Single-chain antibodies and their in vitro synthesis systems and their use

Abstract

This invention provides a single-chain antibody, its in vitro synthesis system, and its use. The antibody simultaneously possesses high expression efficiency, high affinity, and high stability, is expressed efficiently in a single step in a cell-free in vitro synthesis system, does not require the separate secretion and expression of two different protein fragments, the light chain and the heavy chain, is less likely to form homodimers, and has high expression efficiency, making large-scale production possible and offering excellent commercialization potential.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to single-chain antibodies and their in vitro synthesis systems and uses.

Background Art

[0002] As humans deepen their understanding of the mechanisms of tumorigenesis and progression, tumor treatment has entered the era of personalized treatment. In the era of personalized treatment, the most important treatment concept in personalized treatment is targeted therapy. Tumor molecular target therapy is a treatment method that uses specific genes that are expressed in tumor cells and hardly or not expressed in normal cells, or their expression products as treatment targets to kill tumor cells to the greatest extent. Antibodies have high specificity for corresponding antigens and it is possible to produce specific antibodies against specific target molecules (antigens) related to the onset and progression of diseases. Antibodies are used in the treatment of various diseases, and particularly show great potential and application prospects in the treatment of cancer, autoimmune diseases, and viral infections.

[0003] Based on the size of the antibody molecule, recombinant antibodies can be classified into small molecule antibodies, multivalent antibodies, complete antibodies, and antibody gene libraries. Small molecule antibodies are prepared using prokaryotic or eukaryotic expression systems and consist mainly of the antibody light chain variable region and heavy chain variable region, with or without foreign peptide chains, resulting in small molecular weight antibody fragments. Typically, their size is 1 / 3 or 1 / 2, or even less, of a complete IgG molecule, hence the name small molecule antibody. Compared to complete antibodies, small molecule antibodies have advantages such as smaller molecular weight, easier in vitro expression, and easier modification of antibody performance through genetic engineering techniques, leading to expectations of a wider range of applications. Small molecule genetically engineered antibodies mainly include single-chain variable fragments (scFv), bispecific antibodies (BsAb), triabodies, single-domain antibodies (VHH), single-chain disulfide-bond variable fragments (dsFv), antibody Fab fragments (Fab), and antibody F(ab')2 (Hoogenboom HR. Selecting and screening recombinant antibody libraries. Nat Biotechnol. 2005 Sep;23(9):1105-16.). Currently, the small molecule genetically engineered antibody family that receives the most research reports are single-chain antibodies, antibody Fab fragments, disulfide-bonded antibodies, and single-domain antibodies.

[0004] The advantages of small molecule antibodies are as follows: (1) They are easier to prepare than other genetically engineered antibodies. (2) Their immunogenicity is significantly reduced compared to the original antibody. If a modified antibody is constructed as a small molecule antibody, the possibility of eliminating its immunogenicity is further increased. (3) Due to their small molecular weight, small molecule antibodies can easily pass through the blood vessel wall (Bitencourt ALB, Campos RM, Cline EN, Klein WL, Sebollela A. Antibody Fragments as Tools for Elucidating Structure-Toxicity Relationships and for Diagnostic / Therapeutic Targeting of Neurotoxic Amyloid Oligomers. Int J Mol Sci. 2020 Nov 24;21(23):8920.), allowing them to penetrate into solid tumors, which is advantageous for tumor treatment. (4) Because it lacks an Fc region, it does not cause the adverse side effects of CDC and ADCC mediated by Fc, and has a shorter half-life and faster turnover compared to full-length antibodies (Kholodenko RV, Kalinovsky DV, Doronin II, Ponomarev ED, Kholodenko IV. Antibody Fragments as Potential Biopharmaceuticals for Cancer Therapy: Success and Limitations. Curr Med Chem. 2019; 26 (3):396-426.), which is advantageous for tumor detection by radioimmunoimaging and can reach tumor sites more intensively because it cannot bind to receptors on non-target cells. (5) Its small molecular size allows it to bind to antigens distributed in grooves on the surface of viruses, which is advantageous for the treatment of viral diseases. (6) By attaching an appropriate enzyme gene or toxin protein gene to the end of the small molecule antibody gene, enzyme-conjugated antibodies and immunotoxins can be mass-produced.(7) It can be expressed in microbial systems, allowing for antibody production by fermentation, enabling faster manufacturing and higher yields, thus reducing costs (Fernandes JC. Therapeutic application of antibody fragments in autoimmune diseases: current state and prospects. Drug Discov Today. 2018 Dec;23(12):1996-2002.), promoting the widespread use of antibody therapy.

[0005] Conventional small molecule recombinant antibodies, scFv and Fab, have several drawbacks. Firstly, because scFv lacks the CH1 and CL fragments of Fab, the affinity of most scFv is lower than that of its homologous Fab. Furthermore, scFv is prone to forming dimers, resulting in lower stability than its corresponding Fab during long-term storage (Hust M, Jostock T, Menzel C, Voedisch B, Mohr A, Brenneis M, Kirsch MI, Meier D, Dubel S. Single chain Fab (scFab) fragment. BMC Biotechnol. 2007 Mar 8;7:14). Secondly, Fab requires the separate secretion and expression of two different protein fragments, which are linked by disulfide bonds after processing and folding in the periplasmic lumen of E. coli. As a result, the molecular weight of Fab is approximately twice that of scFv, and the Fab light chain tends to form homodimers after expression. Therefore, the secretion and expression efficiency of Fab in the E. coli system is lower than that of scFv. These drawbacks limit the commercial application of antibodies. To overcome these shortcomings, there is a need for the preparation of novel antibodies that simultaneously possess high expression efficiency, high affinity, and high stability.

[0006] Recently reported novel antibodies, single-chain Fab (scFab), are composed of an antibody heavy chain Fd and a light chain κ linked by a linker peptide, and express proteins as single-chain antibodies (Koerber JT, Hornsby MJ, Wells JA. An improved single-chain Fab platform for efficient display and recombinant expression. J Mol Biol. 2015 Jan 30;427(2):576-86; Hanna R, Cardarelli L, Patel N, Blazer LL, Adams JJ, Sidhu SS. A phage-displayed single-chain Fab library optimized for rapid production of single-chain IgGs. Protein Science. 2020 August; 29(10):2075-2084; Koerber JT, Hornsby MJ, Wells JA. An improved single-chain Fab platform for efficient display and recombinant expression. J Mol Biol. 2015 Jan 30;427(2):576-86).

[0007] Antibody single-chain Fab (scFab) combines the advantages of both Fab and scFv. Single-chain Fab proteins reported to date have the following characteristics: Firstly, they exhibit antigen-binding ability equivalent to Fab (Thie H, Binius S, Schirrmann T, Hust M, Dubel S. Multimerization domains for antibody phage display and antibody production. N Biotechnol. 2009 Dec 31;26(6):314-21.); secondly, they have higher expression efficiency than Fab and can be prepared in large quantities as inclusion bodies (Bird RE, Hardman KD, Jacobson JW, Johnson S, Kaufman BM, Lee SM, Lee T, Pope SH, Riordan GS, Whitlow M. Single-chain antigen-binding proteins. Science. 1988 Oct 21;242(4877):423-6); thirdly, they can be prepared in large quantities using E. coli or yeast eukaryotic expression systems (Jordan E, Al-Halabi L, Schirrmann T, Hust M, Dubel S. Production of single chain Fab (scFab) fragments in Bacillus megaterium. Microb Cell Fact. 2007 Nov 27;6:38.), scFab can be used in place of scFv and Fab for screening highly active antibodies in phage display systems (Walker LM, Bowley DR, Burton DR. Efficient recovery of high-affinity antibodies from a single-chain Fab yeast display library. J Mol Biol. 2009 Jun 5;389(2):365-75.).Sixth, because scFab retains the antibody constant regions CH1 and CL, its molecular weight is approximately twice that of scFv, strengthening van der Waals forces in intermolecular interactions and resulting in higher stability than scFv (Hanna R, Cardarelli L, Patel N, Blazer LL, Adams JJ, Sidhu SS. A phage-displayed single-chain Fab library optimized for rapid production of single-chain IgGs. Protein Science. 2020 August; 29(10):2075-2084.).

[0008] Large-scale antibody production depends on the development of high-level production processes, which simultaneously require advantages such as high expression, high stability, and scalability. Large-scale antibody production is a crucial part of the industrialization and commercialization of antibody drugs, and is also a major bottleneck constraining the development of China's antibody industry. [Overview of the project] [Problems that the invention aims to solve]

[0009] To overcome problems that limit the industrialization and commercial application of antibody drugs, such as the low stability of small molecule antibodies and the low secretion and expression efficiency of conventional commercially available monoclonal antibodies, the present invention provides single-chain antibodies, a method for their in vitro synthesis, and their use. These antibodies simultaneously possess high expression efficiency, high affinity, and high stability, and have excellent potential for industrialization and commercialization. [Means for solving the problem]

[0010] A first aspect of the present invention provides a single-chain antibody formed by linking the light chain or light chain fragment of an antibody with the heavy chain or heavy chain fragment of an antibody using a linker peptide.

[0011] Furthermore, single-chain full-length antibodies have equivalent binding activity to the corresponding antigen compared to commercially available full-length antibodies.

[0012] Research has revealed that single-chain antibodies prepared with linkers of different compositions and lengths exhibit different affinity and expression activities. Therefore, the single-chain antibodies provided in this invention optimize the amino acid residues and their number in the linker, achieving both high-efficiency expression and high affinity.

[0013] To achieve both expression efficiency and specific affinity activity, the linker peptide contains at least 20 amino acid residues, preferably 25 to 180 amino acid residues, and more preferably 30 to 120 amino acid residues.

[0014] The amino acids included in the linker peptide are selected from serine (S), glycine (G), glutamic acid (E), threonine (T), alanine (A), arginine (R), tyrosine (Y), isoleucine (I), lysine (K), leucine (L), glutamine (Q), histidine (H), phenylalanine (F), valine (V), proline (P), aspartic acid (D), methionine (M), and asparagine (N).

[0015] Preferably, the amino acids contained in the linker peptide are selected from serine (S), glycine (G), glutamic acid (E), threonine (T), and alanine (A).

[0016] In a preferred embodiment, the percentage of the total number of serine (S), glycine (G), glutamic acid (E), threonine (T), and alanine (A) contained in the linker peptide relative to the total number of amino acids in the linker peptide is 100%, at least 99%, at least 98%, at least 97%, at least 96%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, and at least 50%.

[0017] In another preferred embodiment, the percentage of the total number of serine (S) and glycine (G) atoms in the linker peptide relative to the total number of amino acids in the linker peptide is 100%, at least 99%, at least 98%, at least 97%, at least 96%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, and at least 50%.

[0018] The binding activity of the single-chain antibody to the corresponding antigen is equivalent to that of the full-length antibody. Furthermore, equivalent activity refers to biological equivalence, and for example, the binding activity value of the single-chain antibody to the corresponding antigen reaches 0.1% to 10000%, 1% to 1000%, and 10% to 100% of the activity value of the full-length antibody. More suitable for the present invention is 85% to 115%, preferably 90% to 100%, and even more preferably 95% to 100%, and the activity value is EC 50 The value, etc., can be any numerical value that represents antibody activity in that field.

[0019] The aforementioned single-chain antibody can be synthesized in a single step in an in vitro cell-free synthesis system, eliminating the need to separately secrete and express two different protein fragments, the Fab light chain and heavy chain. It is less likely to form homodimers, has higher expression efficiency and affinity activity than Fab, possesses the antibody constant regions CH1 and CL, has a molecular weight approximately twice that of ScFv, exhibits stronger van der Waals forces in antibody-molecule interactions, and has been shown to be more stable than Scfv and others.

[0020] Furthermore, the antibody is selected from IgM, IgG, IgA, IgD, and / or IgE; More preferably, the IgG antibody is selected from IgG1, IgG2, IgG3 and / or IgG4; In one preferred embodiment, the antibody is selected from IgG1 antibodies, preferably, the IgG1 antibody is any one or more of Trastuzumab, Bevacizumab, Daratumumab, Omalizumab, Cetuximab, Adalimumab, Ustekinumab, Ocrelizumab, Infliximab, Envafolimab, Atlizumab, and Atezolizumab; The IgG2 antibodies are Denosumab, Erenumab, Tremelimumab, and Evocumab.

[0021] The antibody light chain is selected from κ chain and / or λ chain, the antibody light chain fragment is selected from κ chain fragment and λ chain fragment, the heavy chain is selected from μ chain, γ chain, α chain, δ chain, and / or ε chain, and the heavy chain fragment is selected from μ chain fragment, γ chain fragment, α chain fragment, δ chain fragment, and / or ε chain fragment.

[0022] In one preferred embodiment, the antibody light chain is derived from the Tislelizumab light chain TisLC or a fragment thereof, and the antibody heavy chain is derived from the Tislelizumab heavy chain TisHC or a fragment thereof, TisFd.

[0023] In one preferred embodiment, the antibody light chain is derived from the Dupilumab light chain DupLC or a fragment thereof, and the antibody heavy chain is derived from the Dupilumab heavy chain DupHC or a fragment thereof, DupFd.

