WO2019218533A1 - 一种抗体复合物的制备方法 - Google Patents

一种抗体复合物的制备方法 Download PDF

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WO2019218533A1
WO2019218533A1 PCT/CN2018/103120 CN2018103120W WO2019218533A1 WO 2019218533 A1 WO2019218533 A1 WO 2019218533A1 CN 2018103120 W CN2018103120 W CN 2018103120W WO 2019218533 A1 WO2019218533 A1 WO 2019218533A1
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antibody
aldehyde
group
linker
preparation
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PCT/CN2018/103120
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French (fr)
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贾凌云
臧柏林
任军
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大连理工大学
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins

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  • the invention belongs to the field of biomedicine and relates to a bivalent or bispecific C-terminal (carboxy terminal-carboxyl) linkage, and a preparation method of a multivalent or multispecific antibody complex, in particular a nanobody composite The preparation method of the substance.
  • multivalent or multispecific antibodies have higher affinity, so binding target molecules in vivo or in vitro is faster and more stable.
  • multispecific antibodies are able to recognize different antigens, which confers new functions not found in conventional antibodies. For example, it is applied to a bispecific T cell engager (BiTE) to kill tumor cells, and a bispecific antibody (Blinatumomab) constructed by using a single chain fragment variable (scFv) as a unit can be effective.
  • BiTE bispecific T cell engager
  • scFv single chain fragment variable
  • Nanobodies also known as single-domain antibodies, are an antibody molecule found in Camelidae. Its size is only one-tenth of that of conventional monoclonal antibodies, but it has comparable antigen binding ability; it is superior in stability, prokaryotic expression, and binding to epitopes. Because of 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).
  • the present invention provides a method for synthesizing a covalent C-terminal-C-linked bivalent antibody complex using a single domain antibody or a Nanobody as a building block.
  • a method for preparing a C-terminal-C-terminally linked bivalent antibody or bispecific antibody complex comprises the following steps:
  • the freezing condition is -5 ° C to 30 ° C, preferably -10 ° C to 25 ° C, more preferably -20 ° C.
  • the reaction under freezing conditions means that the reaction is carried out 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 10-30 h, more preferably 24 h.
  • the pH of the reaction system reacted under freezing conditions is 4.0 to 7.5, and the pH is preferably 4 5, more preferably 4.
  • the antibody is a Nanobody, a single-chain antibody scFV or a variable region antibody Fab.
  • the FGE recognition amino acid sequence is cysteine-X-valine-X-arginine, and the X is any natural amino acid, preferably The FGE recognizes the amino acid sequence as LCTPSR.
  • the aldehyde-based reactive homobifunctional linker is RLR, wherein R is an amino group, a hydrazide group, an oxyamino group, a benzoquinone group.
  • L is a polymer having -(CH 2 CH 2 -O)n and/or -(O-CH 2 CH 2 )n as a constituent unit, wherein n is 1 An integer of up to 100, n is preferably from 1 to 50, more preferably from 10 to 30.
  • the gene coding sequence of the FGE is derived from Mycobacterium tuberculosis or a human.
  • the reaction product after the reaction under freezing conditions is separated by size exclusion chromatography to obtain a single homobifunctional linker-conjugated antibody.
  • the molar ratio of the antibody obtained in the step (3) to the C-terminal fixed-point aldehyde-modified antibody is 1:1 to 3.
  • the stability of the hydrazone bond, the reducing agent and the amount thereof are not particularly limited, and those skilled in the art can select an appropriate reducing agent and an amount to be added according to a conventional technique.
  • the reducing agent can use sodium borohydride or cyanide. The sodium borohydride or the like is added in an amount of 10 times or more the molar amount of the substance to be reduced.
  • the FGE is a formylglycine generating enzyme
  • the C-terminal-C-terminally linked bivalent antibody complex means that two identical antibodies are each obtained by linking a carboxy terminal to a homobifunctional linker.
  • the C-terminal-C-terminally linked bispecific antibody complex refers to a bispecific antibody complex obtained by linking two antibodies having different variable regions to each other through a carboxy terminal and a homobifunctional linker. Things.
