Vectors and methods used to screen for functional antigen-binding proteins

Abstract

A method for selecting a functional antigen-binding protein, which uses an expression vector constructed with a light chain or a fragment thereof of a reference antigen-binding protein of known sequence and function, or a heavy chain or a fragment thereof of a reference antigen-binding protein, by bacteriophage display and / or cell display, thereby selecting a functional antigen-binding protein that binds to the same epitope as the reference antigen-binding protein. The present application further provides a functional antigen-binding protein obtained by said method.

Description

[Technical field]

[0001] 1.Technical Field The present application relates to the biomedical field, specifically to vectors expressing antigen-binding proteins and methods for screening for functional antigen-binding proteins. [Background technology]

[0002] 2. Description of related technologies At present, in the antibody industry, the methods applied to obtain functional antibodies or antigen-binding fragments thereof with desired properties mainly include hybridoma technology and antibody library technology. However, the hybridoma technology has the problem that the number of candidate clones is small and positive clones are easily lost, and the selection of positive clones, especially the complex functional screening, requires a large amount of time and effort. The antibody library technology uses genetic engineering to clone and express the heavy and light chain variable region genes into plasmids or bacteriophages, and then uses different antigen screening to screen clones with specific antibody genes, which mainly include bacteriophage display and yeast surface display. The antibody library technology has greatly improved the storage capacity, and the target antigen generally has multiple (e.g., several, tens, or even hundreds) antigenic determinants, but only one or a few antigenic determinants may have the desired biological function. When screening for antigen-specific antibodies or antigen-binding fragments of the target target, it is not known which antigenic determinants have the desired biological function. In the past, the technology generally first obtains an antigen-specific antibody or its antigen-binding fragment, and then analyzes and identifies it step by step to find an antibody or its antigen-binding fragment that binds to a specific antigen determinant and has the desired biological function, which requires a long time and a large amount of work, and the screening effect is low. Therefore, it is necessary to use a new antibody or its antigen-binding fragment expression technology to improve the quality, quantity and diversity of lead molecules, accelerate the drug development speed, and improve the development success rate.

[0003] Meanwhile, the present application provides a method for selecting a functional antigen-binding protein, comprising: a) providing a first polynucleotide, wherein the first polynucleotide comprises, in a 5' to 3' direction, R1-nucleic acid fragment I-R2, wherein the nucleic acid fragment I is capable of encoding an antigen-binding fragment I; b) providing a second polynucleotide, wherein the second polynucleotide comprises, in a 5' to 3' direction, R3-reference nucleic acid fragment II-R4, wherein the reference nucleic acid fragment II is capable of encoding a reference antigen-binding fragment II, wherein the reference antigen-binding fragment II can form a reference antigen-binding protein with the reference antigen-binding fragment I encoded by the reference nucleic acid fragment I; c) providing a first vector polynucleotide, wherein the first vector polynucleotide comprises, in a 5' to 3' direction, R2-vector fragment I-R3; d) providing a second vector polynucleotide, wherein the second vector polynucleotide comprises, in a 5' to 3' direction, R4-vector fragment II-R1; e) isolating the first polynucleotide with a restriction endonuclease; cleaving the second polynucleotide, the first vector polynucleotide and the second vector polynucleotide to obtain the cleaved first polynucleotide, the cleaved second polynucleotide, the cleaved first vector polynucleotide and the cleaved second vector polynucleotide; f) mixing the cleaved first polynucleotide, the cleaved second polynucleotide, the cleaved first vector polynucleotide and the cleaved second vector polynucleotide, thereby directional ligating it to circularize the first screening target vector; g) expressing the first screening target vector and selecting as a first replacement vector a first screening target vector that can express an expression product having the following properties: the reference binding protein is capable of binding to a bindable target, and the binding ability of the target is 30% or more of the binding ability of the reference antigen binding protein to the target; h) obtaining the functional antigen binding protein from the first replacement vector; wherein R1, R2, R3 and R4 are each independently a restriction endonuclease recognition site.

[0004] In one embodiment, the method specifically determines R1 and R2. recognition and cleaving said first polynucleotide with a restriction endonuclease which is capable of cleaving said first polynucleotide, thereby obtaining said cleaved first polynucleotide.

[0005] In one embodiment, the method specifically determines R3 and R4. recognition The second polynucleotide is cleaved with a restriction endonuclease which is capable of cleaving the second polynucleotide, and the cleaved second polynucleotide is included.

[0006] In one embodiment, the method specifically determines R2 and R3. recognition and cleaving said first vector polynucleotide with a restriction endonuclease which is capable of cleaving said first vector polynucleotide, thereby obtaining said cleaved first vector polynucleotide.

[0007] In one embodiment, the method uses R4 and R1 specifically. recognition and cleaving said second vector polynucleotide with a restriction endonuclease which is complementary to said restriction endonuclease, to obtain said cleaved second vector polynucleotide.

[0008] In one embodiment, R1 is specific recognition The ends generated by specific cleavage with the restriction endonuclease R2, R3, and R4 are mutually exclusive with any of the ends generated by specific cleavage with the restriction endonuclease corresponding to R2, R3, and R4. recognition or not combined.

[0009] In one embodiment, R2 is specifically recognition The terminus generated by specific cleavage with the restriction endonuclease R1, R3, or R4 is also mutually exclusive with the terminus generated by specific cleavage with the restriction endonuclease R4. recognition or not combined.

[0010] In one embodiment, R3 is specifically recognition The terminus generated by specific cleavage with the restriction endonuclease according to the above formula (I) is mutually exclusive with the terminus generated by specific cleavage with the restriction endonuclease corresponding to any one of R1, R2, and R4. recognition or not combined.

[0011] In one embodiment, R4 is specifically recognition The end generated by specific cleavage with the restriction endonuclease according to the above formula (I) is mutually exclusive with the end generated by specific cleavage with the restriction endonuclease corresponding to any of R1, R2, and R3. recognition or not combined.

[0012] In one embodiment, the restriction endonuclease is selected from SfiI and BsmBI.

[0013] In one embodiment, the method comprises introducing the first vector to be screened into a cell and expressing the first vector to be screened.

[0014] In one embodiment, the method includes introducing the first screening vector into a bacterium, producing a bacteriophage library comprising one or more of the first screening vectors, and obtaining the functional antigen binding protein using the bacteriophage library.

[0015] In one embodiment, the vector fragment I comprises a linker and the vector fragment II is derived from a display vector.

[0016] In one embodiment, the nucleic acid fragment I encodes an antibody light chain or fragment thereof, the vector fragment I comprises the linker, the reference nucleic acid fragment II encodes an antibody heavy chain or fragment thereof, and the vector fragment II is derived from the display vector.

[0017] In one embodiment, R1 comprises the nucleotide sequence shown in SEQ ID NO:1.

[0018] In one embodiment, R2 comprises the nucleotide sequence shown in SEQ ID NO:2.

[0019] In one embodiment, R3 comprises the nucleotide sequence shown in SEQ ID NO:3.

[0020] In one embodiment, R4 comprises the nucleotide sequence shown in SEQ ID NO:4.

[0021] In one embodiment, the vector fragment II comprises a linker and the vector fragment I is derived from a display vector.