[0024] In one preferred embodiment, the antibody light chain is derived from the Adalimumab light chain AdaLC or a fragment thereof, and the antibody heavy chain is derived from the Adalimumab heavy chain AdaHC or a fragment thereof, AdaFd.

[0025] In one preferred embodiment, the antibody light chain is derived from the Bevacizumab light chain BevLC or a fragment thereof, and the antibody heavy chain is derived from the Bevacizumab heavy chain BevHC or a fragment thereof, BevFd.

[0026] In one preferred embodiment, the antibody light chain is derived from the Daratumumab light chain DarLC or a fragment thereof, and the antibody heavy chain is derived from the Daratumumab heavy chain DarHC or a fragment thereof, DarFd.

[0027] In one preferred embodiment, the antibody light chain is derived from the Omalizumab light chain OmaLC or a fragment thereof, and the antibody heavy chain is derived from the Omalizumab heavy chain OmaHC or a fragment thereof, OmaFd.

[0028] In one preferred embodiment, the antibody light chain is derived from the Cetuximab light chain CetLC or a fragment thereof, and the antibody heavy chain is derived from the Cetuximab heavy chain CetHC or a fragment thereof, CetFd. [[ID=第十七]]

[0029] In one preferred embodiment, the antibody light chain is derived from the Denosumab light chain DenLC or a fragment thereof, and the antibody heavy chain is derived from the Denosumab heavy chain DenHC or a fragment thereof, DenFd.

[0030] In one preferred embodiment, the antibody light chain is derived from the trastuzumab light chain TraLC or a fragment thereof, and the antibody heavy chain is derived from the trastuzumab heavy chain TraHC or a fragment thereof. In another preferred embodiment, the antibody light chain is derived from the trastuzumab light chain TraLC, and the antibody heavy chain fragment is the trastuzumab heavy chain fragment TraFd.

[0031] Furthermore, the single-chain antibody contains a fluorescent signal peptide SP, preferably the signal peptide SP is linked to the N-terminus of the light chain or light chain fragment of the single-chain antibody, which contributes to improving the expression efficiency and expression level of the single-chain antibody.

[0032] Furthermore, the single-chain antibody also includes a fluorescent protein tag, preferably the fluorescent protein tag is ligated to the C-terminus of the heavy chain or heavy chain fragment of the single-chain antibody, and more preferably the fluorescent protein tag is EGFP.

[0033] Furthermore, the tag tag is included, preferably the tag tag is ligated to the N-terminus of the light chain or light chain fragment of the single-chain antibody, and preferably the tag tag added to the N-terminus of the light chain or light chain fragment is a His tag.

[0034] Furthermore, preferably, the fluorescent protein tag, tag tag, and signal peptide are linked to the antibody's light chain, light chain fragment, or heavy chain, or heavy chain fragment via single bonds or flexible linker peptides, and preferably, the flexible linker peptide contains 1 to 20 amino acid residues.

[0035] A second aspect of the present invention provides one or more isolated nucleic acids encoding antigen-binding ligand proteins provided in the first aspect.

[0036] Furthermore, the nucleic acid is a DNA sequence and / or an RNA sequence.

[0037] In one possible embodiment, the nucleic acid is linear DNA and a PCR linear fragment.

[0038] Furthermore, the PCR linear fragments can be obtained by amplification techniques, the amplification techniques being not particularly limited and including, but not limited to, PCR amplification techniques, isothermal amplification techniques, room temperature amplification techniques, and room temperature amplification techniques. Here, isothermal amplification techniques are preferably room temperature amplification techniques. This includes, but is not limited to, commercial DNA amplification systems provided by Biocompare, Neta Scientific Inc., ABM, Thermo Fisher Scientific, Expedeon, Vivantis, and others.

[0039] In another embodiment, the nucleic acid is RNA encoding the amino acid sequence of a single-chain antibody.

[0040] Furthermore, the RNA is prepared using in vitro nucleic acid amplification technology. The in vitro nucleic acid amplification technology is not particularly limited and includes, but is not limited to, polymerase chain reaction technology (PCR amplification technology), isothermal amplification technology, room temperature amplification technology, and room temperature amplification technology. Specifically, nucleic acid isothermal amplification methods applicable to the technical means of the present invention include, but are not limited to, loop-mediated isothermal amplification (LAMP), strand displacement amplification (SDA), nucleic acid sequence-based amplification (NASBA), rolling circle amplification (RCA), nick enzyme nucleic acid isothermal amplification, helicase-dependent isothermal amplification (HDA), transcription-dependent amplification, hybrid capture method, transcription-mediated amplification (TMA), recombinase-assisted amplification (RAA), and recombinase polymerase amplification (RPA), and is preferably loop-mediated isothermal amplification.

[0041] A third aspect of the present invention provides one or more expression vectors comprising nucleic acids encoding single-chain antibodies provided in the first aspect.

[0042] Furthermore, the expression vectors include, but are not limited to, eukaryotic plasmid vectors, eukaryotic virus vectors, prokaryotic plasmids, specific vectors, shuttle vectors, minichromosomes, and various other vectors.

[0043] Preferably, the expression vector is a eukaryotic plasmid expression vector or a prokaryotic plasmid expression vector.

[0044] In one feasible embodiment, the expression vector is circular DNA, more preferably plasmid DNA, such as a pET plasmid or a pGEM plasmid.

[0045] A fourth aspect of the present invention provides a host cell characterized by comprising a vector according to the third aspect of the present invention, or having polynucleotides according to the second aspect incorporated into its genome, or expressing a recombinant protein as shown in the first aspect.

[0046] In one preferred embodiment, the host cell is selected from either a prokaryotic host cell or a eukaryotic host cell.

[0047] In another preferred embodiment, the host cell is a prokaryotic cell such as Escherichia coli.

[0048] In another preferred embodiment, the host cell is a eukaryotic cell, such as yeast, progenitor cells, Chinese hamster ovary cells, insect cells, wheat germ cells, or rabbit reticulocytes.

[0049] Preferably, the yeast is a combination of one or more species selected from Saccharomyces cerevisiae and Kluyveromyces yeasts, and in another preferred embodiment, the Kluyveromyces yeast is selected from a combination of one or more species of Kluyveromyces lactis, Kluyveromyces marxianus, and Kluyveromyces dobzhanskii.

[0050] A fifth aspect of the present invention provides an in vitro cell-free protein synthesis system comprising a nucleic acid molecule provided in the second aspect, or an expression vector provided in the third aspect, or a lysate or extract of a host cell described in the fourth aspect of the present invention.

[0051] The extracellular protein synthesis system includes, but is not limited to, an E. coli extracellular protein synthesis system, a bacterial extracellular protein synthesis system, a mammalian extracellular protein synthesis system (e.g., HF9, Hela, CHO, HEK293), a plant extracellular protein synthesis system, a yeast extracellular protein synthesis system, and an insect extracellular protein synthesis system. Preferably, it is a yeast extracellular protein synthesis system, more preferably a Kluyveromyces extracellular protein synthesis system, and most preferably a Kluyveromyces lactis (K. lactis) yeast extracellular protein synthesis system.

[0052] Furthermore, the in vitro protein synthesis system provided by the present invention comprises, but is not limited to, (a) a cell extract, (b) an optional polyethylene glycol, (c) an optional exogenous sucrose, and (d) an optional solvent.

[0053] Furthermore, the reaction system further includes trihydroxymethylaminomethane, potassium acetate, magnesium acetate, a mixture of nucleotide triphosphates (NTPs), a mixture of amino acids, and the like.

[0054] The aforementioned cell extracts are typically used to provide substances such as ribosomes, transfer RNA (tRNA), aminoacyl-tRNA synthetase, initiation and elongation factors necessary for protein synthesis, and termination and release factors. Furthermore, through strain modification, it is also possible to provide other substances such as polymerases (RNA polymerase and / or DNA polymerase) endogenously.

[0055] The protein components required for the extracorporeal cell-free protein synthesis system (e.g., RNA polymerase) can be supplied endogenously or added exogenously. For endogenous supply, gene modification methods described in existing literature such as CN108690139A, CN109423496A, CN106978439A, CN110408635A, CN110551700A, CN110093284A, CN110845622A, CN110938649A, CN111378708A, CN111484998 A, and "Molecular and Cellular Biology, 1990, 10(1):353-360" and the literature they cite can be referenced. Exemplary examples include, but are not limited to, inserting coding sequences into intracellular free plasmids, integrating coding genes into the cellular genome, and combinations thereof. When supplied using an external source, the amount used can be controlled and adjusted according to the system's needs.

[0056] In some preferred embodiments, the yeast cell extract is Kluyveromyces cell extract, Saccharomyces cerevisiae, or a combination thereof. In one preferred embodiment, the Kluyveromyces is Kluyveromyces lactis (K. lactis).

[0057] The technical elements of the present invention, such as the in vitro protein synthesis system, template, plasmid, target protein, in vitro protein synthesis reaction (incubation reaction), various preparation methods, and various detection methods, are as follows: CN111484998A, CN106978349A, CN108535489A, CN108690139A, CN108949801 A, CN108642076A, CN109022478A, CN109423496A, CN109423497A, CN109423509A, CN109837293A, CN109971783A, CN109988801A, Appropriate embodiments or methods of implementation can be independently selected from documents such as CN109971775A, CN110093284A, CN110408635A, CN110408636A, CN110551745A, CN110551700A, CN110551785A, CN110819647A, CN110845622A, CN110938649A, and CN110964736A. Unless they conflict with the objectives of the present invention, these documents and the documents they refer to are cited in their entirety and objectives.

[0058] A sixth aspect of the present invention provides a method for detecting the activity of a single-chain antibody provided in the first aspect of the present invention using an enzyme immunosorbent assay (ELISA), wherein the ELISA method is an indirect antibody measurement or a competitive antibody measurement method for detecting the activity of a single-chain antibody.

[0059] Preferably, the antibody measurement by the indirect method of the ELISA method includes the following steps: The specific detection steps include: (1) binding the specific antigen to the solid support to form a solid antigen, and washing away unbound antigen and impurities; (2) adding diluted test serum, in which the specific antibody binds to the antigen to form a solid antigen-antibody complex. After washing, only the specific antibody remains on the solid support. Other antibodies that cannot bind to the solid antigen and impurities in the serum are removed during the washing process; (3) adding enzyme-labeled secondary antibody: this binds to the antibody in the solid complex, thereby indirectly labeling the antibody with an enzyme, and after washing, the amount of enzyme on the solid support reflects the amount of specific antibody. For example, when measuring human antibodies against a certain disease, enzyme-labeled goat anti-human IgG antibody can be used; (4) color development by substrate addition: the depth of the color reflects the amount of the target antibody in the sample.

[0060] A seventh aspect of the present invention provides for the use of single-chain antibodies provided by the first aspect of the present invention, or single-chain antibodies prepared by the synthetic system provided by the fourth aspect, in the diagnosis, prevention, and treatment of diseases, or in the preparation of pharmaceuticals for the diagnosis, prevention, and treatment of diseases.

[0061] Furthermore, the aforementioned diseases are tumors and / or cancers, and exemplary tumors and / or cancers include, but are not limited to, lung cancer, breast cancer, esophageal cancer, stomach cancer, cervical cancer, colon cancer, rhabdomyosarcoma, liposarcoma, osteosarcoma, lymphoma, leukemia, liver cancer, cervical cancer, ovarian cancer, and breast cancer.

[0062] Preferably, the disease is HER2-positive breast cancer and gastric cancer. [Effects of the Invention]

[0063] The beneficial effects of this invention are: 1) The present invention provides a single-chain antibody that can be expressed with high efficiency in a yeast cell-free system (e.g., D2P system) and has a short production cycle by ligating the light chain or light chain fragment (e.g., trastuzumab light chain) and heavy chain or heavy chain fragment (e.g., trastuzumab heavy chain TraHC or heavy chain fragment TraFd) of an antibody via a linker (linker peptide). 2) The single-chain antibody provided by the present invention has binding activity to the ErbB2 antigen equivalent to that of commercially available monoclonal antibody trastuzumab, and is characterized by unparalleled ease of expression compared to commercially available trastuzumab (natural double-chain structure). Therefore, the single-chain antibody provided by the present invention guarantees antibody activity while solving the drawbacks of conventional antibodies, such as low antibody expression efficiency and the inability to commercialize them. 3) The single-chain antibodies provided by the present invention have a linker that connects the light chain and the heavy chain, making the antibody protein less prone to aggregation and more stable. 4) The single-chain antibodies provided by the present invention simultaneously possess high expression efficiency, high affinity, and high stability, can be mass-produced by combining them with an in vitro cell-free protein synthesis system, specifically bind to the extracellular domain IV of ErbB2, have excellent application prospects in the treatment of HER2-positive breast cancer and gastric cancer, and have good prospects for commercialization. [Brief explanation of the drawing]

[0064] [Figure 1] Figure 1 is a plasmid structure diagram in Example 1 of the present invention.

[0065] Promoter refers to the promoter, 5-UTR to the 5' untranslated region, SP to the signal peptide, 5L to the linker peptide, the portion between SP and 5L to the His tag, TrascFab to the TrascFab sequences linked with different linker lengths, EGFP to the green fluorescent protein sequence, and 3-UTR to the 3' untranslated region and stop.

