditions of -20°C and pH 4.0.
将反应后的溶液用氢氧化钠调节pH至中性，随后通过高效液相色谱法将反应后的混合物进行分离分析并对比相应峰积分面积可计算反应收率。图4A为高效液相色谱法测定二价抗体产率在-20℃冰冻温度下随时间的变化，图4B为在不同温度下二价纳米抗体产率随时间的变化。可见，在反应24小时左右收率可达到最大，-30℃和-5℃均反应较慢。在-10℃到-20℃区间内反应能较快发生。在-10℃到-20℃区间，二价纳米收率为30％至50％之间。The pH of the reacted solution is adjusted to neutral with sodium hydroxide, and then the reacted mixture is separated and analyzed by high performance liquid chromatography, and the reaction yield can be calculated by comparing the corresponding peak integral areas. Fig. 4A is the change with time of the yield of the bivalent antibody determined by high performance liquid chromatography at a freezing temperature of -20° C., and Fig. 4B is the change of the yield of the bivalent nanobody with time at different temperatures. It can be seen that the yield can reach the maximum in about 24 hours of reaction, and the reaction at -30°C and -5°C is relatively slow. The reaction can occur faster in the range of -10°C to -20°C. In the range of -10°C to -20°C, the yield of divalent nanometers is between 30% and 50%.
图5A为上述分别加入不同长度连接头的反应产物在SDS-PAGE上的电泳图，其中泳道M为蛋白Marker，1-4泳道依次别为纳米抗体A0(终浓度100μmol/L)中加入双功能接头(终浓度50μmol/L)，O-linker、HZ-PEG-HZ 400、HZ-PEG-HZ1000和HZ-PEG-HZ2000后的反应产物，5-8依次为纳米抗体B0(终浓度100μmol/L)中加入双功能接头(终浓度50μmol/L)，O-linker、HZ-PEG-HZ400、HZ-PEG-HZ1000和HZ-PEG-HZ2000后的反应产物。图5A中可见，泳道1-8在40kDa左右均出现一定量的二价纳米抗体条带，表明对纳米抗体A0和B0而言，加入不同长度的双功能接头，在冰冻条件下反应后均可以生成一定量的二价纳米抗体。图5B为上述分别加入不同长度连接头的反应产物通过体积排阻层析分离纯化的结果色谱图。图5B中可见，反应后溶液中还会残留有单体，连接了接头的单体和二价抗体，其中二价抗体能够很好的被分离出来进行下一步的研究。Figure 5A is the electrophoresis image on SDS-PAGE of the above-mentioned reaction products with linkers of different lengths added, in which lane M is the protein Marker, and lanes 1-4 are the nanobody A0 (final concentration 100 μmol/L) added bifunctional Linker (final concentration 50μmol/L), reaction products after O-linker, HZ-PEG-HZ 400, HZ-PEG-HZ1000 and HZ-PEG-HZ2000, 5-8 were followed by Nanobody B0 (final concentration 100μmol/L ), the reaction product after adding bifunctional linker (final concentration 50 μmol/L), O-linker, HZ-PEG-HZ400, HZ-PEG-HZ1000 and HZ-PEG-HZ2000. It can be seen in Figure 5A that a certain amount of bivalent Nanobody bands appear at about 40kDa in lanes 1-8, indicating that for Nanobodies A0 and B0, adding bifunctional linkers of different lengths can all be effective after reaction under frozen conditions. Generate a certain amount of bivalent Nanobodies. Fig. 5B is a chromatogram of the result of separation and purification of the reaction products with linkers of different lengths added above by size exclusion chromatography. It can be seen in Figure 5B that monomers remain in the solution after the reaction, and the monomers connected with the linker and the bivalent antibody can be well separated for further research.
实施例3冰冻法制备C-C连接的双特异性纳米抗体Example 3 Preparation of C-C Linked Bispecific Nanobodies by Freezing Method
(1)将纳米抗体B的编码基因序列克隆至表达载体质粒pET23a中，导入大肠杆菌T7Shuffle(DE3)中进行表达，收集菌体，并细胞破碎，从细胞破碎上清中利用金属螯合亲和层析HisTrap HP 5mL(GE Healthcare)纯化纳米抗体，最终产量约为100毫克每升发酵液。将纯化的纳米抗体经超滤浓缩换液至三乙醇胺缓冲液(25mM TEAM，pH 9.0，150mM NaCl，1mM巯基乙醇)，纳米抗体浓度为5mg/mL，加入最终浓度为纳米抗体摩尔量约十分之一即1mg/mL的纯度为>90％FGE酶(FGE酶分子量约为33kDa，纳米抗体分子量约为18kDa)，在18℃条件下温和震荡催化反应20小时，反应后离心去除蛋白沉淀，得到羧基端定点醛基化修饰的纳米抗体B，命名为纳米抗体B0。(1) Cloning the coding gene sequence of Nanobody B into the expression vector plasmid pET23a, introducing it into Escherichia coli T7Shuffle (DE3) for expression, collecting the bacteria, and disrupting the cells. Nanobodies were purified by chromatography on HisTrap HP 5 mL (GE Healthcare) with a final yield of approximately 100 mg per liter of fermentation broth. The purified nanobody was concentrated by ultrafiltration and replaced with triethanolamine buffer solution (25mM TEAM, pH 9.0, 150mM NaCl, 1mM mercaptoethanol), the nanobody concentration was 5mg/mL, and the final concentration was about 10% of the nanobody molar weight. One, that is, the purity of 1mg/mL is >90% FGE enzyme (the molecular weight of FGE enzyme is about 33kDa, and the molecular weight of nanobody is about 18kDa), and the catalytic reaction is gently shaken at 18°C for 20 hours, and the protein precipitate is removed by centrifugation after the reaction. Nanobody B modified by site-directed aldehydes at the carboxy-terminus is named as Nanobody B0.