[0022] In one embodiment, the nucleic acid fragment I encodes an antibody heavy chain or fragment thereof, the vector fragment I is derived from the display vector, the reference nucleic acid fragment II encodes an antibody light chain or fragment thereof, and the vector fragment II comprises the linker.

[0023] In one embodiment, R1 comprises the nucleotide sequence shown in SEQ ID NO:3.

[0024] In one embodiment, R2 comprises the nucleotide sequence shown in SEQ ID NO:4.

[0025] In one embodiment, R3 comprises the nucleotide sequence shown in SEQ ID NO:1.

[0026] In one embodiment, R4 comprises the nucleotide sequence shown in SEQ ID NO:2.

[0027] In one embodiment, the display vector is derived from the pComb3x vector.

[0028] In one embodiment, the linker comprises a nucleic acid sequence encoding the signal peptide pelB or a fragment thereof.

[0029] In one embodiment, the linker is about 50 to about 200 bases in length.

[0030] In one embodiment, the method includes introducing the first screening vector into a bacterium, obtaining DNA of the first screening target vector from the bacterium, and introducing the DNA of the first screening target vector into a cell; and obtaining the functional antigen-specific binding polypeptide by the cell.

[0031] In one embodiment, the cell is a mammalian cell.

[0032] In one embodiment, the Vector Fragment I and / or the Vector Fragment II are derived from a mammalian cell expression vector.

[0033] In one embodiment, the mammalian cell expression vector is derived from pDGB4.

[0034] In certain embodiments, said nucleic acid fragment I encodes an antibody light chain or a fragment thereof, and said reference nucleic acid fragment II encodes an antibody heavy chain or a fragment thereof.

[0035] In one embodiment, R1 comprises the nucleotide sequence shown in SEQ ID NO:7.

[0036] In one embodiment, R2 comprises the nucleotide sequence shown in SEQ ID NO:8.

[0037] In one embodiment, R3 comprises the nucleotide sequence shown in SEQ ID NO:5.

[0038] In one embodiment, R4 comprises the nucleotide sequence shown in SEQ ID NO:6.

[0039] In certain embodiments, said nucleic acid fragment I encodes an antibody heavy chain or a fragment thereof, and said reference nucleic acid fragment II encodes an antibody light chain or a fragment thereof.

[0040] In one embodiment, R1 comprises the nucleotide sequence shown in SEQ ID NO:5.

[0041] In one embodiment, R2 comprises the nucleotide sequence shown in SEQ ID NO:6.

[0042] In one embodiment, R3 comprises the nucleotide sequence shown in SEQ ID NO:7.

[0043] In one embodiment, R4 comprises the nucleotide sequence shown in SEQ ID NO:8.

[0044] In one embodiment, the method comprises: a) providing a third polynucleotide, wherein the third polynucleotide comprises, in a 5' to 3' direction, R5-nucleic acid fragment I'-R6; b) providing a fourth polynucleotide, wherein the fourth polynucleotide comprises, in a 5' to 3' direction, R7-nucleic acid fragment II'-R8, wherein the nucleic acid fragment II' is capable of encoding an antigen binding protein II', and wherein the antigen binding fragment II' is capable of binding to the reference antigen binding fragment I to form an antigen binding protein having the following properties: the reference antigen binding protein is capable of binding to a target capable of binding, and a binding ability of the target is 30% or more of the binding ability of the reference antigen binding protein to the target; c) providing a third vector polynucleotide, wherein the third vector polynucleotide comprises, in a 5' to 3' direction, R6-vector fragment III-R7; d) providing a fourth vector polynucleotide, wherein the fourth vector polynucleotide comprises, in a 5' to 3' direction, R8-vector fragment IV-R5; e) isolating the fourth vector polynucleotide from the first vector polynucleotide with a restriction endonuclease. cleaving the third polynucleotide, the fourth polynucleotide, the third vector polynucleotide and the fourth vector polynucleotide to obtain a cleaved third polynucleotide, a cleaved fourth polynucleotide, a cleaved third vector polynucleotide and a cleaved fourth vector polynucleotide; f) mixing the cleaved third polynucleotide, the cleaved fourth polynucleotide, the cleaved third vector polynucleotide and the cleaved fourth vector polynucleotide, thereby unidirectionally ligating it to circularize a double replacement screening target vector; g) expressing the double replacement screening target vector, and selecting as a double replacement screening target vector a double replacement vector that expresses an expression product having the following properties: it can bind to the target, and the binding ability of the target is 30% or more of the binding ability of the reference antigen binding protein and the target; h) obtaining the functional antigen binding protein from the double replacement vector; wherein R5, R6, R7 and R8 are each independently the restriction endonuclease recognition site.

[0045] In one embodiment, the method specifically determines R5 and R6. recognition and cleaving said third polynucleotide with a restriction endonuclease which is capable of cleaving said third polynucleotide to obtain said cleaved third polynucleotide.

[0046] In one embodiment, the method specifically determines R7 and R8. recognition and cleaving the fourth polynucleotide with a restriction endonuclease that is complementary to the restriction enzyme of the fourth polynucleotide, thereby obtaining the cleaved fourth polynucleotide.

[0047] In one embodiment, the method specifically determines R6 and R7. recognition and cleaving said third vector polynucleotide with a restriction endonuclease which is capable of cleaving said third vector polynucleotide to obtain a cleaved third vector polynucleotide.

[0048] In one embodiment, the method uses R8 and R5 specifically. recognition and cleaving said fourth vector polynucleotide with a restriction endonuclease which is capable of cleaving said fourth vector polynucleotide, thereby obtaining said cleaved fourth vector polynucleotide.

[0049] In one embodiment, R5 is specifically recognition The ends generated by specific cleavage with the restriction endonuclease R6, R7, and R8 are mutually exclusive with any of the ends generated by specific cleavage with the restriction endonuclease R7, R8, or R9. recognition Either it is or it is not combined.

[0050] In one embodiment, R6 is specifically recognition The terminus generated by specific cleavage with the restriction endonuclease R5, R7, or R8 is mutually exclusive with the terminus generated by specific cleavage with the restriction endonuclease R7, R8, or R9. recognition or not combined.

[0051] In one embodiment, R7 is recognition The terminus generated by specific cleavage with the restriction endonuclease R5, R6, or R8 is mutually exclusive with the terminus generated by specific cleavage with the restriction endonuclease R6, R8, or R9. recognition or not combined.

[0052] In one embodiment, R8 is specifically recognition The terminus generated by specific cleavage with the restriction endonuclease R5, R6, or R7 is also mutually exclusive with the terminus generated by specific cleavage with the restriction endonuclease R6, R7, or R8. recognition or not combined.

[0053] In one embodiment, the restriction endonuclease is selected from SfiI and BsmBI.

[0054] In one embodiment, the method comprises introducing the double replacement screened vector into a cell and expressing the double replacement screened vector.

[0055] In one embodiment, the method includes introducing the double replacement screened vector into a bacterium, producing a bacteriophage library comprising one or more of the double replacement screened vectors, and obtaining the functional antigen binding protein using the bacteriophage library.

[0056] In one embodiment, the vector fragment III comprises a linker and the vector fragment IV is derived from a display vector.