[0066] [Figure 2] Figure 2 shows the electrophoretic analysis of plasmids prepared in Examples 2 and 7 of the present invention. In the figure, M represents a marker, and 1 to 7 represent the TrascFab0, TrascFab6, TrascFab34, TrascFab60, TrascFab82, TrascFab120, and TrascIgG60 plasmids, respectively.

[0067] [Figure 3]Figure 3 shows the purified analysis of the AMPI products of Examples 2 and 7 of the present invention. In the figure, M indicates a marker, and 1 to 7 show the AMPI gel electrophoresis diagrams of TrascFab0, TrascFab6, TrascFab34, TrascFab60, TrascFab82, TrascFab120, and scIgG60, respectively.

[0068] [Figure 4] Figure 4 is an analytical diagram of the TrascFab60 protein purified in Example 6 of the present invention. M represents a marker, and 1 represents the purified TrascFab60 protein sample.

[0069] [Figure 5] Figure 5 shows the affinity test results for the TrascFab purified proteins TrascFab0, TrascFab6, TrascFab15, TrascFab25, TrascFab30, TrascFab34, TrascFab60, TrascFab82, TrascFab120, and TrascFab180 with different linker lengths in Example 8. "Concentration" in the figure indicates the concentration. [Modes for carrying out the invention]

[0070] The advantages and features of the present invention will become clearer through the following description. However, these embodiments are merely illustrative and do not constitute any limitation on the scope of the invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and forms of the technical solutions of the invention without departing from the spirit and scope of the invention, all of which fall within the scope of protection of the invention.

[0071] term In this invention, "antibody" refers to a protective protein produced by the body in response to antigen stimulation, and is a type of immunoglobulin that specifically binds to the antigen. These antibodies contain four heterogeneous polypeptide chains, of which the two chains with larger molecular weights are called heavy chains (H), and the two chains with smaller molecular weights are called light chains (L). The amino acid composition of the two H chains and two L chains within the same Ig molecule is completely identical. The heavy chain has a molecular weight of 50,000 to 75,000 and consists of 450 to 550 amino acid residues, while the light chain has a molecular weight of approximately 25,000 and consists of 214 amino acid residues. The two heavy chains and two light chains of a natural antibody molecule are each folded into multiple globular domains, and each domain performs a corresponding function. The light chain has two domains, VL and CL. The heavy chains of IgG, IgA, and IgD have four domains: VH, CH1, CH2, and CH3. The heavy chains of IgM and IgE have five domains, specifically, one additional CH4 domain.

[0072] In this invention, "light chain fragment" refers to a structural fragment capable of exhibiting the corresponding function of a light chain, such as a structural fragment containing a CL domain.

[0073] In this invention, "heavy chain fragment" refers to a structural fragment capable of exhibiting the corresponding function of a heavy chain, such as a structural fragment containing a CH1 domain.

[0074] The in vitro synthesis system in the present invention includes, but is not limited to, the IVTT reaction (in vitro transcription-translation reaction). In the present invention, the IVTT reaction is preferred. The IVTT reaction corresponds to the IVTT system and is a process that transcribes and translates DNA in vitro to produce protein. Therefore, such an in vitro protein synthesis system is also called the D2P system, D-to-P system, D_to_P system, or DNA-to-Protein system. The corresponding in vitro protein synthesis method is also called the D2P method, D-to-P method, D_to_P method, or DNA-to-Protein method, and these have the same meaning as expressions such as "in vitro cell-free protein synthesis system," "in vitro expression system," "in vitro protein synthesis system," "in vitro protein synthesis reaction system," and "cell-free protein synthesis system." There are several ways to describe it, including cell-free systems, cell-free protein synthesis systems, cell-free extracorporeal protein synthesis systems, cell-free extracorporeal protein synthesis systems, cell-free extracorporeal protein synthesis systems, cell-free synthesis systems, CFS systems (cell-free systems), and CFPS systems (cell-free protein synthesis systems). It includes extracorporeal translation systems and extracorporeal transcription-translation systems (IVTT systems). An extracorporeal protein synthesis system is also called a "protein factory." An extracorporeal protein synthesis reaction refers to the reaction that synthesizes proteins in an extracorporeal cell-free synthesis system, and includes at least a translation process.

[0075] A first aspect of the present invention provides a single-chain antibody characterized by linking the light chain or light chain fragment of an antibody with the heavy chain or heavy chain fragment of an antibody by a linker (linker peptide), being expressed with high efficiency in a single step in an in vitro cell-free synthesis system, eliminating the need to secrete and express two different protein fragments, the light chain and the heavy chain, being less likely to form homodimers, having high expression efficiency, and having binding activity to the corresponding antigen equivalent to that of an antibody.

[0076] The aforementioned equivalent activity refers to biological equivalence, for example, the binding activity value of a single-chain antibody to a corresponding antigen is 85% to 115%, preferably 90% to 100%, and more preferably 95% to 100%, of the antibody's activity value, and the aforementioned activity value is EC 50 The value, etc., can be any numerical value that represents antibody activity in that field.

[0077] Research has revealed that single-chain antibodies prepared with linkers of different lengths exhibit different affinity activities. Therefore, by selecting linkers containing the appropriate number of amino acid residues, we have achieved both high-efficiency expression of single-chain antibodies and high affinity.

[0078] To achieve both expression efficiency and specific affinity activity, the linker peptide contains at least 20 amino acid residues, preferably 25 to 180 amino acid residues, and more preferably 34 to 120 amino acid residues; For example, containing 25, 26, 27, 28, 29, 30, 31, 32, and 33 amino acid residues; For example, it also contains 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, and 70 amino acid residues; For example, it contains 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, and 125 amino acid residues.

[0079] Linkers are named based on the number of amino acid residues they contain. For example, a linker with 6 amino acid residues is named linker6, one with 60 is named linker60, and one with 120 is named linker120.

[0080] Single-chain antibodies prepared with linkers of different lengths exhibit different affinity activities. The present invention achieves high-efficiency expression of single-chain antibodies while also achieving high affinity by selecting a linker containing an appropriate number of amino acid residues. The amino acids contained in the linker peptide are selected from serine (S), glycine (G), glutamic acid (E), threonine (T), alanine (A), arginine (R), tyrosine (Y), isoleucine (I), lysine (K), leucine (L), glutamine (Q), histidine (H), phenylalanine (F), valine (V), proline (P), aspartic acid (D), methionine (M), and asparagine (N).

[0081] Preferably, the amino acids contained in the linker peptide are selected from serine (S), glycine (G), glutamic acid (E), threonine (T), and alanine (A).

[0082] In one preferred embodiment, the percentage of serine (S), glycine (G), glutamic acid (E), threonine (T), and alanine (A) in the linker peptide relative to the total number of amino acids in the linker peptide is 100%, at least 99%, at least 98%, at least 97%, at least 96%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, and at least 50%.

[0083] In another preferred embodiment, the percentage of serine (S), glycine (G), and glutamic acid (E) in the linker peptide relative to the total number of amino acids in the linker peptide is 100%, at least 99%, at least 98%, at least 97%, at least 96%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, and at least 50%.

[0084] In another preferred embodiment, the percentage of serine (S), glycine (G), and glutamic acid (E) in the linker peptide relative to the total number of amino acids in the linker peptide is 100%, at least 99%, at least 98%, at least 97%, at least 96%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, and at least 50%.

[0085] In one preferred embodiment, the linker is linker 25 and has the amino acid sequence shown in SEQ ID NO: 11, or a sequence having one or more amino acid substitutions, deletions, or additions to the sequence shown in SEQ ID NO: 11, more preferably the number of substituted, deleted, or added amino acids is 1 to 10, and preferably 1 to 5.

[0086] Sequence ID 11: GGGGSGGGG SGGGGSGGGG SGGGGS

[0087] In one preferred embodiment, the linker is linker 30 and has the amino acid sequence shown in SEQ ID NO: 14, or a sequence having one or more amino acid substitutions, deletions, or additions to the sequence shown in SEQ ID NO: 14, more preferably the number of substituted, deleted, or added amino acids is 1 to 10, and preferably 1 to 5.

[0088] Sequence ID 14: SGGGSGGGSEGGGSEGGGSEGGGSEGGGSG

[0089] In one preferred embodiment, the linker is linker34 and has the amino acid sequence shown in SEQ ID NO: 15, or a sequence having one or more amino acid substitutions, deletions, or additions to the amino acid sequence shown in SEQ ID NO: 15, wherein the number of substituted, deleted, or added amino acids is 1 to 10, preferably 1 to 5.

[0090] Sequence ID 15: SGGGSGGGSEGGGSEGGGSEGGGSEGGGSGGGSG

[0091] In one preferred embodiment, the linker is linker34A and has the amino acid sequence shown in SEQ ID NO: 16, or a sequence having one or more amino acid substitutions, deletions, or additions to the amino acid sequence shown in SEQ ID NO: 16, wherein the number of substituted, deleted, or added amino acids is 1 to 10, preferably 1 to 5.

[0092] Sequence ID 16: RRYEERRYEERRYEERRYEERRYEERRYEERRYE

[0093] In one preferred embodiment, the linker is linker34B and has the amino acid sequence shown in SEQ ID NO: 17, or a sequence having one or more amino acid substitutions, deletions, or additions to the amino acid sequence shown in SEQ ID NO: 17, wherein the number of substituted, deleted, or added amino acids is 1 to 10, preferably 1 to 5.

[0094] Sequence ID 17: YYEYYYYEYYYYEYYYYEYYYYEYYYYEYYEYYE

[0095] In one preferred embodiment, the linker is linker34C and has the amino acid sequence shown in SEQ ID NO: 18, or a sequence having one or more amino acid substitutions, deletions, or additions to the amino acid sequence shown in SEQ ID NO: 18, wherein the number of substituted, deleted, or added amino acids is 1 to 10, preferably 1 to 5.

[0096] Sequence ID 18: IKYLEFISEAIIHVLHSRHPGDFGADAQGAMNKA

[0097] In one preferred embodiment, the linker is linker 60 and has the amino acid sequence shown in SEQ ID NO: 19, or a sequence having one or more amino acid substitutions, deletions, or additions to the amino acid sequence shown in SEQ ID NO: 19, wherein the number of substituted, deleted, or added amino acids is 1 to 50, preferably 1 to 20.

[0098] Sequence ID 19: GGSSGSGSGSTGTSSSGTGTSAGTTGTSASTSGSGSGGGGGSGGGGSAGGTATA GASSGS

[0099] In one preferred embodiment, the linker is linker60A and has the amino acid sequence shown in SEQ ID NO: 20, or a sequence having one or more amino acid substitutions, deletions, or additions to the amino acid sequence shown in SEQ ID NO: 20, wherein the number of substituted, deleted, or added amino acids is 1 to 50, preferably 1 to 20.

[0100] Sequence ID 20: LAQSHATKHKIPIKYLEFISEAIIHVLHSRHPGDFGADAQGAMNKALELFRKDIAAKYKE

[0101] In one preferred embodiment, the linker is linker82 and has the amino acid sequence shown in SEQ ID NO: 21, or a sequence having one or more amino acid substitutions, deletions, or additions to the amino acid sequence shown in SEQ ID NO: 21, wherein the number of substituted, deleted, or added amino acids is 1 to 50, preferably 1 to 20.

[0102] Sequence ID 21: GGSSGSGSGSTGTSSSGTGTSAGTTGTSASTSGSGSGGGGGSGGGGSAGGTATAAGSTGGAGSTGGAGSTGGAGTGSGAGS

[0103] In one preferred embodiment, the linker is linker 120 and has the amino acid sequence shown in SEQ ID NO: 22, or a sequence having one or more amino acid substitutions, deletions, or additions to the amino acid sequence shown in SEQ ID NO: 22, wherein the number of substituted, deleted, or added amino acids is 1 to 50, preferably 1 to 20.

[0104] Sequence ID 22: GGSSGSGSGSTGTSSSGTGTSAGTTGTSASTSGSGSGGGGGSGGGGSAGGTATGGSSGGSGGSSGSGSGSTGTSSSGTGTSAGTTGTSASTSGSGSGGGGGSGGGGSAGGTATAGASSGS

[0105] In one preferred embodiment, the linker is linker 180 and has the amino acid sequence shown in SEQ ID NO: 23, or a sequence having one or more amino acid substitutions, deletions, or additions to the amino acid sequence shown in SEQ ID NO: 23, wherein the number of substituted, deleted, or added amino acids is 1 to 50, preferably 1 to 20.

[0106] Sequence ID 23: GGSSSGSGSTGTSSSGTGTSAGTTGTSASTSGSGSGGGGGSGGGGSAGGTATAGASSGSGGSSGSGSGSTGTSSSGTGTSAGTTGTSASTSGSGSGGGGGSGGGGSAGGTATAGASSGSGGSSGSGSGSTGTSSSGTGTSAGTTGTSASTSGSGSGGGGGSGGGGSAGGTATAGASSGS

[0107] In one preferred embodiment, the linker is Linker36A and has the amino acid sequence shown in SEQ ID NO: 24, or a sequence having one or more amino acid substitutions, deletions, or additions to the amino acid sequence shown in SEQ ID NO: 24, wherein the number of substituted, deleted, or added amino acids is 1 to 50, preferably 1 to 20.