(2)将上一步所得纳米抗体B0超滤浓缩换液至酸性缓冲液(0.1M醋酸缓冲液pH4.0，含150mM NaCl)，终浓度为2mg/mL即100μmol/L。取一定量配置好的纳米抗体B0，分别加入不同长度双功能接头，O-linker、HZ-PEG-HZ400、HZ-PEG-HZ1000和HZ-PEG-HZ2000，双功能接头在反应体系中的终浓度为1mmol/L，即，在反应体系中双功能接头与纳米抗体的摩尔比为10:1，另外加入终浓度1mmol/L的氰基硼氢化钠。将该反应混合物，在-20℃冰冻条件下反应24小时。(2) The nanobody B0 obtained in the previous step was concentrated by ultrafiltration and replaced with an acidic buffer (0.1M acetate buffer pH 4.0, containing 150mM NaCl), with a final concentration of 2mg/mL or 100μmol/L. Take a certain amount of configured nanobody B0, add bifunctional linkers of different lengths, O-linker, HZ-PEG-HZ400, HZ-PEG-HZ1000 and HZ-PEG-HZ2000, the final concentration of bifunctional linkers in the reaction system 1mmol/L, that is, the molar ratio of the bifunctional linker to the nanobody in the reaction system is 10:1, and sodium cyanoborohydride is added at a final concentration of 1mmol/L. The reaction mixture was reacted at -20°C for 24 hours under freezing conditions.
所述体积排阻层析分离步骤如下：将反应产物，放置室温升温，待融化后加入1MNaOH溶液调节pH值为中性，随后通过体积排阻色谱柱Superdex 75Increase 10/300GL(GEHealthcare)，用20mM磷酸盐150mM NaCl pH7.4缓冲液作为运行溶液以0.6mL/min的速度进行分离，收集各个组分，通过SDS-PAGE验证条带。最后保留连接了同双官能接头的纳米抗体B单体，如图6所示。图6A为不同接头过量加入后的产物在体积排阻层析上分离纯化的色谱峰图，图6B为不同色谱峰的SDS-PAGE鉴定(泳道0、3、6、10分别为过量加入双功能接头O-linker、HZ-PEG-HZ400、HZ-PEG-HZ1000和HZ-PEG-HZ2000后的反应产物，其余泳道为图6A中对应的色谱峰组分)。可见，一定量的连接了接头的纳米抗体可以有效得被分离纯化以用于下一步的研究。The separation steps of the size exclusion chromatography are as follows: place the reaction product at room temperature to raise the temperature, add 1M NaOH solution to adjust the pH value to be neutral after melting, then pass through the size exclusion chromatography column Superdex 75Increase 10/300GL (GEHealthcare) with 20mM Phosphate 150mM NaCl pH7.4 buffer was used as the running solution to separate at a speed of 0.6mL/min, and each component was collected, and the bands were verified by SDS-PAGE. Finally, the Nanobody B monomer with the homobifunctional linker attached is retained, as shown in FIG. 6 . Figure 6A is the chromatographic peak diagram of the separation and purification of products after the excess addition of different linkers on size exclusion chromatography, and Figure 6B is the SDS-PAGE identification of different chromatographic peaks (swimming lanes 0, 3, 6, and 10 are respectively the excess addition of bifunctional The reaction products after linker O-linker, HZ-PEG-HZ400, HZ-PEG-HZ1000 and HZ-PEG-HZ2000, and the remaining lanes are the corresponding chromatographic peak components in Figure 6A). It can be seen that a certain amount of linker-linked Nanobodies can be effectively isolated and purified for further research.
(3)上一步所得的连接了同双官能接头的纳米抗体B与等摩尔比的实施例1中的醛基化修饰纳米抗体A混合，并将反应溶液用醋酸调至pH 4.0，另外加入终浓度1mmol/L的氰基硼氢化钠，随后在-20℃下反应至少24小时，所得反应后产物通过体积排阻色谱柱Superdex 75Increase 10/300GL分离可得C-C连接的双特异性纳米抗体A-B。收率为20％到40％。(3) The Nanobody B connected with the homobifunctional linker obtained in the previous step is mixed with the formylation-modified Nanobody A in Example 1 in an equimolar ratio, and the reaction solution is adjusted to pH 4.0 with acetic acid, and the final solution is added in addition. Sodium cyanoborohydride at a concentration of 1 mmol/L, followed by reaction at -20°C for at least 24 hours, and the resulting reaction product was separated by a size exclusion chromatography column Superdex 75Increase 10/300GL to obtain C-C linked bispecific Nanobody A-B. The yield is 20% to 40%.