[0057] In one embodiment, the nucleic acid fragment I' encodes an antibody light chain or fragment thereof, the vector fragment III comprises the linker, the nucleic acid fragment II' encodes an antibody heavy chain or fragment thereof, and the vector fragment IV is derived from the display vector.

[0058] In one embodiment, R5 comprises the nucleotide sequence shown in SEQ ID NO:1.

[0059] In one embodiment, R6 comprises the nucleotide sequence shown in SEQ ID NO:2.

[0060] In one embodiment, R7 comprises the nucleotide sequence set forth in SEQ ID NO:3.

[0061] In one embodiment, R8 comprises the nucleotide sequence shown in SEQ ID NO:4.

[0062] In one embodiment, the vector fragment IV comprises a linker and the vector fragment III is derived from a display vector.

[0063] In one embodiment, the nucleic acid fragment I' encodes an antibody heavy chain or fragment thereof, the vector fragment III is derived from the display vector, the nucleic acid fragment II' encodes an antibody light chain or fragment thereof, and the vector fragment IV comprises the linker.

[0064] In one embodiment, R5 comprises the nucleotide sequence shown in SEQ ID NO:3.

[0065] In one embodiment, R6 comprises the nucleotide sequence set forth in SEQ ID NO:4.

[0066] In one embodiment, R7 comprises the nucleotide sequence set forth in SEQ ID NO:1.

[0067] In one embodiment, R8 comprises the nucleotide sequence shown in SEQ ID NO:2.

[0068] In one embodiment, the display vector is derived from the pComb3x vector.

[0069] In one embodiment, the linker comprises a nucleic acid sequence encoding the signal peptide pelB or a fragment thereof.

[0070] In one embodiment, the linker is about 50 to about 200 bases in length.

[0071] In one embodiment, the method comprises introducing the double replacement screened vector into a bacterium, obtaining DNA of the double replacement screened vector from the bacterium, and introducing DNA of the double replacement screened vector into a cell; and obtaining the functional antigen-specific binding polypeptide by the cell.

[0072] In one embodiment, the cell is a mammalian cell.

[0073] In one embodiment, the Vector Fragment III and / or the Vector Fragment IV are derived from a mammalian expression vector.

[0074] In one embodiment, the mammalian expression vector is derived from pDGB4.

[0075] In one embodiment, said nucleic acid fragment I' encodes an antibody light chain or fragment thereof, and said nucleic acid fragment II' encodes an antibody heavy chain or fragment thereof.

[0076] In one embodiment, R5 comprises the nucleotide sequence shown in SEQ ID NO:7.

[0077] In one embodiment, R6 comprises the nucleotide sequence shown in SEQ ID NO:8.

[0078] In one embodiment, R7 comprises the nucleotide sequence set forth in SEQ ID NO:5.

[0079] In one embodiment, R8 comprises the nucleotide sequence shown in SEQ ID NO:6.

[0080] In one embodiment, said nucleic acid fragment I' encodes an antibody heavy chain or fragment thereof, and said nucleic acid fragment II' encodes an antibody light chain or fragment thereof.

[0081] In one embodiment, R5 comprises the nucleotide sequence shown in SEQ ID NO:5.

[0082] In one embodiment, R6 comprises the nucleotide sequence set forth in SEQ ID NO:6.

[0083] In one embodiment, R7 comprises the nucleotide sequence set forth in SEQ ID NO:7.

[0084] In one embodiment, R8 comprises the nucleotide sequence set forth in SEQ ID NO:8.

[0085] In one embodiment, the method includes: a) providing a fifth polynucleotide, wherein the fifth polynucleotide comprises, in a 5' to 3' direction, an R9-reference nucleic acid fragment I-R10; b) providing a sixth polynucleotide, wherein the sixth polynucleotide comprises, in a 5' to 3' direction, an R11-nucleic acid fragment II-R12; c) providing a fifth vector polynucleotide, wherein the fifth vector polynucleotide comprises, in a 5' to 3' direction, an R10-vector fragment V-R11; d) providing a sixth vector polynucleotide, wherein the sixth vector polynucleotide comprises, in a 5' to 3' direction, an R12-vector fragment VI-R9; e) cleaving the fifth polynucleotide, the sixth polynucleotide, the fifth vector polynucleotide, and the sixth vector polynucleotide with a restriction endonuclease, and obtaining the fifth polynucleotide after cleavage, the sixth polynucleotide after cleavage, and the sixth polynucleotide after cleavage. obtaining a fifth vector polynucleotide and a cleaved sixth vector polynucleotide; f) mixing the cleaved fifth polynucleotide, the cleaved sixth polynucleotide, the cleaved fifth vector polynucleotide and the cleaved sixth vector polynucleotide, thereby directional ligating them to form a second screening target vector into a circular form; g) expressing the second screening target vector, and selecting as a second replacement vector a second screening target vector capable of expressing an expression product having the following properties: the reference binding protein is capable of binding to a target capable of binding, and the binding ability of the target is 30% or more of the binding ability of the reference antigen binding protein to the target; h) selecting as the nucleic acid fragment II of the second replacement vector as the nucleic acid fragment II'; wherein R9, R10, R11 and R12 are each independently a restriction endonuclease recognition site.

[0086] In one embodiment, the method specifically determines R9 and R10. recognition and cleaving said fifth polynucleotide with a restriction endonuclease which is capable of cleaving said fifth polynucleotide, thereby obtaining said cleaved fifth polynucleotide.

[0087] In one embodiment, the method specifically comprises: recognition and cleaving said sixth polynucleotide with a restriction endonuclease which is selected from the group consisting of cleaved sixth polynucleotides.

[0088] In one embodiment, the method specifically comprises: recognition and cleaving said fifth vector polynucleotide with a restriction endonuclease which is capable of cleaving said fifth vector polynucleotide, thereby obtaining said cleaved fifth vector polynucleotide.

[0089] In one embodiment, the method specifically comprises: recognition and cleaving said sixth vector polynucleotide with a restriction endonuclease which is selected from the group consisting of cleaved sixth vector polynucleotides.

[0090] In one embodiment, R9 is recognition The terminus generated by specific cleavage with the restriction endonuclease R10, R11, or R12 is mutually exclusive with the terminus generated by specific cleavage with the restriction endonuclease R11, R12, or R12. recognition or not combined.

[0091] In one embodiment, R10 is a recognition The terminus generated by specific cleavage with the restriction endonuclease R11, R12, or R9 is mutually exclusive with the terminus generated by specific cleavage with the restriction endonuclease R12, R13, or R9. recognition or not combined.

[0092] In one embodiment, R11 is specifically recognition The terminus generated by specific cleavage with the restriction endonuclease R9, R10, or R12 is mutually exclusive with the terminus generated by specific cleavage with the restriction endonuclease R10, R20, or R32. recognition or not combined.

[0093] In one embodiment, R12 is specifically recognition The terminus generated by specific cleavage with the restriction endonuclease R9, R10, or R11 is mutually exclusive with the terminus generated by specific cleavage with the restriction endonuclease R10, R11, or R20. recognition or not combined.

[0094] In one embodiment, the restriction endonuclease is selected from SfiI and BsmBI.

[0095] In one embodiment, the method comprises introducing the second vector to be screened into a cell and expressing the second vector to be screened.