[0108] Sequence ID 24: GSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYSGS

[0109] In one preferred embodiment, the linker is Linker36B and has the amino acid sequence shown in SEQ ID NO: 25, or a sequence having one or more amino acid substitutions, deletions, or additions to the amino acid sequence shown in SEQ ID NO: 25, wherein the number of substituted, deleted, or added amino acids is 1 to 50, preferably 1 to 20.

[0110] Sequence ID 25: GSGEVQLVESGGGLVQPGGSLRLSSAASGFNIKGSG

[0111] In one preferred embodiment, the linker is Linker36C and has the amino acid sequence shown in SEQ ID NO: 26, or a sequence having one or more amino acid substitutions, deletions, or additions to the amino acid sequence shown in SEQ ID NO: 26, wherein the number of substituted, deleted, or added amino acids is 1 to 50, preferably 1 to 20.

[0112] Sequence ID 26: GAGAGSGAGAGSGAGAGSGAGAGSGAGAGSGAGAGS

[0113] The antibody of the present invention is selected from IgM, IgG, IgA, IgD and / or IgE; More preferably, the IgG antibody is selected from IgG1, IgG2, IgG3 and / or IgG4; In one preferred embodiment, the antibody is selected from IgG1, preferably one or more of the following: trastuzumab, bevacizumab, daratumumab, omalizumab, cetuximab, adalimumab, ustekinumab, ocrelizumab, infliximab, envafolimab, atlizumab, and atezolizumab; The IgG2 antibodies mentioned above are denosumab, erenumab, tremelimumab, and evocumab; The IgG4 antibodies mentioned above are tislerizumab, dupilumab, rituximab, pembrolizumab, nivolumab, or palizumab.

[0114] The antibody light chain is selected from a κ chain and / or a λ chain, the antibody light chain fragment is selected from a κ chain fragment and a λ chain fragment, the heavy chain is selected from a μ chain, a γ chain, an α chain, a δ chain and / or an ε chain, and the heavy chain fragment is selected from a μ chain fragment, a γ chain fragment, an α chain fragment, a δ chain fragment and / or an ε chain fragment.

[0115] In one preferred embodiment, the antibody light chain is derived from the trastuzumab light chain TraLC, and the antibody heavy chain is derived from the trastuzumab heavy chain TraHC. In another preferred embodiment, the antibody light chain is derived from the trastuzumab light chain TraLC, and the antibody heavy chain fragment is the trastuzumab heavy chain fragment TraFd.

[0116] Furthermore, the Trastuzumab light chain TraLC includes the amino acid sequence shown in SEQ ID NO: 4, or a sequence having one or more amino acid substitutions, deletions, or additions to the amino acid sequence shown in SEQ ID NO: 4, wherein the number of substituted, deleted, or added amino acids is 1 to 50, preferably 1 to 10 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10).

[0117] Sequence ID 4: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0118] Preferably, the trastuzumab light chain TraLC has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, and most preferably at least 99% homology to the amino acid sequence shown in SEQ ID NO: 4.

[0119] Furthermore, the trastuzumab heavy chain fragment TraFd comprises the amino acid sequence shown in SEQ ID NO: 5, or a sequence having one or more amino acid substitutions, deletions, or additions to the polypeptide shown in SEQ ID NO: 5, wherein the number of substituted, deleted, or added amino acids may be 1 to 100, preferably 1 to 20, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0120] Sequence ID 5: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYT RYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK

[0121] Preferably, the trastuzumab heavy chain fragment TraFd has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, and most preferably at least 99% homology with the amino acid sequence shown in SEQ ID NO: 5.

[0122] Furthermore, the trastuzumab heavy chain TraHC comprises the amino acid sequence described in SEQ ID NO: 6, or a sequence having one or more amino acid substitutions, deletions, or additions to the polypeptide shown in SEQ ID NO: 6, wherein the number of substituted, deleted, or added amino acids may be 1 to 100, preferably 1 to 20, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0123] Sequence ID 6: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

[0124] Preferably, the trastuzumab heavy chain fragment TraHC has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, and most preferably at least 9% homology with the amino acid sequence shown in SEQ ID NO: 6.

[0125] In one preferred embodiment, the antibody light chain is derived from the tislerizumab light chain TisLC or a fragment thereof, and the antibody heavy chain is derived from the tislerizumab heavy chain TisHC or a fragment thereof TisFd.

[0126] Furthermore, the tislerizumab light chain TisLC comprises the amino acid sequence shown in SEQ ID NO: 27, or a sequence having one or more amino acid substitutions, deletions, or additions to the polypeptide of the amino acid sequence shown in SEQ ID NO: 27, wherein the number of substituted, deleted, or added amino acids is 1 to 50, preferably 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0127] Sequence ID 27: DIVMTQSPDSLAVSLGERATINCKSSESVSNDVAWYQQKPGQPPKLLINYAFHRFTGVPDRFSGSGYGTDFTLTISSLQAEDVAVYYCHQAYSSPYTFGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0128] Preferably, the tislerizumab light chain TisLC has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, and most preferably at least 99% homology with the amino acid sequence shown in SEQ ID NO: 27.

[0129] Furthermore, the tislerizumab heavy chain TisHC comprises the amino acid sequence described in SEQ ID NO: 28, or a sequence having one or more amino acid substitutions, deletions, or additions to the polypeptide shown in SEQ ID NO: 28, wherein the number of substituted, deleted, or added amino acids may be 1 to 100, preferably 1 to 20, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0130] Sequence ID 28: QVQLQESGPGLVKPSETLSLTTCTVSGFSLTSYGVHWIRQPPGKGLEWIGVIYADGSTNYNPSLKSRVTISKDTSKNQVSLKLSSVTAADTAVYYCARAYGNYWYIDVWGQGTTV TVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCP APPVAGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPR EPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKTGGAGTGSGA;

[0131] Preferably, the tislerizumab heavy chain TisHC has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, and most preferably at least 99% homology to the amino acid sequence shown in SEQ ID NO: 28.

[0132] Furthermore, the tislerizumab heavy chain fragment TisFd comprises the amino acid sequence shown in SEQ ID NO: 29, or a sequence having one or more amino acid substitutions, deletions, or additions to the polypeptide shown in SEQ ID NO: 29, wherein the number of substituted, deleted, or added amino acids may be 1 to 100, preferably 1 to 20, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0133] Sequence ID 29: QVQLQESGPGLVKPSETLSLTTCTVSGFSLTSYGVHWIRQPPGKGLEWIGVIYADGSTNYNPSLKSRVTISKDTSKNQVSLKLSSVTAADTAVYYCARAYGNYWYIDVWG QGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESK

[0134] Preferably, the trastuzumab heavy chain TraFd has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, and most preferably at least 99% homology with the amino acid sequence shown in SEQ ID NO: 5.

[0135] In one preferred embodiment, the antibody light chain is derived from a dupilumab light chain DupLC or a fragment thereof, and the antibody heavy chain is derived from a dupilumab heavy chain DupHC or a fragment DupFd thereof.

[0136] Furthermore, the dupilumab light chain DupLC comprises the amino acid sequence shown in SEQ ID NO: 30, or a sequence having one or more amino acid substitutions, deletions, or additions to the polypeptide of the amino acid sequence shown in SEQ ID NO: 30, wherein the number of substituted, deleted, or added amino acids is 1 to 50, preferably 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0137] Sequence ID 30: DIVMTQSPLSLPVTPGEPASISCRSSQSLLYSIGYNYLDWYLQKSGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGFYYCMQALQTPYTFGQGTKL EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0138] Preferably, the dupilumab light chain DupLC has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, and most preferably at least 99% homology with the amino acid sequence shown in SEQ ID NO: 30.

[0139] Furthermore, the dupilumab heavy chain DupHC comprises the amino acid sequence described in SEQ ID NO: 31, or a sequence having one or more amino acid substitutions, deletions, or additions to the polypeptide shown in SEQ ID NO: 31, wherein the number of substituted, deleted, or added amino acids may be 1 to 100, preferably 1 to 20, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0140] Sequence ID 31: EVQLVESGGGLEQPGGSLRLSCAGSGFTFRDYAMTWVRQAPGKGLEWVSSISGSGGNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDRLSITIRPRYYGL DVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVES KYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIE KTISKAKGQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG

[0141] Preferably, the dupilumab heavy chain DupHC has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, and most preferably at least 99% homology with the amino acid sequence shown in SEQ ID NO: 31.

[0142] Furthermore, the dupilumab heavy chain fragment DupFd comprises the amino acid sequence shown in SEQ ID NO: 32, or a sequence having one or more amino acid substitutions, deletions, or additions to the polypeptide shown in SEQ ID NO: 32, wherein the number of substituted, deleted, or added amino acids may be 1 to 100, preferably 1 to 20, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0143] Sequence ID 32: EVQLVESGGGLEQPGGSLRLSCAGSGFTFRDYAMTWVRQAPGKGLEWVSSISGSGGNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDRLSITIRPRYYGLDV WGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYG

[0144] Preferably, the dupilumab heavy chain fragment DupFd has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, and most preferably at least 99% homology with the amino acid sequence shown in SEQ ID NO: 32.

[0145] In one preferred embodiment, the antibody light chain is derived from the adalimumab light chain AdaLC or a fragment thereof, and the antibody heavy chain is derived from the adalimumab heavy chain AdaHC or a fragment AdaFd thereof.

[0146] Furthermore, the adalimumab light chain AdaLC comprises the amino acid sequence shown in SEQ ID NO: 33, or a sequence having one or more amino acid substitutions, deletions, or additions to the polypeptide of the amino acid sequence shown in SEQ ID NO: 33, wherein the number of substituted, deleted, or added amino acids is 1 to 50, preferably 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0147] Sequence ID 33: DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0148] Preferably, Adalimumab AdaLC has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, and most preferably at least 99% homology to the amino acid sequence shown in SEQ ID NO: 33.

[0149] Furthermore, the adalimumab heavy chain AdaHC comprises the amino acid sequence shown in SEQ ID NO: 34, or a sequence having one or more amino acid substitutions, deletions, or additions to the polypeptide shown in SEQ ID NO: 34, wherein the number of substituted, deleted, or added amino acids may be 1 to 100, preferably 1 to 20, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0150] Sequence ID 34: EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKTGGAGTGSGA

[0151] Preferably, the adalimumab heavy chain AdaHC has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, and most preferably at least 99% homology to the amino acid sequence shown in SEQ ID NO: 34.

[0152] Furthermore, the adalimumab heavy chain fragment AdaFd comprises the amino acid sequence shown in SEQ ID NO: 35, or a sequence having one or more amino acid substitutions, deletions, or additions to the polypeptide shown in SEQ ID NO: 35, wherein the number of substituted, deleted, or added amino acids may be 1 to 100, preferably 1 to 20, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0153] Sequence ID 35: EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK

[0154] Preferably, the adalimumab heavy chain fragment AdaFd has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, and most preferably at least 99% homology with the amino acid sequence shown in SEQ ID NO: 35.

[0155] In one preferred embodiment, the antibody light chain is derived from the bevacizumab light chain BevLC or a fragment thereof, and the antibody heavy chain is derived from the bevacizumab heavy chain BevHC or a fragment BevFd thereof.

[0156] Furthermore, the bevacizumab light chain BevLC comprises the amino acid sequence shown in SEQ ID NO: 36, or a sequence having one or more amino acid substitutions, deletions, or additions to the polypeptide of the amino acid sequence shown in SEQ ID NO: 36, wherein the number of substituted, deleted, or added amino acids is 1 to 50, preferably 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0157] Sequence ID 36: DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0158] Preferably, the bevacizumab light chain BevLC has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, and most preferably at least 99% homology to the amino acid sequence shown in SEQ ID NO: 36.

[0159] Furthermore, the bevacizumab heavy chain BevHC comprises the amino acid sequence described in SEQ ID NO: 37, or a sequence having one or more amino acid substitutions, deletions, or additions to the polypeptide shown in SEQ ID NO: 37, wherein the number of substituted, deleted, or added amino acids may be 1 to 100, preferably 1 to 20, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0160] Sequence ID 37: EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKTGGAGTGSGA

[0161] Preferably, the bevacizumab heavy chain BevHC has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, and most preferably at least 99% homology to the amino acid sequence shown in SEQ ID NO: 37.

[0162] Furthermore, the bevacizumab heavy chain fragment BevFd comprises the amino acid sequence described in SEQ ID NO: 38, or a sequence having one or more amino acid substitutions, deletions, or additions to the polypeptide shown in SEQ ID NO: 38, wherein the number of substituted, deleted, or added amino acids may be 1 to 100, preferably 1 to 20, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0163] Sequence ID 38: EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWG QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK

[0164] Preferably, the bevacizumab heavy chain fragment BevFd has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, and most preferably at least 99% homology with the amino acid sequence shown in SEQ ID NO: 38.

[0165] In one preferred embodiment, the antibody light chain is derived from the daratumumab light chain DarLC or a fragment thereof, and the antibody heavy chain is derived from the daratumumab heavy chain DarHC or a fragment thereof DarFd.