实施例4二价及双特异性纳米抗体的亲和常数测定Example 4 Determination of Affinity Constants of Bivalent and Bispecific Nanobodies
通过表面等离子体共振(SPR)确定二价或者双特异性纳米抗体与抗原的结合亲和力和动力学参数，所述表面等离子体共振在使用CM5传感器芯片和HBS-EP(10mM HEPES(pH7.4)，150mM NaCl，3mM EDTA，0.05％v/v P20)运行缓冲液的Biacore T200上进行。通过EDC/NHS法将抗原β2微球蛋白通过氨基偶联在芯片表面，最终固载量约为700Ru。各个循环测定由以下步骤组成：首先用120秒注射二价或双特异性纳米抗体，然后监控解离180秒，每个循环后注射60秒glycine-hcl缓冲液(10mM,pH 1.5)以再生。使用Biacore评估软件，通过所得的传感图以标准1：1结合模型全局拟合，确定动力学参数。由表1所示，A0为C末端醛基化修饰的纳米抗体A，B0为C末端醛基化修饰的单体纳米抗体B，A1-A4为采用本发明方法以A0为单元构筑的C-C连接二价纳米抗体，A1采用两端是氧氨基的接头，即O-linker，A2采用两端是酰肼基的PEG400接头即HZ-PEG-HZ 400，A3采用两端是酰肼基的PEG1000接头即HZ-PEG-HZ 1000，A4采用两端是酰肼基的PEG2000接头即HZ-PEG-HZ 2000，其中，连接二价纳米抗体反应温度均为-20℃，反应时间为24h，反应体系pH值为4.0。B1-B5为采用本发明方法以B0为单元构筑的C-C连接二价纳米抗体，具体的含义同A1-A5，反应条件同上。C1-C4为采用本发明方法以纳米抗体A0和B0为单元构筑的C-C连接的双特异性纳米抗体，C1采用两端是氧氨基的接头即O-linker，C2采用两端是酰肼基的PEG400接头即HZ-PEG-HZ 400，C3采用两端是酰肼基的PEG1000接头即HZ-PEG-HZ 1000，C4采用两端是酰肼基的PEG2000接头即HZ-PEG-HZ 2000，其中，连接抗体反应温度均为-20℃，反应时间为24h，反应体系pH值为4.0。A5和B5为基因重组表达C-N连接二价纳米抗体，C5和C6为基因重组表达C-N连接双特异性纳米抗体。所述C-N连接二价或双特异性纳米抗体为本领域常规方法制备得到，即将两段相同或不同的纳米抗体编码基因通过化学合成或PCR技术串联，两段基因之间的连接为编码柔性氨基酸序列的基因，本发明中采用3段重复的甘氨酸-甘氨酸-甘氨酸-甘氨酸-丝氨酸柔性氨基酸序列作为接头，然后将合成后的基因转入表达质粒pET21a中，在大肠杆菌胞内进行表达。A5序列为SEQ ID NO.3，B5序列为SEQ ID NO.4，C5序列为SEQ ID NO.5，C6序列为SEQID NO.6。C5和C6为以纳米抗体A0和B0为单元构筑的基因重组表达C-N连接二价纳米抗体，C5连接顺序为A0-B0，C6的连接顺序为B0-A0，两者都采用3段重复的甘氨酸-甘氨酸-甘氨酸-甘氨酸-丝氨酸柔性氨基酸序列作为接头。表1为，如以上所述.利用表面等离子体共振技术对不同长度C端-C端连接二价纳米抗体，双特异性纳米抗体，以及常规方法生产的C端-N端连接二价纳米抗体，双特异性纳米抗体的结合速率常数Ka，解离速率常数Kd以及所得出的亲和力常数KD。The binding affinities and kinetic parameters of bivalent or bispecific Nanobodies to antigens were determined by surface plasmon resonance (SPR) using a CM5 sensor chip and HBS-EP (10 mM HEPES (pH 7.4) , 150mM NaCl, 3mM EDTA, 0.05% v/v P20) running buffer on Biacore T200. The antigen β2 microglobulin was coupled on the surface of the chip through the EDC/NHS method, and the final immobilized amount was about 700 Ru. Each cycle assay consisted of first 120 s injection of bivalent or bispecific Nanobody, followed by monitoring of dissociation for 180 s, followed by 60 s injection of glycine-hcl buffer (10 mM, pH 1.5) for regeneration after each cycle. Kinetic parameters were determined by global fitting of the resulting sensorgrams with a standard 1:1 binding model using Biacore evaluation software. As shown in Table 1, A0 is Nanobody A modified by C-terminus aldehydeylation, B0 is monomeric Nanobody B modified by C-terminus aldehydeylation, and A1-A4 are CC connections constructed by the method of the present invention with A0 as a unit. For bivalent nanobodies, A1 uses a linker with oxygen amino groups at both ends, that is, O-linker, A2 uses a PEG400 linker with both ends of hydrazide groups, namely HZ-PEG-HZ 400, and A3 uses a PEG1000 linker with both ends of hydrazide groups That is, HZ-PEG-HZ 1000, and A4 uses PEG2000 linkers with hydrazide groups at both ends, that is, HZ-PEG-HZ 2000. The reaction temperature for linking bivalent nanobodies is -20°C, the reaction time is 24 hours, and the pH of the reaction system is The value is 4.0. B1-B5 are CC-linked bivalent nanobodies constructed with B0 as a unit by the method of the present invention, the specific meanings are the same as A1-A5, and the reaction conditions are the same as above. C1-C4 are CC-linked bispecific nanobodies constructed by the method of the present invention with Nanobodies A0 and B0 as units. C1 uses a linker with oxygen amino groups at both ends, that is, an O-linker, and C2 uses a linker with hydrazide groups at both ends. PEG400 connector is HZ-PEG-HZ 