[0096] In one embodiment, the method includes introducing the second screened vector into a bacterium, producing a bacteriophage library comprising one or more of the second screened vectors, and obtaining the functional antigen binding protein using the bacteriophage library.

[0097] In one embodiment, the vector fragment V comprises a linker and the vector fragment VI is derived from a display vector.

[0098] In one embodiment, the reference nucleic acid fragment I encodes an antibody light chain or fragment thereof, the vector fragment V comprises the linker, the nucleic acid fragment II encodes an antibody heavy chain or fragment thereof, and the vector fragment VI is derived from the display vector.

[0099] In one embodiment, R9 comprises the nucleotide sequence set forth in SEQ ID NO:1.

[0100] In one embodiment, R10 comprises the nucleotide sequence shown in SEQ ID NO:2.

[0101] In one embodiment, R11 comprises the nucleotide sequence shown in SEQ ID NO:3.

[0102] In one embodiment, R12 comprises the nucleotide sequence shown in SEQ ID NO:4.

[0103] In one embodiment, the vector fragment VI comprises a linker and the vector fragment V is derived from a display vector.

[0104] In one embodiment, the reference nucleic acid fragment I encodes an antibody heavy chain or fragment thereof, the vector fragment V is derived from the display vector, the nucleic acid fragment II encodes an antibody light chain or fragment thereof, and the vector fragment VI comprises the linker.

[0105] In one embodiment, R9 comprises the nucleotide sequence set forth in SEQ ID NO:3.

[0106] In one embodiment, R10 comprises the nucleotide sequence shown in SEQ ID NO:4.

[0107] In one embodiment, R11 comprises the nucleotide sequence shown in SEQ ID NO:1.

[0108] In one embodiment, R12 comprises the nucleotide sequence shown in SEQ ID NO:2.

[0109] In one embodiment, the display vector is derived from the pComb3x vector.

[0110] In one embodiment, the linker comprises a nucleic acid sequence encoding the signal peptide pelB or a fragment thereof.

[0111] In one embodiment, the linker is about 50 to about 200 bases in length.

[0112] In one embodiment, the method includes introducing the vector to be screened second into a bacterium, obtaining DNA of the vector to be screened second from the bacterium, and introducing DNA of the vector to be screened second into a cell; and obtaining the functional antigen-specific binding polypeptide by the cell.

[0113] In one embodiment, the cell is a mammalian cell.

[0114] In one embodiment, said Vector Fragment V and / or said Vector Fragment VI are derived from a mammalian expression vector.

[0115] In one embodiment, the mammalian expression vector is derived from pDGB4.

[0116] In certain embodiments, the reference nucleic acid fragment I encodes an antibody light chain or a fragment thereof, and the reference fragment II encodes an antibody heavy chain or a fragment thereof.

[0117] In one embodiment, R9 comprises the nucleotide sequence set forth in SEQ ID NO:7.

[0118] In one embodiment, R10 comprises the nucleotide sequence shown in SEQ ID NO:8.

[0119] In one embodiment, R11 comprises the nucleotide sequence shown in SEQ ID NO:5.

[0120] In one embodiment, R12 comprises the nucleotide sequence shown in SEQ ID NO:6.

[0121] In certain embodiments, the reference nucleic acid fragment I encodes an antibody heavy chain or a fragment thereof, and the nucleic acid fragment II encodes an antibody light chain or a fragment thereof.

[0122] In one embodiment, R9 comprises the nucleotide sequence set forth in SEQ ID NO:5.

[0123] In one embodiment, R10 comprises the nucleotide sequence shown in SEQ ID NO:6.

[0124] In one embodiment, R11 comprises the nucleotide sequence shown in SEQ ID NO:7.

[0125] In one embodiment, R12 comprises the nucleotide sequence set forth in SEQ ID NO:8.

[0126] In one embodiment, the antigen binding protein comprises an antibody or an antibody fragment.

[0127] In one embodiment, the antibody fragment comprises an scFv, Fab, Fab', (Fab)2 and / or (Fab')2.

[0128] In one embodiment, the directional ligation comprises using a ligase.

[0129] In one embodiment, the ligase is a DNA ligase.

[0130] On the other hand, the present application provides a method of producing an antigen binding protein, which comprises expressing said first replacement vector, said second replacement vector and / or said double replacement vector under conditions in which said first replacement vector, said second replacement vector and / or said double replacement vector are expressed.

[0131] In another aspect, the present application provides a first replacement vector produced according to the method.

[0132] Alternatively, the present application provides a second replacement vector prepared according to the above method.

[0133] On the other hand, the present application provides a double replacement vector prepared according to the above method.

[0134] On the other hand, the present application provides functional antigen-binding proteins produced according to the above-mentioned methods.

[0135] [Brief Summary of the Invention] The present application provides a method for selecting a functional antigen-binding protein, the method comprising constructing a vector (e.g., the double replacement screened vector, the first screened vector and / or the second screened vector) using a reference antigen-binding fragment (e.g., reference antigen-binding fragment I and / or reference antigen-binding fragment II) of a reference antigen-binding protein having a known sequence and / or function to express the vector, and then detecting whether the expression product of the vector and the reference antigen-binding protein are identical. recognition The present application also provides a method for constructing the vector, and a functional antigen-binding protein obtained by the vector. The present application combines antibody bacteriophage display or cell surface display screening with a light and heavy chain or a fragment thereof of a reference antigen-binding protein with a sequence and / or function known in the prior art to obtain a novel functional antigen-binding protein with desired properties; the functional antigen-binding protein obtained by screening and the reference antigen-binding protein are likely to bind to the same or a similar epitope, are likely to have biological activity, have high screening efficiency, and are likely to have a short screening time.

[0136] Those skilled in the art can easily understand other aspects and advantages of the present application from the following detailed description. In the following detailed description, only exemplary embodiments of the present application are shown and described. As will be understood by those skilled in the art, the contents of the present application may be modified by those skilled in the art to the specific embodiments disclosed without departing from the spirit and scope of the invention. In addition, the drawings and descriptions in the present application are merely illustrative and are not intended to be limiting. Specific features of the invention related to this application are set forth in the appended claims. A better understanding of the features and advantages of the invention related to this application can be obtained by reference to the exemplary implementations in the following detailed description and drawings, the accompanying drawings of which are briefly described as follows: [Brief description of the drawings]

[0137] [Figure 1] Shown in FIG. 1 is an exemplary structure of the first screened vector of the present application; [Diagram 2] Shown in FIG. 2 is an exemplary structure of the double replacement screening vector of the present application; [Diagram 3] Shown in FIG. 3 is an exemplary structure of the second screening vector of the present application. [Figure 4] Shown in FIG. 4 are the results of an analysis of the antibody agonist activity of the antibodies obtained by the above-mentioned method of the present application. [Diagram 5] Shown in FIG. 5 are the results of analyzing the antibody endocytosis activity of the antibodies obtained by the above-mentioned method of the present application. [Figure 6] FIG. 6 shows the antitumor effect of the antibody obtained by the above method of the present application when used alone. [Figure 7] FIG. 7 shows the antitumor effect of using a combination of antibodies obtained by the above-mentioned method of the present application.