[0166] Furthermore, the daratumumab light chain DarLC comprises the amino acid sequence shown in SEQ ID NO: 39, or a sequence having one or more amino acid substitutions, deletions, or additions to the polypeptide of the amino acid sequence shown in SEQ ID NO: 39, wherein the number of substituted, deleted, or added amino acids is 1 to 50, preferably 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0167] Sequence ID 39: EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0168] Preferably, the daratumumab light chain DarLC has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, and most preferably at least 99% homology to the amino acid sequence shown in SEQ ID NO: 39.

[0169] Furthermore, the daratumumab heavy chain DarHC comprises the amino acid sequence described in SEQ ID NO: 40, or a sequence having one or more amino acid substitutions, deletions, or additions to polypeptide 1 shown in SEQ ID NO: 40, wherein the number of substituted, deleted, or added amino acids may be 1 to 100, preferably 1 to 20, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0170] Sequence ID 40: EVQLLESGGGLVQPGGSLRLSCAVSGFTFNSFAMSWVRQAPGKGLEWVSAISGSGGGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCAKDKILWFGEPVFDYWG QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0171] Preferably, the daratumumab heavy chain DarHC has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, and most preferably at least 99% homology to the amino acid sequence shown in SEQ ID NO: 40. Furthermore, the daratumumab heavy chain fragment DarFd comprises the amino acid sequence described in SEQ ID NO: 41, or a sequence having one or more amino acid substitutions, deletions, or additions to polypeptide 1 shown in SEQ ID NO: 41, wherein the number of substituted, deleted, or added amino acids may be 1 to 100, preferably 1 to 20, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0172] Sequence ID 41: EVQLLESGGGLVQPGGSLRLSCAVSGFTFNSFAMSWVRQAPGKGLEWVSAISGSGGGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCAKDKILWFGEPVFDYWG QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK

[0173] Preferably, the daratumumab heavy chain fragment DarFd has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, and most preferably at least 99% homology with the amino acid sequence shown in SEQ ID NO: 41.

[0174] In one preferred embodiment, the antibody light chain is derived from the omalizumab light chain OmaLC or a fragment thereof, and the antibody heavy chain is derived from the omalizumab heavy chain OmaHC or a fragment OmaFd thereof.

[0175] Furthermore, the omalizumab light chain OmaLC comprises the amino acid sequence shown in SEQ ID NO: 42, or a sequence having one or more amino acid substitutions, deletions, or additions to the polypeptide of the amino acid sequence shown in SEQ ID NO: 42, wherein the number of substituted, deleted, or added amino acids is 1 to 50, preferably 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0176] Sequence ID 42: DIQLTQSPSSLSASVGDRVTITCRASQSVDYDGDSYMNWYQQKPGKAPKLLIYAASYLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSHEDPYTFGQGTK VEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR

[0177] Preferably, the omalizumab light chain OmaLC has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, and most preferably at least 99% homology to the amino acid sequence shown in SEQ ID NO: 42.

[0178] Furthermore, the omalizumab heavy chain OmaHC comprises a sequence having one or more amino acid substitutions, deletions, or additions to the amino acid sequence described in SEQ ID NO: 43, or to the polypeptide shown in SEQ ID NO: 43, wherein the number of substituted, deleted, or added amino acids may be 1 to 100, preferably 1 to 20, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0179] Sequence ID 43: EVQLVESGGGLVQPGGSLRLSCAVSGYSITSGYSWNWIRQAPGKGLEWVASITYDGSTNYADSVKGRFTISRDDSKNTFYLQMNSLRAEDTAVYYCARGSHYFGHWHFAVW GQGTLVTVSSGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKAEPKSCDKT HTCPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0180] Preferably, the omalizumab heavy chain OmaHC has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, and most preferably at least 99% homology to the amino acid sequence shown in SEQ ID NO: 43.

[0181] Furthermore, the omalizumab heavy chain fragment OmaFd comprises the amino acid sequence shown in SEQ ID NO: 44, or a sequence having one or more amino acid substitutions, deletions, or additions to the polypeptide shown in SEQ ID NO: 44, wherein the number of substituted, deleted, or added amino acids may be 1 to 100, preferably 1 to 20, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0182] Sequence ID 44: EVQLVESGGGLVQPGGSLRLSCAVSGYSITSGYSWNWIRQAPGKGLEWVASITYDGSTNYADSVKGRFTISRDDSKNTFYLQMNSLRAEDTAVYYCARGSHYFGHWHFAVW GQGTLVTVSSGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKAEPKSCDK

[0183] Preferably, the omalizumab heavy chain fragment OmaFd has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, and most preferably at least 99% homology to the amino acid sequence shown in SEQ ID NO: 44.

[0184] In one preferred embodiment, the antibody light chain is derived from the cetuximab light chain CetLC or a fragment thereof, and the antibody heavy chain is derived from the cetuximab heavy chain CetHC or a fragment CetFd thereof.

[0185] Furthermore, the cetuximab light chain CetLC comprises the amino acid sequence shown in SEQ ID NO: 45, or a sequence having one or more amino acid substitutions, deletions, or additions to the polypeptide of the amino acid sequence shown in SEQ ID NO: 45, wherein the number of substituted, deleted, or added amino acids is 1 to 50, preferably 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0186] Sequence ID 45: DVQVTQSPSSLSASVGDRVTITCRSSQSLANSYGNTFLSWYLHKPGKAPQLLIYGISNRFSGVPDRFSGSGSGTDFTLTISSLQPEDFATYYCLQGTHQPYTFGQGTKV EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0187] Preferably, the cetuximab light chain CetLC has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, and most preferably at least 99% homology with the amino acid sequence shown in SEQ ID NO: 45.

[0188] Furthermore, the cetuximab heavy chain CetHC comprises a sequence having one or more amino acid substitutions, deletions, or additions to the amino acid sequence described in SEQ ID NO: 46, or to the polypeptide shown in SEQ ID NO: 46, wherein the number of substituted, deleted, or added amino acids may be 1 to 100, preferably 1 to 20, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0189] Sequence ID 46: EVQLVQSGAEVKKPGASVKVSCKASGYRFTNYWIHWVRQAPGQGLEWIGGINPGNNYATYRRKFQGRVTMTADTSTSTVYMELSSLRSEDTAVYYCTREGYGNYGAWFAYWG QGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYG PPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKT ISKAKGQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0190] Preferably, the cetuximab heavy chain CetHC has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, and most preferably at least 99% homology to the amino acid sequence shown in SEQ ID NO: 46.

[0191] Furthermore, the cetuximab heavy chain fragment CetFd comprises the amino acid sequence described in SEQ ID NO: 47, or a sequence having one or more amino acid substitutions, deletions, or additions to the polypeptide shown in SEQ ID NO: 47, wherein the number of substituted, deleted, or added amino acids may be 1 to 100, preferably 1 to 20, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0192] Sequence ID 47: EVQLVQSGAEVKKPGASVKVSCKASGYRFTNYWIHWVRQAPGQGLEWIGGINPGNNYATYRRKFQGRVTMTADTSTSTVYMELSSLRSEDTAVYYCTREGYGNYGAWFAY WGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVES

[0193] Preferably, the cetuximab heavy chain fragment CetFd has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, and most preferably at least 99% homology to the amino acid sequence shown in SEQ ID NO: 47.

[0194] In one preferred embodiment, the antibody light chain is derived from a denosumab light chain DenLC or a fragment thereof, and the antibody heavy chain is derived from a denosumab heavy chain DenHC or a fragment thereof.

[0195] Furthermore, the denosumab light chain DenLC comprises the amino acid sequence shown in SEQ ID NO: 48, or a sequence having one or more amino acid substitutions, deletions, or additions to the polypeptide of the amino acid sequence shown in SEQ ID NO: 48, wherein the number of substituted, deleted, or added amino acids is 1 to 50, preferably 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0196] Sequence ID 48: EIVLTQSPGTLSLSPGERATLSCRASQSVRGRYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVFYCQQYGSSPRTFGQGTKVEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0197] Preferably, the denosumab light chain DenLC has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, and most preferably at least 99% homology with the amino acid sequence shown in SEQ ID NO: 48.

[0198] Furthermore, the denosumab heavy chain DenHC comprises the amino acid sequence described in SEQ ID NO: 49, or a sequence having one or more amino acid substitutions, deletions, or additions to the polypeptide shown in SEQ ID NO: 49, wherein the number of substituted, deleted, or added amino acids may be 1 to 100, preferably 1 to 20, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0199] Sequence ID 49: EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSGITGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDPGTTVIMSWFDPWG QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0200] Preferably, the denosumab heavy chain DenHC has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, and most preferably at least 99% homology to the amino acid sequence shown in SEQ ID NO: 49.

[0201] Furthermore, the denosumab heavy chain fragment DenFd comprises the amino acid sequence shown in SEQ ID NO: 50, or a sequence having one or more amino acid substitutions, deletions, or additions to the polypeptide shown in SEQ ID NO: 50, wherein the number of substituted, deleted, or added amino acids may be 1 to 100, preferably 1 to 20, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0202] Sequence ID 50: EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSGITGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDPGTTVIMSWFDPWG QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK

[0203] Preferably, the denosumab heavy chain fragment DenFd has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, and most preferably at least 99% homology to the amino acid sequence shown in SEQ ID NO: 50.

[0204] Furthermore, the single-chain antibody is used to improve the expression efficiency and expression level of the single-chain antibody by ligating a signal peptide SP to the N-terminus of the light chain or light chain fragment.

[0205] Preferably, SP includes the amino acid sequence shown in SEQ ID NO: 1, or a sequence having one or more amino acid substitutions, deletions, or additions to the sequence shown in SEQ ID NO: 1, wherein the number of substituted, deleted, or added amino acids is 1 to 10, preferably 1 to 5.

[0206] Sequence ID 1: MITETSSPFRSIFSHSGK

[0207] Furthermore, a fluorescent protein tag is ligated to the C-terminus of the heavy chain or heavy chain fragment of the single-chain antibody, and more preferably, the fluorescent protein tag is eGFP or an improved protein tag based on eGFP, such as mTagBFP2, moxCerulean3, AmCyanl, MiCy, ZsGreen, Clover, mVenus, ZsYellow 1, mKO2, TurboRFP, tdTomato, eqFP611, mKate1.3, mNeptune2, miRFP670, mAmetrine, PAmCherry 2, mEos3.2, etc.

[0208] Furthermore, the eGFP includes the amino acid sequence shown in SEQ ID NO: 8, or a sequence having one or more amino acid substitutions, deletions, or additions to the amino acid sequence shown in SEQ ID NO: 8, wherein the number of substituted, deleted, or added amino acids may be 1 to 20, preferably 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0209] Sequence ID 8: VSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPW PTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPYVQERTISFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYK

[0210] Furthermore, a tag is ligated to the N-terminus of the light chain or light chain fragment of the single-chain antibody, preferably the tag added to the N-terminus of the light chain or light chain fragment is called a His tag, and the number of histidine residues in the His tag is 3 to 15, preferably 5 to 10. The name of the His tag is determined based on the number of histidine residues. For example, if there are 8 histidine residues, it is named 8His, and if there are 10 histidine residues, it is named 10His.

[0211] Furthermore, preferably, the fluorescent protein tag, tag tag, and signal peptide are linked to the light chain, light chain fragment, or heavy chain, and heavy chain fragment of the antibody via a flexible linker peptide, and preferably, the flexible linker peptide contains 1 to 20 amino acid residues.

[0212] Examples of flexible linker peptides may include 5L (SEQ ID NO: 3: GSGGS) and 10L (SEQ ID NO: 7: TGGAGTGSGA).

[0213] In one preferred embodiment, the single-chain antibody is TrascFab and comprises the structure of formula (I): TraLC-linker-TraFd (I)

[0214] TraLC is a trastuzumab light chain TraLC, which is a sequence having one or more amino acid substitutions, deletions, or additions to the amino acid sequence shown in SEQ ID NO: 4, or the polypeptide shown in SEQ ID NO: 4, and having at least 60%, preferably at least 65%, more preferably at least 70%, even more preferably at least 75%, even more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, and most preferably at least 99% homology, and preferably the trastuzumab light chain TraLC has at least 60%, preferably at least 65%, more preferably at least 70%, even more preferably at least 75%, even more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, and most preferably at least 99% homology to the amino acid sequence shown in SEQ ID NO: 4.

[0215] TraFd refers to the trastuzumab heavy chain fragment TraFd, which is the amino acid sequence shown in SEQ ID NO: 5, or a sequence having one or more amino acid substitutions, deletions, or additions to the amino acid sequence shown in SEQ ID NO: 5, the number of such substitutions, deletions, or additions being 1 to 20, preferably 1 to 10, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and preferably the TraFd has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, and most preferably at least 99% homology with the sequence shown in SEQ ID NO: 5; The linker is a sequence that has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, and most preferably at least 99% homology with linker34, linker60, linker82, linker120, or any of the sequences described above.

[0216] In a preferred embodiment, the single-chain antibody is scIgG and comprises the structure shown in formula (II): TraLC-linker-TraHC

[0217] TraHC is a trastuzumab heavy chain, which is a sequence having one or more amino acid substitutions, deletions, or additions to the amino acid sequence shown in SEQ ID NO: 6, or the polypeptide shown in SEQ ID NO: 6, wherein the number of substituted, deleted, or added amino acids is 1 to 100, preferably 1 to 20, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and preferably, the TraHC has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, and most preferably at least 99% homology to the sequence shown in SEQ ID NO: 6.