  • the antibody linked to the single homobifunctional linker refers to a complex formed by attaching an antibody to only one end of the same bifunctional linker.
  • the C-terminal fixed-point aldehyde-modified antibody can be prepared as follows:
  • the gene sequence of the recognition peptide of formylglycine generating enzyme (FGE) is added to the 3' end of the gene sequence of the antibody, and the recombinant gene is ligated to the vector and transferred to an Escherichia coli expressing host strain or Pichia or mammalian cells. Recombinant expression is carried out in the host cell, and the expressed antibody is extracted and purified to obtain a recombinant antibody having a FGE recognition amino acid sequence at the 3' end (C-terminus), and the extraction and purification can be carried out by a method conventional in the art, such as purification.
  • the PGE recognition peptide is preferably LCTPSR, and the plasmid is preferably pET21a or pET23a;
  • the recombinant antibody and a certain amount of FGE are mixed, and the C-terminal tag of the FGE-catalyzed recombinant antibody is converted into a side chain carrying an aldehyde group under appropriate buffer conditions and temperature, thereby obtaining the present invention.
  • the C-terminal fixed-aldehyde-modified antibody wherein the recombinant antibody and FGE are preferably added in a molar ratio of 1:5 to 10, and the buffer is preferably triethanolamine-hydrochloric acid buffer, pH 7.0-9.0, in a buffer. 1-5 mM mercaptoethanol is added, the ionic strength is 50-150 mM sodium chloride, and the catalytic temperature is preferably 18 ° C to 30 ° C.
  • the C-terminal-C-terminally linked bivalent antibody complex can be prepared by reacting the C-terminally fixed aldehyde-modified antibody obtained above with an aldehyde group.
  • the linker is mixed at a molar ratio of 1:0.5-1.0, and reacted at -5 ° C to -30 ° C, pH 4-5 for 2-24 h to complete the aldehyde-reactive homobifunctional linker and the C-terminal aldehyde group of the antibody.
  • the C-terminal-C-terminally linked bispecific antibody complex can be prepared as follows:
  • the C-terminal fixed-aldehyde-modified antibody obtained above is reacted with an aldehyde-based homobifunctional linker at a molar ratio of 1:5-15, at a temperature of -5 ° C to -30 ° C, and a pH of 4-5.
  • the lower reaction can be carried out for 2-24 hours to complete the attachment of the aldehyde-reactive homobifunctional linker to the C-terminal aldehyde group of the antibody to obtain a single homobifunctional linker; wherein, preferably, the reaction product can be separated by size exclusion chromatography.
  • a purified single homobifunctional linker antibody was obtained for the next reaction.
  • the antibody linked to the single homobifunctional linker obtained in the above step (1) is mixed with the C-terminal fixed-point aldehyde-modified antibody at a molar ratio of 1:1 to 3, at -5 ° C to -30 ° C, pH 4- Reaction under conditions of 5 to 2 h, wherein the antibody linked to the single homobifunctional linker is different from the variable region of the C-terminally fixed aldehyde-modified antibody, thereby obtaining a C-terminal-C-linked bispecific antibody complex
  • the final yield is about 30%.
  • the invention realizes a C-terminal-C-terminally linked bivalent or bispecific antibody complex under freezing conditions, the conditions are mild, the reaction speed is fast, and the yield is high.
  • the present invention is capable of obtaining higher affinity (Fig. 1) than the C-terminal-N-terminally linked bivalent or bispecific antibody complex produced by conventional methods, which is based on the equilibrium measured by surface plasmon resonance. Dissociation constant.
  • the present invention has better specificity and fewer steps, and is only generated at the C-terminus.
  • the aldehyde groups are linked, and the resulting hydrazone or hydrazone bond is stable under physiological conditions and irreversible after reduction with a reducing agent, and the blood half-life can be prolonged due to the addition of a polyethylene glycol linker.
  • FIG. 1 is a schematic diagram showing the structure of a Nanobody, the structure of a C-N-linked bivalent or bispecific Nanobody prepared by a conventional method, and the structure of a C-C-linked bivalent or bispecific Nanobody prepared by the present invention.