[0138] [Detailed Description of the Invention] The following specific illustrative examples illustrate embodiments of the present invention, and those skilled in the art will appreciate other advantages and benefits of the present invention from the disclosure herein.

[0139] [ Definition of Terms ]

[0140] In the present application, the term "reference antigen binding protein" generally refers to an antigen binding protein that has at least one desired biological function, such as being able to specifically bind to a target with high affinity, being able to block the binding of a protein to a specific ligand, and / or being able to stimulate immune cells to secrete cytokines. The reference antigen binding protein may be derived from an amino acid sequence and / or functional antigen binding protein (e.g., an antibody) known in the art. In some embodiments, the reference antigen binding protein is derived from a commercialized antibody product and / or an antibody in clinical trials. The choice of which reference antigen binding protein (and "reference antigen binding fragment I" and / or "reference antigen binding fragment II") depends on which biological function of the antigen binding protein is desired. For example, if one wishes to obtain an antigen binding protein that specifically binds to PD-1, one may choose the PD-1 antibodies Nivolumab, Pembrolizumab, or Cemiplimab as the reference antigen binding protein. For example, if one wishes to obtain an antigen binding protein that specifically binds to PD-L1, one can select the PD-1 antibodies Atezolizumab, Avelumab, or Durvalumab as the reference antigen binding protein.

[0141] In this application, the term "reference nucleic acid fragment I" generally refers to a nucleic acid fragment capable of encoding a reference antigen-binding fragment I.

[0142] In this application, the term "reference nucleic acid fragment II" generally refers to a nucleic acid fragment capable of encoding a reference antigen-binding fragment II.

[0143] In the present application, the term "reference antigen-binding fragment I" generally refers to a polypeptide fragment capable of forming a reference antigen-binding protein. The "reference antigen-binding fragment I" may be an antibody heavy chain or heavy chain variable region or heavy chain fragment, or an antibody light chain or light chain variable region or light chain fragment.

[0144] In the present application, the term "reference antigen-binding fragment II" generally refers to a polypeptide fragment capable of forming a reference antigen-binding protein. The "reference antigen-binding fragment II" may be an antibody heavy chain or heavy chain variable region or heavy chain fragment, or an antibody light chain or light chain variable region or light chain fragment.

[0145] The "reference antigen-binding fragment I" can form an antigen-binding protein with the "reference antigen-binding fragment II" (eg, a reference antigen-binding protein).

[0146] In this application, the term "antigen-binding protein" generally refers to a protein that comprises a binding antigen portion, and optionally a conformational scaffold or framework portion that allows the binding antigen portion to promote binding of the antigen-binding protein to the antigen. Antigen-binding proteins typically comprise an antibody light chain variable region (VL), an antibody heavy chain variable region (VH), or both, and functional fragments thereof. The variable regions of the heavy and light chains comprise the binding domains that interact with the antigen. Examples of antigen-binding proteins include, but are not limited to, antibodies, antigen-binding fragments, immunoconjugates, multispecific antibodies (e.g., bispecific antibodies), antibody fragments, antibody derivatives, antibody mimetics, or fusion proteins, etc., as long as they exhibit the required antigen-binding activity.

[0147] In this application, the term "antibody" generally refers to an immunoglobulin reactive to a specified protein or peptide or fragment thereof. The antibody may be of any origin, including, but not limited to, IgG, IgA, IgM, IgD and IgE, and antibodies of any subclass (e.g., IgG1, IgG2, IgG3, and IgG4). The antibody may have a heavy chain constant region selected from, for example, IgG1, IgG2, IgG3, or IgG4. The antibody may further have a light chain selected from, for example, kappa (κ) or lambda (λ). The antibody of the present application may be of any species origin.

[0148] In the present application, the term "antigen-binding protein" may refer to a portion of an antibody molecule, which includes amino acid residues that interact with an antigen to confer specificity and affinity to the antibody for the antigen. In the present application, the term "antigen-binding protein" may include an antibody or an antibody fragment, particularly the antibody portions of the antibody light chain or fragment thereof (e.g., VL) and the antibody heavy chain or fragment thereof (e.g., VH). In the antigen-binding protein, the antibody light chain or fragment thereof (e.g., VL) and the antibody heavy chain or fragment thereof (e.g., VH) may be linked to the N-terminus or C-terminus by a linker (e.g., a peptide linker) to form a polypeptide chain. In the antigen-binding protein, the antibody light chain or fragment thereof (e.g., VL) and the antibody heavy chain or fragment thereof (e.g., VH) may be linked to a dimer by a chemical bond (e.g., disulfide bond) between the chains. Examples of antigen-binding proteins include, but are not limited to, Fab, Fab', F(ab)2, Fv fragments, F(ab')2, scFv, di-scFv, and / or dAb. For example, the antigen-binding protein may be an scFv or Fab.

[0149] As used herein, the term "Fab" generally refers to digestion of an intact antibody with papain (e.g., removing the Fc region and hinge region) to produce two identical antigen-binding fragments. An Fab may be composed of a complete light chain, a heavy chain variable region (VH), and the first constant region (CH1) of the heavy chain. Each Fab may have a single antigen-binding site.

[0150] As used herein, the term "scFv" generally refers to a monovalent molecule in which one heavy chain variable region and one light chain variable region of an antibody are paired together by covalent ligation via a flexible peptide linker.

[0151] In the present application, the term "functional antigen-binding protein" generally refers to an antigen-binding protein having a desired function selected based on the method of the present application, such as being able to specifically bind to a target with high affinity, being able to block the binding of a protein to a specific ligand, and / or being able to stimulate immune cells to secrete cytokines. The functional antigen-binding protein has the following properties: it can bind to a target to which the reference binding protein of the present application can bind, and the binding capacity of the target is 30% or more (e.g., 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% or more) of the binding capacity of the reference antigen-binding protein to the target. The functional antigen-binding protein may comprise a reference antigen-binding fragment I and an antigen-binding fragment II'. The functional antigen-binding protein may comprise a reference antigen-binding fragment II and an antigen-binding fragment I'. The functional antigen-binding protein may comprise an antigen-binding fragment I' and an antigen-binding fragment II'.

[0152] In this application, the term "nucleic acid fragment I" generally refers to a nucleic acid molecule capable of expressing antigen-binding fragment I. In nucleic acid fragment I, a nucleic acid fragment I having the following properties is selected as nucleic acid fragment I': the antigen-binding fragment I expressed by it can form an antigen-binding protein with a reference antigen-binding fragment II expressed by a reference nucleic acid fragment II, and the antigen-binding protein has one or more of the following properties: it can bind to a target that the reference antigen-binding protein specifically binds to, and the binding ability of the target is 30% or more (e.g., 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% or more) of the binding ability of the reference antigen-binding protein to the target. Not all nucleic acid fragments I are nucleic acid fragment I'. All antigen-binding fragments I expressed by each nucleic acid fragment I can form an antigen-binding protein with a reference antigen-binding fragment II expressed by a reference nucleic acid fragment II.