[0218] The present invention provides single-chain antibodies formed by the relinking expression of light and heavy chains via a linker peptide. These antibodies can be synthesized in a single step in an in vitro cell-free synthesis system. Compared to Fab, they do not require the separate secretion and expression of two different protein fragments, the light and heavy chains, making them less prone to homodimerization and resulting in higher expression efficiency and affinity activity than Fab.

[0219] A second aspect of the present invention provides one or more isolated nucleic acids encoding light chain antibodies provided in the first aspect.

[0220] The nucleic acid is a DNA sequence and / or an RNA sequence.

[0221] In one possible embodiment, the nucleic acid is linear DNA and a PCR linear fragment.

[0222] Furthermore, the PCR linear fragments can be obtained by amplification techniques. The amplification techniques are not particularly limited and include, but are not limited to, PCR amplification techniques, isothermal amplification techniques, room temperature amplification techniques, and room temperature amplification techniques. Here, isothermal amplification techniques are preferably room temperature amplification techniques. This includes, but is not limited to, commercial DNA amplification systems provided by Biocompare, Neta Scientific Inc., ABM, Thermo Fisher Scientific, Expedeon, Vivantis, and others.

[0223] In one feasible embodiment, the nucleic acid is RNA containing an amino acid sequence encoding a single-chain antibody.

[0224] Furthermore, the RNA is prepared using in vitro nucleic acid amplification techniques. These in vitro nucleic acid amplification techniques are not particularly limited and include, but are not limited to, polymerase chain reaction (PCR) amplification techniques, isothermal amplification techniques, room temperature amplification techniques, and room temperature amplification techniques. Specific examples include loop-mediated isothermal amplification (LAMP), strand-displacement amplification (SDA), nucleic acid sequence-based amplification (NASBA), rolling circle amplification (RCA), nick enzyme nucleic acid isothermal amplification, helicase-dependent isothermal amplification (HDA), transcription-dependent amplification, hybrid capture, transcription-mediated amplification (TMA), recombinase-assisted amplification (RAA), and recombinase polymerase amplification (RPA).

[0225] A third aspect of the present invention provides one or more expression vectors comprising nucleic acids encoding single-chain antibodies provided in the first aspect.

[0226] The expression vectors include, but are not limited to, eukaryotic plasmid vectors, eukaryotic virus vectors, prokaryotic plasmids, specific vectors, shuttle vectors, minichromosomes, and various other vectors.

[0227] Preferably, the expression vector is a eukaryotic plasmid expression vector or a prokaryotic plasmid expression vector.

[0228] In one preferred embodiment, the expression vector is circular DNA, and more preferably plasmid DNA, such as a pET plasmid or a pGEM plasmid.

[0229] A fourth aspect of the present invention provides a host cell characterized by comprising a vector according to the third aspect of the present invention, or having polynucleotides according to the second aspect incorporated into its genome, or expressing a recombinant protein as shown in the first aspect.

[0230] In one preferred embodiment, the host cell is selected from either a prokaryotic host cell or a eukaryotic host cell.

[0231] In another preferred embodiment, the host cell is a prokaryotic cell such as Escherichia coli.

[0232] In another preferred embodiment, the host cells are eukaryotic cells, such as yeast, progenitor cells, Chinese hamster ovary cells, insect cells, wheat germ cells, and rabbit reticulocytes.

[0233] Preferably, the yeast is a combination of one or more species selected from Saccharomyces cerevisiae and Kluyveromyces yeasts. In another preferred embodiment, the Kluyveromyces yeast is selected from a combination of one or more species selected from Kluyveromyces lactis, Kluyveromyces marxianus, and Kluyveromyces dobzhanskii.

[0234] A fifth aspect of the present invention provides an in vitro cell-free protein synthesis system comprising a nucleic acid molecule provided in the second aspect, or an expression vector provided in the third aspect, or a lysate or extract of a host cell described in the fourth aspect of the present invention.

[0235] Furthermore, the extracellular protein synthesis system includes, but is not limited to, an Escherichia coli extracellular protein synthesis system, a bacterial extracellular protein synthesis system, a mammalian extracellular protein synthesis system (e.g., HF9, Hela, CHO, HEK293), a plant extracellular protein synthesis system, a yeast extracellular protein synthesis system, and an insect extracellular protein synthesis system. Preferably, it is a yeast extracellular protein synthesis system, more preferably a Kluyveromyces extracellular protein synthesis system, and most preferably a Kluyveromyces lactis (K. lactis) or Kluyveromyces marxianus yeast extracellular protein synthesis system.

[0236] Furthermore, the extracorporeal protein synthesis system provided by the present invention includes, but is not limited to, cell extracts, trihydroxymethylaminomethane, potassium acetate, magnesium acetate, nucleotide triphosphate mixtures (NTPs), amino acid mixtures, and the like.

[0237] The aforementioned cell extracts are typically used to provide substances such as ribosomes, transfer RNA (tRNA), aminoacyl-tRNA synthetase, initiation and elongation factors necessary for protein synthesis, and termination and release factors. Furthermore, through strain modification, it is also possible to provide other substances such as polymerases (RNA polymerase and / or DNA polymerase) endogenously.

[0238] The protein components required for the extracorporeal cell-free protein synthesis system (e.g., RNA polymerase) can be supplied endogenously or added exogenously. For endogenous supply, gene modification methods described in existing literature such as CN108690139A, CN109423496A, CN106978439A, CN110408635A, CN110551700A, CN110093284A, CN110845622A, CN110938649A, CN111378708A, CN111484998 A, and "Molecular and Cellular Biology, 1990, 10(1):353-360" and the literature they cite can be referenced. Specifically, these methods include, but are not limited to, inserting coding sequences into intracellular free plasmids, integrating coding genes into the cellular genome, and combinations thereof. When supplied using an external source, usage can be controlled and adjusted according to the system's needs.

[0239] In some preferred examples, the yeast cell extract is a Kluyveromyces cell extract. In one preferred example, the Kluyveromyces is Kluyveromyces lactis (K. lactis).

[0240] The technical elements of the present invention, such as the in vitro protein synthesis system, template, plasmid, target protein, in vitro protein synthesis reaction (incubation reaction), various preparation methods, and various detection methods, are as follows: CN111484998A, CN106978349A, CN108535489A, CN108690139A, CN108949801 A, CN108642076A, CN109022478A, CN109423496A, CN109423497A, CN109423509A, CN109837293A, CN109971783A, CN109988801A, Appropriate embodiments or methods of implementation can be independently selected from documents such as CN109971775A, CN110093284A, CN110408635A, CN110408636A, CN110551745A, CN110551700A, CN110551785A, CN110819647A, CN110845622A, CN110938649A, and CN110964736A. Unless they conflict with the objectives of the present invention, these documents and the documents they refer to are cited in their entirety and objectives.

[0241] A sixth aspect of the present invention provides a use of the single-chain antibody of the present invention for the diagnosis, prevention, and treatment of diseases, or for the preparation of formulations for the diagnosis, prevention, and treatment of diseases.

[0242] Furthermore, the aforementioned diseases are tumors and / or cancers. Exemplary tumors and / or cancers include, but are not limited to, lung cancer, breast cancer, esophageal cancer, stomach cancer, cervical cancer, colon cancer, rhabdomyosarcoma, liposarcoma, osteosarcoma, lymphoma, leukemia, liver cancer, cervical cancer, ovarian cancer, and breast cancer.

[0243] In one preferred embodiment, the disease is a disease caused by abnormalities in HER2, TNF-α, VEGF, PD-1, IL-4, IL-13, or CD38. [Examples]

[0244] The present invention will be described in further detail below with reference to specific examples and drawings. It should be understood that these examples are presented for illustrative purposes only and do not limit the scope of the present invention.

[0245] In the following examples, experimental methods where specific conditions are not explicitly stated are generally carried out under conventional conditions, such as those described in literature such as "Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989)" or "Cell-free protein synthesis: methods and protocols[M]. 2008" (Edited by Alexander S. Spirin and James R. Swartz), or those recommended by the manufacturer, or those based on the specific implementation methods described above. Unless otherwise specified, percentages and parts referred to in this invention refer to weight percentages and weight parts.

[0246] First, single-chain antibody experiments based on the construction of trastuzumab.

[0247] To verify the present invention, the following plasmids were synthesized using an in vitro synthesis system: TrascFab0, TrascFab6, TrascFab15, TrascFab25, TrascFab30, TrascFab34, TrascFab60, TrascFab82, TrascFab120, TrascFab180, TrascFab34A, TrascFab34B, TrascFab34C, TrascFab60A, TrascFab36A, TrascFab36B, TrascFab36BHL, TrascIgG60A, and TrascIgG60B. The specific plasmid structures are shown in Table 1.

[0248] [Table 1]

[0249] Example 1: Plasmid Construction 1. Gene synthesis Here, TrascFab34, TrascFab60, TrascFab120, and TrascIgG60B were synthesized by gene synthesis. TrascFab0, TrascFab6, TrascFab15, TrascFab25, TrascFab30, TrascFab180, TrascFab34A, TrascFab34B, TrascFab34C, TrascFab60A, TrascFab36A, TrascFab36B, TrascFab36B, and TrascFab82 were obtained from TrascFab60 by PCR construction using primers. The primer sequences are shown in Table 2 below.

[0250] [Table 2]

[0251] 2. Plasmid construction PCR was performed using TrascFab60 as a template and corresponding primers. DpnI was added to the PCR product, and after enzymatic treatment in a 37°C incubator for at least 4 hours, transformation (DH5α) was performed. Finally, a single colony was collected, and the correct sequence was identified by sequencing validation. The plasmid structure is shown in Figure 1.

[0252] Example 2: Conversion of the TrascFab60 plasmid 1 μl of the target plasmid was added to 20 μl of DH5α, left to stand on ice for 30 minutes, then subjected to heat shock treatment at 42°C for 45 seconds, cooled on ice for 2 minutes, 500 μl of medium was added, and cultured with shaking at 200 rpm at 37°C for 1 hour. 100 μl was spread onto LB (antibiotic-containing) medium, incubated upside down at 37°C for 12-16 hours, then removed and stored in a refrigerator at 4°C.

[0253] Example 3: Extraction of TrascFab60 plasmid (using a 10 ml bacterial solution as an example) (1) Cell collection: The bacterial suspension cultured for 12-16 hours was centrifuged and then centrifuged again at 4°C, 4000 rpm for 10 minutes. The supernatant was discarded, and after a short centrifugation, the remaining liquid on the surface of the precipitate was removed with a pipette. (2) Add 400 μl of P1 to the precipitate and vortex mix for at least 2 minutes to thoroughly disperse the bacterial cell precipitate in P1. (3) Add 500 μl of P2 to each and gently invert the 2 ml EP tube eight times to mix. (4) 700 μl of P3 was added to each sample. The 2 ml EP tube was gently inverted and mixed eight times. (5) Centrifuge at 4°C and 12000 rpm for 15 minutes and remove the precipitate that formed. (6) The supernatant was mixed with 100 μl of pre-suspended magnetic beads and incubated at room temperature for 5 minutes, inverting the EP tube at 1-minute intervals to ensure that the magnetic beads and supernatant were thoroughly mixed. (7) Magnetic beads were attracted using a magnetic stand, and the supernatant was removed. (Method: The magnetic attraction time was approximately 5-10 seconds. After confirming that the supernatant was pale yellow, the supernatant was immediately discarded, and the stand was promptly removed. Care should be taken to avoid excessive adhesion of the magnetic beads, which can make dispersion difficult in the next step. Since the plasmid structure is susceptible to damage from physical force, vortex treatment in the washing step should be avoided as much as possible, and the integrity of the plasmid structure affects IVTT activity.) (8) The magnetic beads were washed with 1 ml of PR. Since the magnetic beads were not tightly bonded in the previous step, they could be dispersed by shaking by hand for 1 minute. The supernatant was removed using a magnetic stand, in the same manner as described above. (9) The magnetic beads were washed with 1.2 ml of W. Since the magnetic beads were not tightly bonded in the previous step, they could be dispersed by shaking by hand for 2 minutes. The supernatant was removed using a magnetic stand, in the same manner as described above. (10) Repeat step 9. (11) The EP tube was placed on a magnetic stand, and the beads were dried with a blower (minimum output). After about 12 minutes, the completely dried magnetic beads were cracked, and there were no visible traces of moisture on the surface in contact with the EP tube. (12) The dried magnetic beads were lightly scraped off with the tip of the pipette and collected at the bottom of the EP tube, and 400 μl of eluent was added. Vortexed for 15 seconds, and the EP tube was gently shaken at 5-minute intervals to promote sufficient elution. The supernatant was collected using a magnetic stand. (13) The residual magnetic beads were removed by centrifuging at 4°C, 12000 rpm for 5 minutes. (14) The supernatant was collected using a pipette and centrifuged at 4°C and 12000 rpm for 10 minutes to remove residual magnetic beads. (15) The supernatant was collected and the plasmid concentration was measured. (16) Agarose gel (1%) electrophoresis was performed and plasmid morphology was observed. The results are shown in Figure 2.