  • FIG. 2A shows the results of labeling aldehyde-modified Nanobodies using fluorescent molecules and separating them on SDS-PAGE.
  • Figure 2B shows the modification of aldehydes by high performance liquid chromatography coupled with point-spray ionization orbital ion trap mass spectrometry. The Nanobody was accurately quantified and the map shown is the molecular weight-abundance map after deconvolution.
  • Figure 3 shows the results of the separation of the product on SDS-PAGE 24 hours after the reaction of the dihydrazide-functionalized PEG400 connector at different pH values and temperatures.
  • FIG. 4A is a graph showing the change of bivalent antibody yield with time at a freezing temperature of -20 ° C by high performance liquid chromatography
  • FIG. 4B is a graph showing the change of the yield of divalent Nanobodies over time at different temperatures.
  • FIG. 5A shows the results of separation of bivalent Nanobodies with different lengths of linkers on SDS-PAGE
  • FIG. 5B shows the separation and purification of bivalent Nanobodies containing different lengths of linker.
  • Figure 6 shows the separation of Nanobody B monomers linked to homobifunctional linkers by size exclusion chromatography.
  • Nanobody A the coding gene sequence is SEQ ID NO. 1, wherein the sequence 511-528 of ctgtgcaccccgtctcgt is a FGE recognition sequence;
  • Nanobody B The coding gene sequence is SEQ ID NO. 2, wherein the sequence 499-516 ctgtgcaccccgtctcgt is the FGE recognition sequence.
  • Nanobody A The coding sequence of Nanobody A was cloned into the expression vector plasmid pET23a, introduced into E. coli T7Shuffle (DE3) for expression, the cells were collected, and the cells were disrupted, and the metal chelate affinity chromatography HisTrap was used from the cell disrupted supernatant.
  • HP 5mL (GE Healthcare) purified Nanobody the final yield is around 100 mg per liter of fermentation broth.
  • the purified Nanobody was concentrated by ultrafiltration to triethanolamine buffer (25 mM TEAM, pH 9.0, 150 mM NaCl, 1 mM mercaptoethanol), the nanobody concentration was 5 mg/mL, and the final concentration was added to the nano-antibody molar amount of about ten.
  • One of the fractions, 1 mg/mL, has a purity of >90% FGE (the FGE enzyme has a molecular weight of about 33 kDa and the nanobody has a molecular weight of about 18 kDa), and the reaction is gently shaken at 18 ° C for 20 hours. After the reaction, the protein precipitate is removed by centrifugation. A Nanobody A modified at the carboxy terminus of the aldehyde group was obtained and designated as Nanobody A0.
  • FIG. 2B shows the molecular weight-abundance map after deconvolution of the protein mass spectrum.
  • the pre- and post-modification proteins were visible in the map ( Nano-antibody) peak, after aldehyde modification, the protein component is reduced by about 18 Da, and the aldehyde 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 to an acidic buffer (such as 0.1 M acetate buffer pH 4.0 containing 150 mM NaCl), followed by addition of a fluorescent molecule Lucifer with a hydrazide group at a final concentration of 500 ⁇ mol/L.
  • an acidic buffer such as 0.1 M acetate buffer pH 4.0 containing 150 mM NaCl
  • Lucifer with a hydrazide group at a final concentration of 500 ⁇ mol/L.
  • Yellow CH lithium slat (Thermo Fisher) marks the aldehyde group
  • SDS-PAGE analysis can also preliminarily quantify the C-terminal aldehyde group modification efficiency, as shown in Figure 2A, due to the labeling of Lucifer Yellow fluorescent molecules, the protein is in UV imaging.
  • Nanobody A0 The carboxy-terminated aldehyde-modified Nanobody A (Nanobody A0) prepared in Example 1 was subjected to ultrafiltration and concentrated to an acidic buffer, 0.1 M acetate buffer pH 4.0 or 0.1 M MES buffer pH 5.5, Or neutral buffer 0.2 M PBS pH 7.4, the concentration of Nanobody A0 is 2 mg / mL, ie 100 ⁇ mol / L.