400, C3 adopts PEG1000 connector with hydrazide groups at both ends, namely HZ-PEG-HZ 1000, and C4 adopts PEG2000 connector with hydrazide groups at both ends, namely HZ-PEG-HZ 2000. Among them, The reaction temperature of the linked antibody was -20°C, the reaction time was 24 hours, and the pH value of the reaction system was 4.0. A5 and B5 express CN-linked bivalent nanobodies through gene recombination, and C5 and C6 express CN-linked bispecific nanobodies through gene recombination. The CN-linked bivalent or bispecific nanobody is prepared by a conventional method in the art, that is, two identical or different nanobody coding genes are connected in series by chemical synthesis or PCR technology, and the connection between the two genes is encoded by a flexible amino acid. The sequence of the gene, the present invention uses three sections of repeated glycine-glycine-glycine-glycine-serine flexible amino acid sequence as a linker, and then the synthesized gene is transferred into the expression plasmid pET21a, and expressed in Escherichia coli cells. The A5 sequence is SEQ ID NO.3, the B5 sequence is SEQ ID NO.4, the C5 sequence is SEQ ID NO.5, and the C6 sequence is SEQ ID NO.6. C5 and C6 are gene recombination expression CN-linked bivalent nanobodies constructed with Nanobodies A0 and B0 as units, the connection sequence of C5 is A0-B0, and the connection sequence of C6 is B0-A0, both of which use 3 repeats of glycine - Glycine-glycine-glycine-serine flexible amino acid sequence as linker. Table 1 is, as mentioned above. The use of surface plasmon resonance technology for different lengths of C-terminal-C-terminal linked bivalent Nanobodies, bispecific Nanobodies, and C-terminal-N-terminal linked bivalent Nanobodies produced by conventional methods , the on-rate constant Ka , the off-rate constant Kd and the resulting affinity constant KD of the bispecific Nanobody.
表1.Table 1.
表1的结果显示：用表面等离子体共振检测，C-N构建的及C-C构建的二价纳米抗体或双特异性抗体在亲和力上相比其单体均有不同程度提升，其中C-C连接的A1和A2相比C-N连接的A5亲和力高出20倍到30倍，同样的，B3和B4相比B5亲和力也高出20到30倍。C-C连接的双特异性抗体C1-C4亲和力要高出C-N连接的C5和C6约40到50倍。综上所述，C-C连接的二价或双特异性纳米抗体，在抗原结合能力上要优于C-N连接的抗体。The results in Table 1 show that: detected by surface plasmon resonance, the affinity of C-N-constructed and C-C-constructed bivalent nanobodies or bispecific antibodies has different degrees of improvement compared with their monomers, and C-C-linked A1 and A2 The affinity of A5 is 20 to 30 times higher than that of the C-N link, and similarly, the affinity of B3 and B4 is 20 to 30 times higher than that of B5. The affinity of C-C-linked bispecific antibodies C1-C4 is about 40 to 50 times higher than that of C-N-linked C5 and C6. In summary, C-C-linked bivalent or bispecific nanobodies are superior to C-N-linked antibodies in terms of antigen binding ability.
最后应说明的是：显然，上述实施例仅仅是为清楚地说明本申请所作的举例，是优选的实施例。而并非对实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引申出的显而易见的变化或变动仍处于本申请型的保护范围之中。Finally, it should be noted that obviously, the above-mentioned embodiments are only examples for clearly illustrating the present application, and are preferred embodiments. Rather than limiting the implementation. For those of ordinary skill in the art, on the basis of the above description, other changes or changes in different forms can also be made. It is not necessary and impossible to exhaustively list all the implementation manners here. However, the obvious changes or variations derived therefrom are still within the scope of protection of the present application.