[0254] Example 4: Preparation of AMPI product using TrascFab60 (10 ml scale) (1) Prepare 10 ml of AMPI buffer (the AMPI buffer used was the commercially available product AMPiX (10x DNA Amplifier) ​​from Kangma (Shanghai) Biotechnology Co., Ltd., product model number PROTN_AMPiN10V03500). (2) A fixed amount of plasmid was added to achieve a final concentration of 4 ng / μl. (3) 5 μl of AMPI enzyme was added. (4) After thorough mixing, the mixture was reacted at 37°C and 30 rpm for 2 hours, and the product mixture was analyzed and identified by gel electrophoresis (1% agar gel). Using TrascFab60 as an example, the protein purification results are shown in Figure 3.

[0255] Example 5: TrascFab60 protein expression (250 mL scale) 8.3 ml of the AMPI product was added to 250 ml of fast (the volume of the AMPI product used was 1 / 30 of that of fast), and the reaction was carried out at 100 rpm for 3 - 6 hours in a constant temperature bath at 30 ± 2 °C. (fast is an in vitro cell-free synthesis system product commercially available from Yuankang Code (Shanghai) Biotechnology Co., Ltd., product model number: profac_fast0510000, CoPure: FAST5 + His-Monster Beads + General Magnetic Rack) After the reaction, 10 μL of the reaction solution was added to a 384-well black plate, and each sample was carried out in 3 replicates, immediately placed on a Tecan infinite F200, and measured with an excitation wavelength of 488 nm, a fluorescence wavelength of 507 nm, and the manual parameter of gain set to 32. The measured values were read, the expression activity was detected, and the relative light unit value (Relative Light Unit, RLU) was adopted as the activity unit. As a result of the measurement, the RFU of TrascFab60 was 1279.

[0256] Example 6 Purification of TrascFab60 Protein (1) Determination of the amount of magnetic beads used: The amount of magnetic beads used was 1% of the volume of fast used. (2) Washing of magnetic beads: Before use, they were washed 3 times with water, vortexed for 15 seconds each time, and the magnetic beads were collected with a magnetic stand. The amount of water used each time was 5 - 10 times the amount of magnetic beads used. (3) Binding of the target protein to magnetic beads: The washed beads and the IVTT product were mixed in a large beaker, and after a 1-hour binding reaction under the conditions of 4 °C and 100 rpm, the magnetic beads were collected in a centrifuge tube. (4) Washing of impurities: The magnetic beads bound with the protein with 10 mM imidazole were washed 3 times at 180 rpm for 10 minutes each time. The amount of the washing solution used was 3 - 5 times the volume of the magnetic beads. (5) Elution was carried out with a 250 mM imidazole eluent. The amount of the first eluent used was 2 times the amount of magnetic beads used, and the amount of the second eluent used was the same as the amount of magnetic beads used. (6) The residual magnetic beads were removed by centrifugation, initially at 12,000 rpm for 5 minutes. The second time was at 12,000 rpm for 10 minutes, and SDS-PAGE gel purification was performed. The results are shown in Figure 4.

[0257] Example 7 The experimental methods of Examples 1 to 6 were repeated, and TrascFab0, TrascFab6, TrascFab15, TrascFab25, TrascFab30, TrascFab180, TrascFab34A, TrascFab34B, TrascFab34C, TrascFab60A, TrascFab36A, TrascFab36B, TrascFab36B, TrascFab34, TrascFab82, TrascFab120, TrascFab180, TraFabHL, Tra2Fab, Tra3Fab, TrascIgG60A and TrascIgG60A proteins were prepared, respectively. Figure 2 is an electrophoretic analysis diagram of the plasmids prepared in Examples 2 and 7 of the present invention. In the figure, M indicates a marker, and 1 to 7 indicate TrascFab0, TrascFab6, TrascFab34, TrascFab60, TrascFab82, TrascFab120, and TrascIgG60 plasmids, respectively. Figure 3 is a purification analysis diagram of the AMPI products of Examples 2 and 7 of the present invention. In the figure, M indicates a marker, and 1 to 7 indicate the AMPI gel electrophoresis diagrams of TrascFab0, TrascFab6, TrascFab34, TrascFab60, TrascFab82, TrascFab120, and scIgG60, respectively.

[0258] After the reaction was completed, the expression activity was detected, and the relative light unit value (Relative Light Unit, RLU) was measured as the activity unit. As a result, the RFU detection results are as shown in Table 3.

[0259]

Table 3

[0260] The results confirmed that the single-chain antibody of the present invention is efficiently expressed even in an in vitro cell-free expression system.

[0261] Example 8 Detection of the binding activity of TrascFab or scIgG to ErbB2 antigen by ELISA method 1. Establishment of an ELISA detection method 1. The trastuzumab-specific antigen was diluted to 0.25 μg / ml with the coating solution, added to an enzyme-labeled plate at a rate of 100 μl / well, and left to stand overnight at 4°C. 2. The plate was washed three times with PBST at 300 μl / wash, 200 μl of blocking solution was added, and the plate was blocked at 37°C for 1 hour. 3. Wash the plate three times with 300 μl / wash using PBST. Dilute the sample and commercial monoclonal antibody to 20 μg / ml with diluent (PBST, containing 1% BSA), then perform 11 three-fold serial dilutions. Add 100 μl / well to each microplate, simultaneously add the diluent as a blank control, and incubate at 37°C for 1 hour. 4. The microplate was washed three times with 300 μl / wash using PBST, 100 μl / well of HRP-labeled rabbit anti-human IgG (Fab-specific) (1:5000 dilution) was added, and the plate was incubated at 37°C for 1 hour. 5. After washing the plate three times, 100 μl / well of TMB chromogenic solution was added, and the plate was allowed to develop color at 37°C for 10 minutes. Then, 50 μl / well of stop solution (2M sulfuric acid) was added, and the absorbance at a wavelength of 450 nm was measured using a microplate reader. 6. Using the prism software, create a calibration curve with 4 parameters, plotting the sample concentration on the x-axis and the A450 value on the y-axis, and the EC 50 The value was calculated.

[0262] 2. Detection Results 1. Using the ELISA detection method established based on this embodiment, the binding activity of purified TrascFab0, TrascFab6, TrascFab15, TrascFab25, TrascFab30, TrascFab34, TrascFab60, TrascFab82, TrascFab120, and TrascFab180 proteins to the ErbB2 antigen was detected, and calibration curves were created for four parameters. The results are shown in Figure 5.

[0263] With sample concentration on the x-axis and A450 on the y-axis, EC 50 The values ​​were calculated and are shown in Table 4: [Table 4]

[0264] Experimental results showed that the activity of the single-chain antibodies of the present invention is closely related to the linker length. When the number of amino acid residues in the linker was less than 25, the activity of the corresponding single-chain antibody was significantly lower than that of single-chain antibodies with more than 25 amino acid residues in the linker. In particular, when the number of amino acid residues in the linker of the single-chain antibody was 82, the activity of the corresponding single-chain antibody was more than three times higher than that of TrascFab0 and TrascFab6, and reached up to more than nine times higher.

[0265] 2. Activity measurement of purified TrascFab proteins of different lengths and types Using the ELISA assay method established in this example, the binding activity of purified proteins of TrascFab34A, TrascFab34B, TrascFab34C, TrascFab60A, TrascFab36A, TrascFab36B, TrascFab36BHL, TrascIgG60A, and TrascIgG60B obtained in Example 7, as well as the binding activity of the commercially available monoclonal antibody Trastuzumab to the ErbB2 antigen, were measured. A calibration curve was created using four parameters, with sample concentration on the x-axis and A450 on the y-axis, and EC 50 The values ​​were calculated as shown in Table 5:

[0266]

Table 5

[0267] The experimental results show that the activities of TrascFab34A, TrascFab34B, and TrascFab34C are significantly lower compared to TrascFab34, suggesting that the activity of the single-chain antibody is closely related not only to the length of the linker but also to the amino acid residue composition of the linker. When the linker is mainly composed of amino acid residues such as G, S, and E, the activity of the corresponding single-chain antibody is clearly higher than that of the single-chain antibody corresponding to a linker containing amino acid residues such as R, Y, E, I, and K. The affinity activity of TrascFab60A and TrascFab60 also supports this point.

[0268] In addition, the affinity activities of TrascFab36A, TrascFab36B, and TrascFab36C were also tested, but these showed satisfactory results. This demonstrates that even when the linker mainly contains amino acid residues such as G, S, E, and A and contains a small amount of other amino acid residues such as V, P, L, R, Y, and N, it has little effect on the affinity activity of the single-chain antibody.

[0269] TrascFabHL has almost the same activity as TrascFab36C, confirming that the influence of the linking order of LC and Fd on the activity of the single-chain antibody is small. That is, in the single-chain antibody of the present invention, any of the different linking orders of the light chain and the heavy chain can realize the present invention.

[0270] The affinity activities of Tra3Fab and Tra2Fab are also satisfactory. It has been shown that the present invention can also be achieved by tandem expression of Scfab, which is also included in the protection scope of the present invention.

[0271] Furthermore, when single-chain antibodies TrascIgG60A and TrascIgG60B, similar to the full-length antibody, were prototyped, their activity was significantly improved, demonstrating that TrascIgG60A and TrascIgG60B obtained by the present invention have affinity activity equivalent to commercially available trastuzumab antibodies. This confirms that the present invention is also applicable to single-chain antibodies prepared by linking full-length heavy and light chains.

[0272] The experimental results showed that TrascIgG60A and TrascIgG60B exhibited high binding activity to the ErbB2 antigen, reaching activity equivalent to that of the commercially available monoclonal antibody Trastuzumab. Furthermore, the single-chain antibody possessed unparalleled ease of expression compared to the commercially available monoclonal antibody Trastuzumab (natural double-chain structure). Therefore, the single-chain antibody provided by the present invention overcomes the drawbacks of conventional antibodies, such as low expression efficiency and limitations on their commercial application, in ensuring antibody activity. The single-chain antibody provided by the present invention simultaneously possesses high expression efficiency, high affinity, and high stability, and can be mass-produced by combining it with an in vitro cell-free protein synthesis system, giving it excellent potential for industrialization and commercialization.

[0273] It should be noted that although the single-chain antibody of the Fab fragment did not reach the same activity as the commercially available monoclonal antibody Trastuzumab, this is because it is natural that the antibody activity of the commercially available full-length antibody would exceed that of Fab. For example, when using the same linker60, the antibody activity of Fab (TrascFab60) was 0.49, while the activity of the full-length antibodies (TrascIgG60A and TrascIgG60B) was close to that of the commercially available monoantibody Trastuzumab (0.055). This indicates that using linker60 to link the light and heavy chains is rational for both the linking of full-length antibodies and the linking of Fab.

[0274] Therefore, if the antibody activity of Fab fragment antibodies is relatively similar, their full-length antibody activity will also be relatively similar. For example, in Table 4, if the antibody activity is close to or less than the result for (TrascFab60)linker60, it can be seen that their full-length antibody activity is at least comparable to the activity of the commercially available full-length monoclonal antibody Trastuzumab.

[0275] Second, experiments with single-chain antibodies constructed based on other types of antibodies.

[0276] To verify the present invention, the present invention synthesized TisIgG60 and TisscFab60 single-chain antibodies designed based on Tislelizumab using an in vitro synthesis system, and prepared and synthesized the following single-chain antibodies based on Dupilumab: DupIgG60 and DupscFab60; Adalimumab: AdaIgG60 and AdascFab60; Bevacizumab: BevIgG60 and BevscFab60; Dartumumab: DarIgG60 and DarscFab60; Omalizumab: OmaIgG60 and OmascFab60; Cetuximab: CetIgG60 and CetscFab60; and DenscFab36 single-chain antibody designed based on Denosumab. The specific plasmid structures are shown in Table 6:

[0277] [Table 6]

[0278] Example 9 The preparation methods of Examples 1 to 6 were repeated to prepare TisIgG60, TisscFab60, DupIgG60, DupscFab60, AdaIgG60, AdascFab60, BevIgG60, BevscFab60, DarIgG60, DarscFab60, OmaIgG60, OmascFab60, CetIgG60, and CetscFab60, respectively. After the reaction was complete, the expression activity and affinity of each reaction solution were detected.

[0279] Expression activity was measured using relative light units (RLU) as the activity unit. The detection results are shown in Table 7. [Table 7]

[0280] The results demonstrated that the single-chain antibody scheme constructed in this invention is applicable to various antibody types. Antibody types such as IgG1, IgG2, and / or IgG4 all conformed to the single-chain antibody scheme of this invention, all showed good expression activity, and were confirmed to be efficiently expressed even in an in vitro cell-free expression system.