  • the bifunctional linker HZ-PEG-HZ 400 dihydrazide polyethylene glycol-400
  • the reducing agent sodium cyanoborohydride among which HZ-PEG-HZ 400 and cyano
  • the final concentration of sodium borohydride in the reaction system is 50 ⁇ mol/L and 1 mmol/L, respectively, that is, the molar ratio of PEG to Nanobody in the reaction system is 1:2, and the molar ratio of sodium cyanoborohydride to the nanobody is 10:1.
  • the good reaction mixture will be added, placed in a low temperature bath and cooled to -30 ° C, the reaction mixture will be frozen, and then adjusted to different temperatures, respectively -30 ° C, -20 ° C, -10 ° C and -5 ° C, the reaction 0 ⁇ 25h.
  • the reaction mixture is brought into a frozen state and then adjusted to each reaction temperature, so that the time for freezing the sample can be shortened, thereby shortening the reaction time, and the reaction mixture can be directly placed in the corresponding temperature in the present invention.
  • FIG. 4A is a graph showing the change of bivalent antibody yield with time at a freezing temperature of -20 ° C by high performance liquid chromatography
  • FIG. 4B is a graph showing the change of the yield of divalent Nanobodies over time at different temperatures. It can be seen that the yield can reach the maximum in the reaction for about 24 hours, and the reaction is slow at -30 ° C and -5 ° C. The reaction occurs faster in the interval of -10 ° C to -20 ° C. In the interval of -10 ° C to -20 ° C, the yield of divalent nanometers is between 30% and 50%.
  • Fig. 5A is an electrophoresis diagram of the above reaction products respectively fed with different lengths of linkers on SDS-PAGE, wherein lane M is a protein Marker, and lanes 1-4 are sequentially added to a nanobody A0 (final concentration 100 ⁇ mol/L).
  • the reaction product of the linker (final concentration 50 ⁇ mol/L), O-linker, HZ-PEG-HZ 400, HZ-PEG-HZ1000 and HZ-PEG-HZ2000, 5-8 followed by Nanobody B0 (final concentration 100 ⁇ mol/L)
  • the reaction product after the addition of a bifunctional linker (final concentration 50 ⁇ mol/L), O-linker, HZ-PEG-HZ400, HZ-PEG-HZ1000 and HZ-PEG-HZ2000 was added.
  • lanes 1-8 exhibit a certain amount of bivalent nanobody strips around 40 kDa, indicating that for the Nanobodies A0 and B0, bifunctional linkers of different lengths can be added, and the reaction can be carried out under freezing conditions. A certain amount of bivalent Nanobody is produced.
  • Fig. 5B is a chromatogram of the results of separation and purification of the reaction products of the above-mentioned different length connectors by size exclusion chromatography. As can be seen in Fig. 5B, the monomer remains in the solution after the reaction, and the monomer of the linker and the bivalent antibody are linked, and the bivalent antibody can be well separated for further study.
  • the coding sequence of the Nanobody B gene was cloned into the expression vector plasmid pET23a, introduced into E. coli T7Shuffle (DE3) for expression, the cells were collected, and the cells were disrupted, and the metal chelate affinity was utilized from the cell disrupted supernatant.
  • the Nanobody was purified by chromatography 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 to triethanolamine buffer (25 mM TEAM, pH 9.0, 150 mM NaCl, 1 mM mercaptoethanol), the concentration of the Nanobody was 5 mg/mL, and the final concentration was added to the nanomolecule.
  • Nanobody B0 The carboxy-terminal fixed-point aldehyde-modified Nanobody B was named Nanobody B0.
  • the nanobody B0 obtained in the previous step was subjected to ultrafiltration and concentrated to an acidic buffer (0.1 M acetate buffer pH 4.0, containing 150 mM NaCl) to a final concentration of 2 mg/mL, that is, 100 ⁇ mol/L.
  • an acidic buffer 0.1 M acetate buffer pH 4.0, containing 150 mM NaCl
  • a final concentration of 2 mg/mL that is, 100 ⁇ mol/L.