序列表 sequence listing
<110> 大连理工大学<110> Dalian University of Technology
<120> 一种抗体复合物的制备方法<120> A kind of preparation method of antibody complex
<130> 2011<130> 2011
<160> 6<160> 6
<170> SIPOSequenceListing 1.0<170> SIPOSequenceListing 1.0
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<211> 537<211> 537
<212> DNA<212>DNA
<213> 人工合成()<213> artificial synthesis ()
<400> 1<400> 1
catatggccc aggtgcagct cgtggagtct gggggagggt tggtgcaggc tggggggtca 60catatggccc aggtgcagct cgtggagtct gggggagggt tggtgcaggc tggggggtca 60
ctgagactct cctgtgcagc ctctggatcc actttggatt cttattacat aggctggttc 120ctgagactct cctgtgcagc ctctggatcc actttggatt cttattacat aggctggttc 120
cgccaggccc caggcaaaga gcgcgagggg gtctcatgta ttagtagtag tggtaatagc 180cgccaggccc caggcaaaga gcgcgagggg gtctcatgta ttagtagtag tggtaatagc 180
atacgttatg tagattccgt gaaggaccga ttcaccatct ctagagacaa cggcaagaac 240atacgttatg tagattccgt gaaggaccga ttcaccatct ctagagacaa cggcaagaac 240
acggcctatc tccacatcaa cagcctgaaa cctgaggaca cggccgttta ttactgtgca 300acggcctatc tccacatcaa cagcctgaaa cctgaggaca cggccgttta ttactgtgca 300
gcgagtcgtc gagggcgcat accgggccta ccttgtagtt tagtacgtga acgctatgcc 360gcgagtcgtc gagggcgcat accgggccta ccttgtagtt tagtacgtga acgctatgcc 360
tattggggcc aggggaccca ggtcaccgtg agctcagaac ccaagacacc aaaaccacaa 420tattggggcc aggggaccca ggtcaccgtg agctcagaac ccaagacacc aaaaccacaa 420
ccacaaccac aaccacaacc ccaaccggat cctacaacag aaggaggcgg tgggagccac 480ccacaaccac aaccaacacc ccaaccggat cctacaacag aaggaggcgg tgggagccac 480
caccaccacc accacggagg cggtgggagc ctgtgcaccc cgtctcgtta actcgag 537caccaccacc accacggagg cggtgggagc ctgtgcaccc cgtctcgtta actcgag 537
<210> 2<210> 2
<211> 525<211> 525
<212> DNA<212>DNA
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catatggccc aagttcaact gcaagaatct ggcggcggtt ctgttcaagc aggcggtagt 60catatggccc aagttcaact gcaagaatct ggcggcggtt ctgttcaagc aggcggtagt 60
ctgcgtctga gttgtgcagc aagcggttat accgattccc gctattgtat ggcctggttt 120ctgcgtctga gttgtgcagc aagcggttat accgattccc gctattgtat ggcctggttt 120
cgtcaagctc cgggtaaaga acgcgagtgg gttgcacgta tcaacagcgg tcgcgatatc 180cgtcaagctc cgggtaaaga acgcgagtgg gttgcacgta tcaacagcgg tcgcgatatc 180
acctactacg cagatagcgt taaaggccgc tttaccttca gccaggataa cgcgaaaaac 240acctactacg cagatagcgt taaaggccgc tttacccttca gccaggataa cgcgaaaaac 240
accgtctacc tgcagatgga tagtctggaa ccggaagata ccgcgaccta ttattgcgca 300accgtctacc tgcagatgga tagtctggaa ccggaagata ccgcgaccta ttattgcgca 300
accgatatcc cgctgcgttg tcgcgatatt gtagcaaaag gcggcgacgg ttttcgttat 360accgatatcc cgctgcgttg tcgcgatatt gtagcaaaag gcggcgacgg ttttcgttat 360
tggggtcaag gtacccaagt taccgtgagc tcagaaccca agacaccaaa accacaacca 420tggggtcaag gtacccaagt taccgtgagc tcagaaccca agacaccaaa accaccaacca 420
caaccacaac cacaacccca acccaatcct acaacagaag aattccacca tcaccaccat 480caaccacaac cacaacccca acccaatcct acaacagaag aattccacca tcaccaccat 480
catggtggcg gtggttcgct gtgcaccccg tcccgttgac tcgag 525catggtggcg gtggttcgct gtgcaccccg tcccgttgac tcgag 525
<210> 3<210> 3
<211> 1017<211> 1017
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<213> 人工合成()<213> artificial synthesis ()
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catatggccc aggtgcagct cgtggagtct gggggagggt tggtgcaggc tggggggtca 60catatggccc aggtgcagct cgtggagtct gggggagggt tggtgcaggc tggggggtca 60
ctgagactct cctgtgcagc ctctggatcc actttggatt cttattacat aggctggttc 120ctgagactct cctgtgcagc ctctggatcc actttggatt cttattacat aggctggttc 120