[0153] In the present application, the term "nucleic acid fragment I'" generally refers to a nucleic acid molecule capable of expressing antigen-binding fragment I', wherein antigen-binding fragment I' expressed by nucleic acid fragment I' can express a functional antigen-binding protein together with a reference antigen-binding fragment II expressed by a reference nucleic acid fragment II, and wherein the antigen-binding protein has one or more of the following properties: it can bind to a target that the reference antigen-binding protein specifically binds to, and the binding ability of the target is 30% or more (e.g., 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% or more) of the binding ability of the reference antigen-binding protein to the target.

[0154] In this application, the term "nucleic acid fragment II" generally refers to a nucleic acid molecule capable of expressing an antigen-binding fragment II. In the nucleic acid fragment II, a nucleic acid fragment II having the following properties is selected as a nucleic acid fragment II': the antigen-binding fragment II expressed by it can form an antigen-binding protein with the reference antigen-binding fragment I expressed by the reference nucleic acid fragment I, and the antigen-binding protein has one or more of the following properties: it can bind to a target that the reference antigen-binding protein specifically binds to, and the binding ability of the target is 30% or more (e.g., 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% or more) of the binding ability of the reference antigen-binding protein to the target. Not all nucleic acid fragments II are nucleic acid fragments II'. All antigen-binding fragments II expressed by each nucleic acid fragment II can form an antigen-binding protein with the reference antigen-binding fragment I expressed by the reference nucleic acid fragment I.

[0155] In the present application, the term "nucleic acid fragment II' " generally refers to a nucleic acid molecule capable of expressing an antigen-binding fragment II', wherein the antigen-binding protein II' expressed by the nucleic acid fragment II' can form a functional antigen-binding protein with a reference antigen-binding fragment I expressed by a reference nucleic acid fragment I, and the antigen-binding protein has one or more of the following properties: it can bind to a target that the reference antigen-binding protein specifically binds to, and the binding ability of the target is 30% or more (e.g., 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% or more) of the binding ability of the reference antigen-binding protein to the target.

[0156] In the present application, the term "antigen-binding fragment I" generally refers to an expression product of nucleic acid fragment I, which is encoded by nucleic acid fragment I. In antigen-binding fragment I, an antigen-binding fragment I having the following properties can be selected as antigen-binding fragment I': it can form an antigen-binding protein with a reference antigen-binding fragment II, and the antigen-binding protein has one or more of the following properties: it can bind to a target that the reference antigen-binding protein specifically binds to, and the binding ability of the target is 30% or more (e.g., 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% or more) of the binding ability of the reference antigen-binding protein to the target. Not all antigen-binding fragments I are antigen-binding fragment I'. The "antigen-binding fragment I" may be an antibody heavy chain or heavy chain variable region, or a heavy chain fragment, or an antibody light chain or light chain variable region, or a light chain fragment.

[0157] In the present application, the term "antigen-binding fragment I' " generally refers to the expression product of nucleic acid fragment I', which is encoded by nucleic acid fragment I'. Antigen-binding fragment I' can bind to a reference antigen-binding fragment II in antigen-binding fragment I to form a functional antigen-binding protein, and said antigen-binding protein has one or more of the following properties: it can bind to a target that the reference antigen-binding protein specifically binds to, and the binding ability of said target is 30% or more (e.g., 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% or more) of the binding ability of said reference antigen-binding protein to said target.

[0158] In the present application, the term "antigen-binding fragment II" generally refers to an expression product of nucleic acid fragment II, which is encoded by nucleic acid fragment II. In the antigen-binding fragment II, an antigen-binding fragment II having the following properties can be selected as antigen-binding fragment II': it can form an antigen-binding protein with a reference antigen-binding fragment I, and the antigen-binding protein has one or more of the following properties: it can bind to a target that the reference antigen-binding protein specifically binds to, and the binding ability of the target is 30% or more (e.g., 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% or more) of the binding ability of the reference antigen-binding protein to the target. Not all antigen-binding fragments II are antigen-binding fragment II'. The "antigen-binding fragment II" may be an antibody heavy chain or heavy chain variable region, or a heavy chain fragment, or an antibody light chain or light chain variable region, or a light chain fragment.

[0159] In the present application, the term "antigen-binding fragment II'" generally refers to the expression product of nucleic acid fragment II', which is encoded by nucleic acid fragment II'. Antigen-binding fragment II' can bind to reference antigen-binding fragment I in antigen-binding fragment II to form a functional antigen-binding protein, and the antigen-binding protein has one or more of the following properties: it can bind to a target that the reference antigen-binding protein specifically binds to, and the binding ability of the target is 30% or more (e.g., 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% or more) of the binding ability of the reference antigen-binding protein to the target.

[0160] In the present application, the term "first polynucleotide" generally refers to a polynucleotide comprising a nucleic acid fragment I. In the present application, the first polynucleotide further comprises an endonuclease (e.g., a restriction endonuclease) recognition site at the 5'-end and / or 3'-end. For example, the first polynucleotide comprises, from 5' to 3', the structure R1-nucleic acid fragment I-R2, where R1 and R2 can be recognition sites for a restriction endonuclease. For example, the endonuclease recognition site in the first polynucleotide can be recognition An endonuclease that recognition The first polynucleotide may be enzymatically digested with a restriction endonuclease that is capable of cleaving the nucleic acid fragment I, such as SfiI or BsmBI, and the enzymatically digested first polynucleotide may comprise the nucleic acid fragment I.

[0161] In the present application, the term "first vector polynucleotide" generally refers to a polynucleotide comprising vector fragment I. In the present application, the first vector polynucleotide may further comprise a recognition site for an endonuclease (e.g., a restriction endonuclease) at the 5'-end and / or 3'-end. For example, the first vector polynucleotide may comprise the structure R2-vector fragment I-R3 from 5' to 3', where R2, R3 may be recognition sites for a restriction endonuclease. For example, the endonuclease recognition site in the first vector polynucleotide may be recognition An endonuclease that recognition The first vector polynucleotide may be enzymatically digested with an endonuclease that inhibits the synthesis of the first vector polynucleotide, such as SfiI or BsmBI, and the enzymatically digested first vector polynucleotide may comprise the vector fragment I.

[0162] In the present application, the term "second polynucleotide" generally refers to a polynucleotide comprising a reference nucleic acid fragment II. In the present application, the second polynucleotide may further comprise a recognition site for an endonuclease (e.g., a restriction endonuclease) at the 5'-end and / or 3'-end. For example, the second polynucleotide may comprise the structure R3-reference nucleic acid fragment II-R4 in the 5' to 3' direction, where R3 and R4 may be recognition sites for a restriction endonuclease. For example, the endonuclease recognition site in the second polynucleotide may be recognition An endonuclease that recognition The second polynucleotide may be enzymatically cleaved with an endonuclease that selectively cleaves the reference nucleic acid fragment II, such as SfiI or BsmBI, and the enzymatically cleaved second polynucleotide may comprise the reference nucleic acid fragment II.

[0163] In the present application, the term "second vector polynucleotide" generally refers to a polynucleotide comprising vector fragment II. In the present application, the second vector polynucleotide may further comprise a recognition site for an endonuclease (e.g., a restriction endonuclease) at the 5'-end and / or 3'-end. For example, the second vector polynucleotide may comprise the structure R4-vector fragment II-R1 from 5' to 3', where R4, R1 may be a recognition site for a restriction endonuclease. For example, the endonuclease recognition site in the second vector polynucleotide may be recognition An endonuclease that recognition The second vector polynucleotide may be enzymatically digested with an endonuclease that inhibits the transcription of the ribozyme, such as SfiI or BsmBI, and the enzymatically digested second vector polynucleotide may comprise the vector fragment II.