  • the size exclusion chromatography separation step is as follows: the reaction product is allowed to stand at room temperature, and after being melted, 1 M NaOH solution is added to adjust the pH to neutrality, and then passed through a size exclusion chromatography column Superdex 75 Increase 10/300 GL (GE Healthcare). Separation was carried out with 20 mM phosphate 150 mM NaCl pH 7.4 buffer as a running solution at a rate of 0.6 mL/min, and each fraction was collected, and the bands were verified by SDS-PAGE. Finally, the Nanobody B monomer to which the homobifunctional linker is attached is retained, as shown in FIG. Fig.
  • FIG. 6A is a chromatogram of the separation and purification of the products after the excessive addition of different linkers on the size exclusion chromatography
  • Fig. 6B is the SDS-PAGE identification of the different chromatographic peaks (the lanes 0, 3, 6, and 10 are respectively added with double function).
  • the reaction products after the linker O-linker, HZ-PEG-HZ400, HZ-PEG-HZ1000 and HZ-PEG-HZ2000, the remaining lanes are the corresponding chromatographic peak components in Figure 6A). It can be seen that a certain amount of nano-antibody linked to the linker can be effectively isolated and purified for use in the next study.
  • Nanobody B linked to the homobifunctional link obtained in the previous step is mixed with the aldehyde-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 addition is added.
  • Sodium cyanoborohydride at a concentration of 1 mmol/L was then reacted at -20 ° C for at least 24 hours, and the resulting product was isolated by size exclusion chromatography column Superdex 75 Increase 10/300 GL to obtain a CC-linked bispecific Nanobody AB.
  • the yield is 20% to 40%.
  • Binding affinities and kinetic parameters of divalent 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) , 150 mM NaCl, 3 mM EDTA, 0.05% v/v P20) run buffer on Biacore T200.
  • SPR surface plasmon resonance
  • HBS-EP 10 mM HEPES (pH 7.4) , 150 mM NaCl, 3 mM EDTA, 0.05% v/v P20
  • the antigen ⁇ 2 microglobulin was coupled to the surface of the chip by an EDC/NHS method via an amino group, and the final immobilization amount was about 700 Ru.
  • Each cycle assay consisted of first injection of a bivalent or bispecific Nanobody with 120 seconds followed by monitoring for 180 seconds of dissociation, followed by 60 hours of glycine-hcl buffer (10 mM, pH 1.5) after each cycle for regeneration.
  • Kinetic parameters were determined by global fitting of the resulting sensorgrams using a standard 1:1 binding model using Biacore evaluation software.
  • A0 is a C-terminal aldehyde-modified Nanobody A
  • B0 is a C-terminal aldehyde-modified monomeric Nanobody B
  • A1-A4 is a CC-ligation constructed by A0 as a unit using the method of the present invention.
  • A1 uses a linker with an oxyamino group at both ends, ie O-linker
  • A2 uses a PEG400 linker with a hydrazide group at both ends, ie HZ-PEG-HZ 400
  • A3 uses a PEG1000 linker with a hydrazide group at both ends. That is, HZ-PEG-HZ 1000, A4 adopts PEG2000 linker which is hydrazide group at both ends, ie HZ-PEG-HZ2000, wherein the reaction temperature of the linked bivalent nano-antibody is -20 ° C, the reaction time is 24 h, the pH value of the reaction system Is 4.0.
  • B1-B5 is a CC-linked bivalent Nanobody constructed by B0 as a unit by the method of the present invention, and the specific meaning is the same as A1-A5, and the reaction conditions are the same as above.
  • C1-C4 is a CC-linked bispecific Nanobody constructed by the nano-antibody A0 and B0 using the method of the present invention, and C1 adopts an O-linker which is an oxyamino group at both ends, and C2 adopts a hydrazide group at both ends.
  • PEG400 linker is HZ-PEG-HZ 400
  • C3 adopts PEG1000 linker which is hydrazide group at both ends, ie HZ-PEG-HZ 1000
  • C4 adopts PEG2000 linker which is hydrazide group at both ends, ie HZ-PEG-HZ 2000
  • the reaction temperature of the conjugated antibody was -20 ° C
  • the reaction time was 24 h
  • the pH of the reaction system was 4.0.