cgccaggccc caggcaaaga gcgcgagggg gtctcatgta ttagtagtag tggtaatagc 180cgccaggccc caggcaaaga gcgcgagggg gtctcatgta ttagtagtag tggtaatagc 180
atacgttatg tagattccgt gaaggaccga ttcaccatct ctagagacaa cggcaagaac 240atacgttatg tagattccgt gaaggaccga ttcaccatct ctagagacaa cggcaagaac 240
acggcctatc tccacatcaa cagcctgaaa cctgaggaca cggccgttta ttactgtgca 300acggcctatc tccacatcaa cagcctgaaa cctgaggaca cggccgttta ttactgtgca 300
gcgagtcgtc gagggcgcat accgggccta ccttgtagtt tagtacgtga acgctatgcc 360gcgagtcgtc gagggcgcat accgggccta ccttgtagtt tagtacgtga acgctatgcc 360
tattggggcc aggggaccca ggtcaccgtg gaacccaaga caccaaaacc acaaccacaa 420tattggggcc aggggaccca ggtcaccgtg gaacccaaga caccaaaacc acaaccacaa 420
ccacaaccac aaccccaacc ggatcctaca acagaagaat tcggtggtgg aggctccggc 480ccacaaccac aaccccaacc ggatcctaca acagaagaat tcggtggtgg aggctccggc 480
ggagggggta gtggcggcgg tggaagtaag cttgcccagg tgcagctcgt ggagtctggg 540ggagggggta gtggcggcgg tggaagtaag cttgcccagg tgcagctcgt ggagtctggg 540
ggagggttgg tgcaggctgg ggggtcactg agactctcct gtgcagcctc tggatccact 600ggagggttgg tgcaggctgg ggggtcactg agactctcct gtgcagcctc tggatccact 600
ttggattctt attacatagg ctggttccgc caggccccag gcaaagagcg cgagggggtc 660ttggattctt attacatagg ctggttccgc caggccccag gcaaagagcg cgagggggtc 660
tcatgtatta gtagtagtgg taatagcata cgttatgtag attccgtgaa ggaccgattc 720tcatgtatta gtagtagtgg taatagcata cgttatgtag attccgtgaa ggaccgattc 720
accatctcta gagacaacgg caagaacacg gcctatctcc acatcaacag cctgaaacct 780accatctcta gagacaacgg caagaacacg gcctatctcc acatcaacag cctgaaacct 780
gaggacacgg ccgtttatta ctgtgcagcg agtcgtcgag ggcgcatacc gggcctacct 840gaggacacgg ccgtttatta ctgtgcagcg agtcgtcgag ggcgcatacc gggcctacct 840
tgtagtttag tacgtgaacg ctatgcctat tggggccagg ggacccaggt caccgtgagc 900tgtagtttag tacgtgaacg ctatgcctat tggggccagg ggacccaggt caccgtgagc 900
tcagaaccca agacaccaaa accacaacca caaccacaac cacaacccca accggatcct 960tcagaaccca agacaccaaa accacaacca caaccacaac cacaacccaaccggatcct 960
acaacagaag gaggcggtgg gagccaccac caccaccacc accaccacta actcgag 1017acaacagaag gaggcggtgg gagccaccac caccaccacc accacacta actcgag 1017
<210> 4<210> 4
<211> 1005<211> 1005
<212> DNA<212>DNA
<213> 人工合成()<213> artificial synthesis ()
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catatggccc aagttcaact gcaagaatct ggcggcggtt ctgttcaagc aggcggtagt 60catatggccc aagttcaact gcaagaatct ggcggcggtt ctgttcaagc aggcggtagt 60
ctgcgtctga gttgtgcagc aagcggttat accgattccc gctattgtat ggcctggttt 120ctgcgtctga gttgtgcagc aagcggttat accgattccc gctattgtat ggcctggttt 120
cgtcaagctc cgggtaaaga acgcgagtgg gttgcacgta tcaacagcgg tcgcgatatc 180cgtcaagctc cgggtaaaga acgcgagtgg gttgcacgta tcaacagcgg tcgcgatatc 180
acctactacg cagatagcgt taaaggccgc tttaccttca gccaggataa cgcgaaaaac 240acctactacg cagatagcgt taaaggccgc tttacccttca gccaggataa cgcgaaaaac 240
accgtctacc tgcagatgga tagtctggaa ccggaagata ccgcgaccta ttattgcgca 300accgtctacc tgcagatgga tagtctggaa ccggaagata ccgcgaccta ttattgcgca 300
accgatatcc cgctgcgttg tcgcgatatt gtagcaaaag gcggcgacgg ttttcgttat 360accgatatcc cgctgcgttg tcgcgatatt gtagcaaaag gcggcgacgg ttttcgttat 360
tggggtcaag gtacccaagt taccgtggaa cccaagacac caaaaccaca accacaacca 420tggggtcaag gtacccaagt taccgtggaa cccaagacac caaaaccaca accaccaacca 420
caaccacaac cccaacccaa tcctacaaca gaagaattcg gaggcggtgg gagcggaggc 480caaccacaac cccaacccaa tcctacaaca gaagaattcg gaggcggtgg gagcggaggc 480