[0164] In the present application, the term "third polynucleotide" generally refers to a polynucleotide comprising a nucleic acid fragment I'. In the present application, the third polynucleotide may further comprise a recognition site for an endonuclease (e.g., a restriction endonuclease) at the 5'-end and / or 3'-end. For example, the third polynucleotide may comprise, from 5' to 3', the structure R5-nucleic acid fragment I'-R6, where R5 and R6 may be recognition sites for a restriction endonuclease. For example, the endonuclease recognition site in the third polynucleotide may be recognition Endonucleases that bind to R5 and R6 recognition The enzymatically cleaved third polynucleotide may comprise nucleic acid fragment I'.

[0165] In the present application, the term "third vector polynucleotide" generally refers to a polynucleotide comprising vector fragment III. In the present application, the third vector polynucleotide may further comprise a recognition site for an endonuclease (e.g., a restriction endonuclease) at the 5'-end and / or 3'-end. For example, the third vector polynucleotide may comprise the structure R6-vector fragment III-R7 from 5' to 3', where R6 and R7 may be recognition sites for a restriction endonuclease. For example, the endonuclease recognition site in the third vector polynucleotide may be recognition Endonucleases that bind to R6 and R7 recognition The third vector polynucleotide may be enzymatically digested with an endonuclease that inhibits the transcription of the ribozyme, such as SfiI or BsmBI, and the enzymatically digested third vector polynucleotide may comprise the vector fragment III.

[0166] In the present application, the term "fourth polynucleotide" generally refers to a polynucleotide comprising nucleic acid fragment II'. In the present application, the fourth polynucleotide may further comprise a recognition site for an endonuclease (e.g., a restriction endonuclease) at the 5'-end and / or 3'-end. For example, the fourth polynucleotide may comprise the structure R7-nucleic acid fragment II'-R8 from 5' to 3', where R7 and R8 may be recognition sites for a restriction endonuclease. For example, the endonuclease recognition site in the fourth polynucleotide may be recognition Endonucleases that bind to the nuclease (e.g., R7 and R8) recognition The second polynucleotide may be enzymatically cleaved with an endonuclease that selectively cleaves the nucleic acid fragment II', such as SfiI or BsmBI, and the enzymatically cleaved second polynucleotide may comprise the nucleic acid fragment II'.

[0167] In the present application, the term "fourth vector polynucleotide" generally refers to a polynucleotide comprising vector fragment IV. In the present application, the fourth vector polynucleotide may further comprise a recognition site for an endonuclease (e.g., a restriction endonuclease) at the 5'-end and / or 3'-end. For example, the fourth vector polynucleotide may comprise the structure R8-vector fragment IV-R5 from 5' to 3', where R8 and R5 may be recognition sites for a restriction endonuclease. For example, the endonuclease recognition site in the fourth vector polynucleotide may be recognition Endonucleases that bind to the nuclease (e.g., R8 and R5) recognition The fourth vector polynucleotide may be enzymatically cleaved with an endonuclease that inhibits the transcription of the ribozyme, such as SfiI or BsmBI, and the enzymatically cleaved fourth vector polynucleotide may comprise the vector fragment IV.

[0168] In the present application, the term "fifth polynucleotide" generally refers to a polynucleotide comprising a reference nucleic acid fragment I. In the present application, the fifth polynucleotide may further comprise a recognition site for an endonuclease (e.g., a restriction endonuclease) at the 5'-end and / or 3'-end. For example, the fifth polynucleotide may comprise the structure R9-reference nucleic acid fragment I-R10 in the 5' to 3' direction, where R9, R10 may be a recognition site for a restriction endonuclease. For example, the endonuclease recognition site in the fifth polynucleotide may be recognition Endonucleases that bind to the nuclease (e.g., R9 and R10) recognition The fifth polynucleotide may be enzymatically cleaved with an endonuclease that selectively cleaves the reference nucleic acid fragment I, for example, SfiI or BsmBI, and the enzymatically cleaved fifth polynucleotide may comprise the reference nucleic acid fragment I.

[0169] In the present application, the term "fifth vector polynucleotide" generally refers to a polynucleotide comprising a vector fragment V. In the present application, the fifth vector polynucleotide may further comprise a recognition site for an endonuclease (e.g., a restriction endonuclease) at the 5'-end and / or 3'-end. For example, the fifth vector polynucleotide may comprise, from 5' to 3', the structure R10-vector fragment V-R11, wherein R10 and R11 may be recognition sites for a restriction endonuclease. For example, the endonuclease recognition site in the fifth vector polynucleotide may be recognition Endonucleases that bind to R10 and R11 recognition The fifth vector polynucleotide may be enzymatically cleaved with an endonuclease which selectively cleaves the fifth vector polynucleotide, for example SfiI or BsmBI, and the enzymatically cleaved fifth vector polynucleotide may comprise the vector fragment V.

[0170] In the present application, the term "sixth polynucleotide" generally refers to a polynucleotide comprising nucleic acid fragment II. In the present application, the sixth polynucleotide may further comprise a recognition site for an endonuclease (e.g., a restriction endonuclease) at the 5'-end and / or 3'-end. For example, the sixth polynucleotide may comprise the structure R11-nucleic acid fragment II-R12 in the 5' to 3' direction, where R11 and R12 may be recognition sites for a restriction endonuclease. For example, the endonuclease recognition site in the sixth polynucleotide may be recognition Endonucleases that react with R11 and R12 recognition The sixth polynucleotide may be enzymatically cleaved with an endonuclease that inhibits the synthesis of the nucleic acid fragment II, such as SfiI or BsmBI, and the enzymatically cleaved sixth polynucleotide may comprise the nucleic acid fragment II.

[0171] In the present application, the term "sixth vector polynucleotide" generally refers to a polynucleotide comprising vector fragment VI. In the present application, the sixth vector polynucleotide may further comprise a recognition site for an endonuclease (e.g., a restriction endonuclease) at the 5'-end and / or 3'-end. For example, the sixth vector polynucleotide may comprise, from 5' to 3', the structure R12-vector fragment VI-R9, wherein R12, R9 may be a recognition site for a restriction endonuclease. For example, the endonuclease recognition site in the sixth vector polynucleotide may be recognition Endonucleases that bind to the nuclease (e.g., R12 and R9) recognition The sixth vector polynucleotide may be enzymatically digested with an endonuclease that inhibits the transcription of the ribozyme, such as SfiI or BsmBI, and the enzymatically digested sixth vector polynucleotide may comprise the vector fragment VI.

[0172] In this application, the term "first polynucleotide after cleavage" generally refers to a nucleic acid molecule obtained by treating the first polynucleotide with a restriction endonuclease. The "first polynucleotide after cleavage" includes nucleic acid fragment I and ends having a specific sequence obtained by treating both ends with a restriction endonuclease.

[0173] In the present application, the term "cleaved first vector polynucleotide" generally refers to a nucleic acid molecule obtained by treating the first vector polynucleotide with a restriction endonuclease. The "cleaved first vector polynucleotide" includes vector fragment I and ends having specific sequences obtained by treating both ends with the restriction endonuclease.