  • A5 and B5 are recombinantly expressed CN-linked bivalent Nanobodies
  • C5 and C6 are recombinantly expressed CN-linked bispecific Nanobodies.
  • 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 encodes a flexible amino acid.
  • a 3-step repeat glycine-glycine-glycine-glycine-glycine-serine flexible amino acid sequence is used as a linker, and then the synthesized gene is transferred into the expression plasmid pET21a and expressed in E. 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
  • the C6 sequence is SEQ ID NO.
  • C5 and C6 are recombinantly expressed CN-linked bivalent Nanobodies constructed by Nanobodies A0 and B0.
  • the C5 linkage sequence is A0-B0
  • the C6 linkage sequence is B0-A0. Both use 3 repeats of glycine.
  • a glycine-glycine-glycine-serine flexible amino acid sequence is used as a linker.
  • Table 1 is a C-terminal-N-terminally linked bivalent Nanobody produced by a surface plasmon resonance technique using a surface plasmon resonance technique to link C-terminal C-terminal bivalent Nanobodies, bispecific Nanobodies, and conventional methods. , bispecific Nanobodies binding rate constant K a, K d dissociation rate constant and affinity constant K D derived.

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Abstract

一种抗体复合物的制备方法，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的制备，条件温和，反应速度快，收率较高。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，能够得到更高的亲和力，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，具有更好的特异性和稳定性，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。

Description

一种抗体复合物的制备方法 技术领域
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。
背景技术
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."Nature Biotechnology 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。
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 National Academy of Sciences of the United States of America109(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。
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,single domain 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)。
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。
发明内容
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。
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：
(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；
(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；
(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；
(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。
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℃。
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。
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。
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。
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-(CH 2CH 2-O)n和/或-(O-CH 2CH 2)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。
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。
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。
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。
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。
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。
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：
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；
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℃。
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。
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：
(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。
(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。
本发明相对于现有技术具有如下优点和效果：
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。
附图说明
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。
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。
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。
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。
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。
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
具体实施方式
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。
下述实施例中所用的材料：
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；
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。
实施例1纳米抗体的C末端定点修饰
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。
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。
实施例2在冰冻条件下实现制备C-C连接二价纳米抗体
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。
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。
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。
实施例3冰冻法制备C-C连接的双特异性纳米抗体
(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。
(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pH 4.0，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。
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1M NaOH溶液调节pH值为中性，随后通过体积排阻色谱柱Superdex 75Increase 10/300GL(GE Healthcare)，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。
(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％。
实施例4二价及双特异性纳米抗体的亲和常数测定
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-HZ2000，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SEQ ID 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K a，解离速率常数K d以及所得出的亲和力常数K D。
表1.
Figure PCTCN2018103120-appb-000001
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。
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。
Figure PCTCN2018103120-appb-000002
Figure PCTCN2018103120-appb-000003
Figure PCTCN2018103120-appb-000004
Figure PCTCN2018103120-appb-000005

Claims (10)

  1. 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：
    (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；
    (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；
    (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；
    (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。
  2. 根据权利要求1所述的制备方法，其特征在于，在步骤(2)、步骤(3)以及步骤(4)中，所述的冰冻条件为-5℃～-30℃。
  3. 根据权利要求1所述的制备方法，其特征在于，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℃下放置2～48h。
  4. 根据权利要求1～3的任一项所述的制备方法，其特征在于，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pH值为4.0～7.5。
  5. 根据权利要求1所述的制备方法，其特征在于，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。
  6. 根据权利要求1所述的制备方法，其特征在于，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。
  7. 根据权利要求1所述的制备方法，其特征在于，在步骤(2)和步骤(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-(CH 2CH 2-O)n和/或-(O-CH 2CH 2)n作为构成单元的聚合物，其中，n为1至100的整数。
  8. 根据权利要求1所述的制备方法，其特征在于，所述FGE的基因编码序列来源于结核分枝杆菌或人类。
  9. 根据权利要求1所述的制备方法，其特征在于，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。
  10. 根据权利要求1所述的制备方法，其特征在于，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。
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