ggtgggagcg gaggcggtgg atccgcccaa gttcaactgc aagaatctgg cggcggttct 540ggtgggagcg gaggcggtgg atccgcccaa gttcaactgc aagaatctgg cggcggttct 540
gttcaagcag gcggtagtct gcgtctgagt tgtgcagcaa gcggttatac cgattcccgc 600gttcaagcag gcggtagtct gcgtctgagt tgtgcagcaa gcggttatac cgattcccgc 600
tattgtatgg cctggtttcg tcaagctccg ggtaaagaac gcgagtgggt tgcacgtatc 660tattgtatgg cctggtttcg tcaagctccg ggtaaagaac gcgagtgggt tgcacgtatc 660
aacagcggtc gcgatatcac ctactacgca gatagcgtta aaggccgctt taccttcagc 720aacagcggtc gcgatatcac ctactacgca gatagcgtta aaggccgctt taccttcagc 720
caggataacg cgaaaaacac cgtctacctg cagatggata gtctggaacc ggaagatacc 780caggataacg cgaaaaacac cgtctacctg cagatggata gtctggaacc ggaagatacc 780
gcgacctatt attgcgcaac cgatatcccg ctgcgttgtc gcgatattgt agcaaaaggc 840gcgacctatt attgcgcaac cgatatcccg ctgcgttgtc gcgatattgt agcaaaaggc 840
ggcgacggtt ttcgttattg gggtcaaggt acccaagtta ccgtgagctc agaacccaag 900ggcgacggtt ttcgttattg gggtcaaggt acccaagtta ccgtgagctc agaacccaag 900
acaccaaaac cacaaccaca accacaacca caaccccaac ccaatcctac aacagaagga 960acaccaaaac cacaaccaca accacaacca caacccaac ccaatcctac aacagaagga 960
ggcggtggga gccaccacca ccaccaccac caccactaac tcgag 1005ggcggtggga gccaccacca ccaccaccac caccactaac tcgag 1005
<210> 5<210> 5
<211> 1008<211> 1008
<212> DNA<212>DNA
<213> 人工合成()<213> artificial synthesis ()
<400> 5<400> 5
catatggccc aggtgcagct cgtggagtct gggggagggt tggtgcaggc tggggggtca 60catatggccc aggtgcagct cgtggagtct gggggagggt tggtgcaggc tggggggtca 60
ctgagactct cctgtgcagc ctctggatcc actttggatt cttattacat aggctggttc 120ctgagactct cctgtgcagc ctctggatcc actttggatt cttattacat aggctggttc 120
cgccaggccc caggcaaaga gcgcgagggg gtctcatgta ttagtagtag tggtaatagc 180cgccaggccc caggcaaaga gcgcgagggg gtctcatgta ttagtagtag tggtaatagc 180
atacgttatg tagattccgt gaaggaccga ttcaccatct ctagagacaa cggcaagaac 240atacgttatg tagattccgt gaaggaccga ttcaccatct ctagagacaa cggcaagaac 240
acggcctatc tccacatcaa cagcctgaaa cctgaggaca cggccgttta ttactgtgca 300acggcctatc tccacatcaa cagcctgaaa cctgaggaca cggccgttta ttactgtgca 300
gcgagtcgtc gagggcgcat accgggccta ccttgtagtt tagtacgtga acgctatgcc 360gcgagtcgtc gagggcgcat accgggccta ccttgtagtt tagtacgtga acgctatgcc 360
tattggggcc aggggaccca ggtcaccgtg gaacccaaga caccaaaacc acaaccacaa 420tattggggcc aggggaccca ggtcaccgtg gaacccaaga caccaaaacc acaaccacaa 420
ccacaaccac aaccccaacc ggatcctaca acagaagaat tcggaggcgg tgggagcgga 480ccacaaccac aaccccaacc ggatcctaca acagaagaat tcggaggcgg tgggagcgga 480
ggcggtggga gcggaggcgg tggatccgcc caagttcaac tgcaagaatc tggcggcggt 540ggcggtggga gcggaggcgg tggatccgcc caagttcaac tgcaagaatc tggcggcggt 540
tctgttcaag caggcggtag tctgcgtctg agttgtgcag caagcggtta taccgattcc 600tctgttcaag caggcggtag tctgcgtctg agttgtgcag caagcggtta taccgattcc 600
cgctattgta tggcctggtt tcgtcaagct ccgggtaaag aacgcgagtg ggttgcacgt 660cgctattgta tggcctggtt tcgtcaagct ccgggtaaag aacgcgagtg ggttgcacgt 660
atcaacagcg gtcgcgatat cacctactac gcagatagcg ttaaaggccg ctttaccttc 720atcaacagcg gtcgcgatat cacctactac gcagatagcg ttaaaggccg ctttaccttc 720
agccaggata acgcgaaaaa caccgtctac ctgcagatgg atagtctgga accggaagat 780agccaggata acgcgaaaaa caccgtctac ctgcagatgg atagtctgga accggaagat 780
accgcgacct attattgcgc aaccgatatc ccgctgcgtt gtcgcgatat tgtagcaaaa 840accgcgacct attattgcgc aaccgatatc ccgctgcgtt gtcgcgatat tgtagcaaaa 840
ggcggcgacg gttttcgtta ttggggtcaa ggtacccaag ttaccgtgag ctcagaaccc 900ggcggcgacg gttttcgtta ttggggtcaa ggtacccaag ttaccgtgag ctcagaaccc 900