[0281] Example 10 ELISA measurement of recombinant human PD-1 protein binding activity of TisIgG60 and TisscFab60 1. ELISA Test Method 1. Recombinant human PD-1 protein was diluted to 0.25 μg / ml with the coating solution, added to an enzyme-labeled plate at a rate of 100 μl / well, and left to stand overnight at 4°C. 2. The plate was washed three times with PBST at 300 μl / wash, 200 μl of blocking solution was added, and the plate was blocked at 37°C for 1 hour. 3. Wash the plate three times with 300 μl / wash using PBST. Dilute the sample and commercial monoclonal antibody to 20 μg / ml with diluent (PBST, containing 1% BSA), then perform 11 three-fold serial dilutions. Add 100 μl / well to each microplate, simultaneously add the diluent as a blank control, and incubate at 37°C for 1 hour. 4. The microplate was washed three times with 300 μl / wash using PBST, 100 μl / well of HRP-labeled rabbit anti-human IgG (Fab-specific) (1:5000 dilution) was added, and the plate was incubated at 37°C for 1 hour. 5. After washing the plate three times, 100 μl / well of TMB colorant was added, and the plate was allowed to develop color at 37°C for 10 minutes. After adding 50 μl / well of stop solution (2M sulfuric acid), the absorbance was measured at a wavelength of 450 nm using a microplate reader. 6. Using Prism software, create a calibration curve with 4 parameters, plotting the sample concentration on the x-axis and the A450 value on the y-axis, and then calculate the EC. 50 The value was calculated.

[0282] 2. Detection Results 1. Using the ELISA detection method established based on this embodiment, the binding activity of TisIgG60, TisscFab60, Tislelizumab, and ErbB2 antigen was detected, and a calibration curve was created using the four parameters. The sample concentration was plotted on the x-axis and A450 on the y-axis, and the EC 50 The values ​​were calculated and are shown in Table 8: [Table 8]

[0283] As the experimental results above demonstrate, the single-chain antibody design of the present invention is applicable to various types of antibodies. Antibodies of types such as IgG1, IgG2, and / or IgG4 can all be applied to the single-chain antibody construction design of the present invention, possessing appropriate affinity activity and unparalleled ease of expression. Therefore, the single-chain antibodies provided by the present invention overcome the drawbacks of conventional antibodies, such as low antibody expression efficiency and limitations on their commercial application, in ensuring antibody activity. The single-chain antibodies provided by the present invention simultaneously possess high expression efficiency, high affinity, and high stability, and can be mass-produced by combining them with an in vitro cell-free protein synthesis system, offering excellent prospects for industrialization and commercialization.

[0284] The sequences, their names, and sequence numbers mentioned herein are summarized in Table 9: [Table 9-1] [Table 9-2] [Table 9-3] [Table 9-4] [Table 9-5]

[0285] The preferred specific embodiments of the present invention have been described in detail above. It should be understood that a number of modifications and changes can be made by those skilled in the art based on the concept of the present invention without requiring any creative effort. Accordingly, all technical solutions that a person skilled in the art can obtain by logical analysis, reasoning, or limited experimentation based on the prior art in accordance with the concept of the present invention should be included within the scope of protection defined by the claims.

Claims

1. A single-chain antibody comprising a linker peptide that links the light chain or light chain fragment of the antibody to the heavy chain or heavy chain fragment of the antibody, wherein the linker peptide comprises at least 20 amino acid residues, preferably 25 to 180 amino acid residues, and more preferably 34 to 120 amino acid residues; Preferably, the antibody is a single-chain antibody, characterized in that it is selected from IgM, IgG, IgA, IgD, or IgE.

2. The amino acids contained in the linker peptide are selected from serine (S), glycine (G), glutamic acid (E), threonine (T), alanine (A), arginine (R), tyrosine (Y), isoleucine (I), lysine (K), leucine (L), glutamine (Q), histidine (H), phenylalanine (F), valine (V), proline (P), aspartic acid (D), methionine (M), and asparagine (N), and preferably the amino acids contained in the linker peptide are selected from serine (S), glycine (G), glutamic acid (E), threonine (T), and alanine (A); More preferably, the total number of serine (S), glycine (G), glutamic acid (E), threonine (T), and alanine (A) contained in the linker peptide is 100%, at least 99%, at least 98%, at least 97%, at least 96%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, and at least 50% of the total number of amino acids in the linker peptide; more preferably, the total number of serine (S), glycine (G), and glutamic acid (E) contained in the linker peptide is 100%, at least 99%, and at least The percentage of the total number of serine (S), glycine (G), and glutamic acid (E) contained in the linker peptide relative to the total number of amino acids in the linker peptide is 100%, at least 99%, at least 98%, at least 97%, at least 96%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, and at least 50%; More preferably, the linker peptide is one or more selected from linkser25 (SEQ ID NO: 11), linkser30 (SEQ ID NO: 14), linkser34 (SEQ ID NO: 15), linkser34A (SEQ ID NO: 16), linkser34B (SEQ ID NO: 17), linkser34C (SEQ ID NO: 18), linkser60 (SEQ ID NO: 19), linkser60A (SEQ ID NO: 20), linkser82 (SEQ ID NO: 21), linkser120 (SEQ ID NO: 22), linkser180 (SEQ ID NO: 23), linkser36A (SEQ ID NO: 24), linkser36B (SEQ ID NO: 25), linkser36C (SEQ ID NO: 26), and / or one or more sequences having one or more amino acid substitutions, deletions, or additions to any of the above amino acid sequences, wherein the number of amino acids substituted, deleted, or added is 1 to 10, preferably 1 to 5, as described in claim 1.

3. The IgG antibody is selected from any one or more of IgG1, IgG2, IgG3, and IgG4. The antibody is selected from IgG1 antibodies, preferably one or more of the following: trastuzumab, bevacizumab, daratumumab, omalizumab, cetuximab, adalimumab, ustekinumab, ocrelizumab, infliximab, emborizumab, atlizumab, and atezolizumab. The IgG2 antibody is one or more of the following: denosumab, erenumab, tremelimumab, and evocumab; The single-chain antibody according to claim 1 or 2, characterized in that the IgG4 antibody is one or more of the following: tislerizumab, dupilumab, rituximab, pembrolizumab, nivolumab, and palizumab.

4. The antibody light chain is selected from a κ chain and / or a λ chain, and the antibody light chain fragment is selected from a κ chain fragment and / or a λ chain fragment; the heavy chain is selected from one or more of the μ chain, γ chain, α chain, δ chain and ε chain; the heavy chain fragment is selected from one or more of the μ chain fragment, γ chain fragment, α chain fragment, δ chain fragment and ε chain fragment; Preferably, the antibody light chain is derived from the tislerizumab light chain TisLC or a fragment thereof, and the antibody heavy chain is derived from the tislerizumab heavy chain TisHC or a fragment thereof TisFd; or, The antibody light chain is derived from the dupilumab light chain DupLC or a fragment thereof, and the antibody heavy chain is derived from the dupilumab heavy chain DupHC or a fragment DupFd; or, The antibody light chain is derived from the adalimumab light chain AdaLC or a fragment thereof, and the antibody heavy chain is derived from the adalimumab heavy chain AdaHC or a fragment AdaFd; or, The antibody light chain is derived from the bevacizumab light chain BevLC or a fragment thereof, and the antibody heavy chain is derived from the bevacizumab heavy chain BevHC or a fragment BevFd; or, The antibody light chain is derived from the daratumumab light chain DarLC or a fragment thereof, and the antibody heavy chain is derived from the daratumumab heavy chain DarHC or a fragment thereof DarFd; or, The antibody light chain is derived from the omalizumab light chain OmaLC or a fragment thereof, and the antibody heavy chain is derived from the omalizumab heavy chain OmaHC or a fragment OmaFd; or, The antibody light chain is derived from the cetuximab light chain CetLC or a fragment thereof, and the antibody heavy chain is derived from the cetuximab heavy chain CetHC or a fragment CetFd; or, The antibody light chain is derived from the denosumab light chain DenLC or a fragment thereof, and the antibody heavy chain is derived from the denosumab heavy chain DenHC or a fragment DenFd; or The antibody light chain is derived from the trastuzumab light chain TraLC or a fragment thereof, and the antibody heavy chain is derived from the trastuzumab heavy chain TraHC or a fragment thereof; or The single-chain antibody according to any one of claims 1 to 3, characterized in that the antibody light chain is a trastuzumab light chain (TraLC) and the antibody heavy chain fragment is a trastuzumab heavy chain fragment (TraFd).

5. The single-chain antibody further comprises a signal peptide SP, preferably the single-chain antibody has the signal peptide SP linked to the N-terminus of a light chain or light chain fragment, and is used to improve the expression efficiency and expression level of the single-chain antibody, preferably the SP comprises the amino acid sequence shown in SEQ ID NO: 1, or a sequence having one or more amino acid substitutions, deletions, or additions to the sequence shown in SEQ ID NO: 1, more preferably the number of amino acids substituted, deleted, or added is 1 to 10, characterized in that the single-chain antibody according to any one of claims 1 to 4.

6. The single-chain antibody further comprises a fluorescent protein tag, preferably the single-chain antibody having the fluorescent protein tag ligated to the C-terminus of the heavy chain or heavy chain fragment, and more preferably the fluorescent protein tag is eGFP or a protein tag modified based on eGFP, such as mTagBFP2, moxCerulean3, AmCyanl, MiCy, ZsGreen, Clover, mVenus, ZsYellow1, mKO2, TurboRFP, tdTomato, eqFP611 A single-chain antibody according to any one of claims 1 to 5, comprising mKate1.3, mNeptune2, miRFP670, mAme-traine, PAmCherry2, and mEos3.2, more preferably the eGFP comprising the amino acid sequence shown in SEQ ID NO: 8, or a sequence having one or more amino acid substitutions, deletions, or additions to the amino acid sequence shown in SEQ ID NO: 8, and preferably the number of amino acids substituted, deleted, or added is 1 to 20.

7. The single-chain antibody according to any one of claims 1 to 6, further comprising a tag, preferably having the tag ligated to the N-terminus of a light chain or light chain fragment, more preferably the tag being a His tag, and preferably having 3 to 15 histidine residues.

8. The fluorescent protein tag, tag tag, and signal peptide are linked to the light chain, light chain fragment, or heavy chain, or heavy chain fragment of the antibody via a single bond or a flexible linker peptide, preferably the flexible linker peptide comprises 1 to 20 amino acid residues, and preferably the flexible linker peptide is 5L (SEQ ID NO: 3: GSGGS) and / or 10L (SEQ ID NO: 7: TGGAGTGSGA), characterized in that the single-chain antibody is according to any one of claims 1 to 7.

9. One or more isolated nucleic acids, wherein the nucleic acid encodes a single-chain antibody according to any one of claims 1 to 12, and preferably the nucleic acid is a DNA sequence and / or an RNA sequence.

10. An expression vector comprising one or more types of expression vectors, wherein the vector comprises a nucleic acid encoding a single-chain antibody as described in any one of claims 1 to 12, and preferably the expression vector includes, but is not limited to, eukaryotic plasmid vectors, eukaryotic virus vectors, prokaryotic plasmids, specific vectors, shuttle vectors, minichromosomes, and various other vectors, and preferably the expression vector is a eukaryotic plasmid expression vector and a prokaryotic plasmid expression vector.

11. A host cell comprising the vector described in claim 10 or having the nucleic acid described in claim 9 incorporated in its genome, wherein the host cell is selected from prokaryotic host cells and / or eukaryotic host cells, preferably the host cell is a eukaryotic cell, such as yeast, progenitor cells, Chinese hamster ovary cells, insect cells, wheat germ cells, rabbit reticulocytes, and more preferably the yeast is one or more selected from Saccharomyces cerevisiae and Kluiveromyces yeasts, in another preferred embodiment the Kluiveromyces yeasts are Kluiveromyces lactis, Kluiveromyces marxianus, and Kluiveromyces dobzanskyi A host cell characterized by being selected from any one or a combination of any of the following (dobzhanskii).

12. An extracorporeal cell-free protein synthesis system comprising the nucleic acid described in claim 9, or the expression vector described in claim 10, or the lysate or extract of the host cell described in claim 17, The in vitro cell-free protein synthesis system is capable of expressing the single-chain antibody described in any one of claims 1 to 8 under appropriate expression conditions. Preferably, the extracorporeal cell-free protein synthesis system includes, but is not limited to, an Escherichia coli extracellular protein synthesis system, a bacterial extracellular protein synthesis system, a mammalian extracellular protein synthesis system, a plant extracellular protein synthesis system, a yeast extracellular protein synthesis system, and an insect extracellular protein synthesis system; more preferably, it is a yeast extracellular protein synthesis system, even more preferably a Kluyveromyces yeast extracellular protein synthesis system, and most preferably a Kluyveromyces lactis or Kluyveromyces marcianus extracellular protein synthesis system.

13. The extracorporeal cell-free protein synthesis system according to claim 12, characterized in that the extracorporeal cell-free protein synthesis system comprises a cell extract, trihydroxymethylaminomethane, potassium acetate, magnesium acetate, a mixture of nucleoside triphosphates (NTPs), and an amino acid mixture.

14. Use of a single-chain antibody prepared using a single-chain antibody according to any one of claims 1 to 8, or a single-chain antibody prepared using a synthetic system according to any one of claims 12 to 13, in the diagnosis, prevention and treatment of a disease, or in the manufacture of a pharmaceutical for the diagnosis, prevention and treatment of a disease, wherein the disease is a tumor and / or cancer, and the tumor and / or cancer includes diseases such as lung cancer, breast cancer, esophageal cancer, gastric cancer, cervical cancer, colon cancer, rhabdomyosarcoma, liposarcoma, osteosarcoma, lymphoma, leukemia, liver cancer, cervical cancer, ovarian cancer, and breast cancer; more preferably, the disease is a disease caused by abnormalities in HER2, TNF-α, VEGF, PD-1, IL-4, IL-13, or CD38.

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