aagacaccaa aaccacaacc acaaccacaa ccacaacccc aacccaatcc tacaacagaa 960aagacaccaa aaccacaacc acaaccacaa ccacaaccc aacccaatcc tacaacagaa 960
ggaggcggtg ggagccacca ccaccaccac caccaccact aactcgag 1008ggaggcggtg ggagccacca ccaccaccac caccaccact aactcgag 1008
<210> 6<210> 6
<211> 1008<211> 1008
<212> DNA<212>DNA
<213> 人工合成()<213> artificial synthesis ()
<400> 6<400> 6
catatggccc aagttcaact gcaagaatct ggcggcggtt ctgttcaagc aggcggtagt 60catatggccc aagttcaact gcaagaatct ggcggcggtt ctgttcaagc aggcggtagt 60
ctgcgtctga gttgtgcagc aagcggttat accgattccc gctattgtat ggcctggttt 120ctgcgtctga gttgtgcagc aagcggttat accgattccc gctattgtat ggcctggttt 120
cgtcaagctc cgggtaaaga acgcgagtgg gttgcacgta tcaacagcgg tcgcgatatc 180cgtcaagctc cgggtaaaga acgcgagtgg gttgcacgta tcaacagcgg tcgcgatatc 180
acctactacg cagatagcgt taaaggccgc tttaccttca gccaggataa cgcgaaaaac 240acctactacg cagatagcgt taaaggccgc tttacccttca gccaggataa cgcgaaaaac 240
accgtctacc tgcagatgga tagtctggaa ccggaagata ccgcgaccta ttattgcgca 300accgtctacc tgcagatgga tagtctggaa ccggaagata ccgcgaccta ttattgcgca 300
accgatatcc cgctgcgttg tcgcgatatt gtagcaaaag gcggcgacgg ttttcgttat 360accgatatcc cgctgcgttg tcgcgatatt gtagcaaaag gcggcgacgg ttttcgttat 360
tggggtcaag gtacccaagt taccgtggaa cccaagacac caaaaccaca accacaacca 420tggggtcaag gtacccaagt taccgtggaa cccaagacac caaaaccaca accaccaacca 420
caaccacaac cccaacccaa tcctacaaca gaagaattcg gaggcggtgg gagcggaggc 480caaccacaac cccaacccaa tcctacaaca gaagaattcg gaggcggtgg gagcggaggc 480
ggtgggagcg gaggcggtgg atccgcccag gtgcagctcg tggagtctgg gggagggttg 540ggtgggagcg gaggcggtgg atccgcccag gtgcagctcg tggagtctgg gggagggttg 540
gtgcaggctg gggggtcact gagactctcc tgtgcagcct ctggatccac tttggattct 600gtgcaggctg gggggtcact gagactctcc tgtgcagcct ctggatccac tttggattct 600
tattacatag gctggttccg ccaggcccca ggcaaagagc gcgagggggt ctcatgtatt 660tattacatag gctggttccg ccaggcccca ggcaaagagc gcgaggggggt ctcatgtatt 660
agtagtagtg gtaatagcat acgttatgta gattccgtga aggaccgatt caccatctct 720agtagtagtg gtaatagcat acgttatgta gattccgtga aggaccgatt caccatctct 720
agagacaacg gcaagaacac ggcctatctc cacatcaaca gcctgaaacc tgaggacacg 780agagacaacg gcaagaacac ggcctatctc cacatcaaca gcctgaaacc tgaggacacg 780
gccgtttatt actgtgcagc gagtcgtcga gggcgcatac cgggcctacc ttgtagttta 840gccgtttatt actgtgcagc gagtcgtcga gggcgcatac cgggcctacc ttgtagtta 840
gtacgtgaac gctatgccta ttggggccag gggacccagg tcaccgtgag ctcagaaccc 900gtacgtgaac gctatgccta ttggggccag gggacccagg tcaccgtgag ctcagaaccc 900
aagacaccaa aaccacaacc acaaccacaa ccacaacccc aaccggatcc tacaacagaa 960aagacaccaa aaccacaacc acaaccacaa ccacaaccccc aaccggatcc tacaacagaa 960
ggaggcggtg ggagccacca ccaccaccac caccaccact aactcgag 1008ggaggcggtg ggagccacca ccaccaccac caccaccact aactcgag 1008
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810461883.4ACN108610415A (en) | 2018-05-15 | 2018-05-15 | A kind of preparation method of antibody complex |
| PCT/CN2018/103120WO2019218533A1 (en) | 2018-05-15 | 2018-08-30 | Method for preparing antibody complex |
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810461883.4ACN108610415A (en) | 2018-05-15 | 2018-05-15 | A kind of preparation method of antibody complex |
| Publication Number | Publication Date |
|---|---|
| CN108610415Atrue CN108610415A (en) | 2018-10-02 |
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810461883.4APendingCN108610415A (en) | 2018-05-15 | 2018-05-15 | A kind of preparation method of antibody complex |
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