[0174] In this application, the term "cleaved second polynucleotide" generally refers to a nucleic acid molecule obtained by treating the second polynucleotide with a restriction endonuclease. The "cleaved second polynucleotide" includes the reference nucleic acid fragment II and ends having a specific sequence obtained by treating both ends with a restriction endonuclease.

[0175] In the present application, the term "cleaved second vector polynucleotide" generally refers to a nucleic acid molecule obtained by treating the second vector polynucleotide with a restriction endonuclease. The "cleaved second vector polynucleotide" includes vector fragment II and ends having specific sequences obtained by treating both ends with the restriction endonuclease.

[0176] In the present application, the term "cleaved third polynucleotide" generally refers to a nucleic acid molecule obtained by treating the third polynucleotide with a restriction endonuclease. The "cleaved third polynucleotide" includes a nucleic acid fragment I' and ends having a specific sequence obtained by treating both ends with a restriction endonuclease.

[0177] In the present application, the term "cleaved third vector polynucleotide" generally refers to a nucleic acid molecule obtained by treating the third vector polynucleotide with a restriction endonuclease. The "cleaved third vector polynucleotide" includes the vector fragment III and ends having specific sequences obtained by treating both ends with the restriction endonuclease.

[0178] In the present application, the term "cleaved fourth polynucleotide" generally refers to a nucleic acid molecule obtained by treating the fourth polynucleotide with a restriction endonuclease. The "cleaved fourth polynucleotide" includes the reference nucleic acid fragment II' and ends having a specific sequence obtained by treating both ends with a restriction endonuclease.

[0179] In the present application, the term "cleaved fourth vector polynucleotide" generally refers to a nucleic acid molecule obtained by treating the fourth vector polynucleotide with a restriction endonuclease. The "cleaved fourth vector polynucleotide" includes vector fragment IV and ends having specific sequences obtained by treating both ends with the restriction endonuclease.

[0180] In the present application, the term "cleaved fifth polynucleotide" generally refers to a nucleic acid molecule obtained by treating the fifth polynucleotide with a restriction endonuclease. The "cleaved fifth polynucleotide" comprises the reference nucleic acid fragment I and ends having a specific sequence obtained by treating both ends with a restriction endonuclease.

[0181] In the present application, the term "cleaved fifth vector polynucleotide" generally refers to a nucleic acid molecule obtained by treating the fifth vector polynucleotide with a restriction endonuclease. The "cleaved fifth vector polynucleotide" includes a vector fragment V and ends having specific sequences obtained by treating both ends with the restriction endonuclease.

[0182] In the present application, the term "cleaved sixth polynucleotide" generally refers to a nucleic acid molecule obtained by treating the sixth polynucleotide with a restriction endonuclease. The "cleaved sixth polynucleotide" includes nucleic acid fragment II and ends having a specific sequence obtained by treating both ends with a restriction endonuclease.

[0183] In the present application, the term "cleaved sixth vector polynucleotide" generally refers to a nucleic acid molecule obtained by treating the sixth vector polynucleotide with a restriction endonuclease. The "cleaved sixth vector polynucleotide" includes vector fragment VI and ends having specific sequences obtained by treating both ends with restriction endonucleases.

[0184] In the present application, the term "first screened vector" generally refers to nucleic acid fragment I, reference nucleic acid fragment II, vector fragment I, and vector fragment II circular nucleic acid molecule. In one embodiment, the "first screened vector" refers to a unidirectional ligation of a cleaved first polynucleotide, a cleaved second polynucleotide, a cleaved first vector polynucleotide, and a cleaved second vector polynucleotide. In some cases, the "first screened vector" can be expressed to form an antigen-binding protein. In other cases, the "first screened vector" cannot be expressed to form an antigen-binding protein. If the antigen-binding protein formed by expression of the "first screening target vector" has one or more of the following properties: it can bind to a target that specifically binds to a reference antigen-binding protein, and the binding ability of the target is 30% or more (e.g., 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% or more) of the binding ability of the reference antigen-binding protein to the target, then the "first screening target vector" can be referred to as a "first replacement vector", and nucleic acid fragment I of the "first screening target vector" can be referred to as nucleic acid fragment I'. The "first screening target vector library" comprises one or more first screening target vectors.

[0185] In the present application, the term "first replacement vector" generally includes a circular nucleic acid molecule of nucleic acid fragment I', reference nucleic acid fragment II, vector fragment I and vector fragment II, and refers to a "first replacement vector" that can form an antigen binding protein upon expression, and the antigen binding protein has one or more of the following properties: it can bind to a target that specifically binds to the reference antigen binding protein, and the binding ability of the target is 30% or more of the binding ability of the reference antigen binding protein to the target (e.g., 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% or more). A "first replacement vector library" includes one or more "first replacement vectors", and does not include a first screening subject vector whose expression to form an antigen-binding protein does not have one or more of the following properties: capable of binding to a target that the reference antigen-binding protein specifically binds to, and has a binding ability to the target that is 30% or more of the binding ability of the reference antigen-binding protein to the target (e.g., 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% or more).

[0186] In the present application, the term "second screened vector" generally refers to a circular nucleic acid molecule comprising nucleic acid fragment II, reference nucleic acid fragment I, vector fragment V and vector fragment VI. In one embodiment, the "second screened vector" is directionally ligated to the cleaved fifth polynucleotide, the cleaved sixth polynucleotide, the cleaved fifth vector polynucleotide and the cleaved sixth vector polynucleotide. The "second screened vector" can be expressed to form an antigen binding protein. When the antigen-binding protein formed by expression of the "second screening target vector" has the following properties: it can bind to a target that the reference antigen-binding protein specifically binds to, and the binding ability of the target is 30% or more of the binding ability of the reference antigen-binding protein to the target (e.g., 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% or more), the "second screening target vector" can be referred to as a "second replacement vector", and nucleic acid fragment II in the "second screening target vector" can be referred to as nucleic acid fragment II'. The "second screening target vector library" comprises one or more second screening target vectors.

[0187] In the present application, the term "second replacement vector" generally refers to a circular nucleic acid molecule comprising nucleic acid fragment II', reference nucleic acid fragment I, vector fragment V and vector fragment VI, wherein the "second replacement vector" can be expressed to form an antigen binding protein, and the antigen binding protein has the following properties: it can bind to a target that specifically binds to the reference antigen binding protein, and the binding ability of the target is 30% or more of the binding ability of the reference antigen binding protein to the target (e.g., 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% or more). In the present application, the "second replacement vector library" includes one or more "second replacement vectors", and the antigen binding proteins formed by the expression thereof do not include second screening subject vectors that do not have the following properties: capable of binding to a target that the reference antigen binding protein specifically binds to, and having a binding ability to the target that is 30% or more of the binding ability of the reference antigen binding protein to the target (e.g., 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% or more).

[0188] In this application, the term "double replacement screened vector" generally refers to a circular nucleic acid molecule comprising nucleic acid fragment I', nucleic acid fragment II', vector fragment I and vector fragment II. The "double replacement screened vector" generally refers to a unidirectional ligation of the first polynucleotide after cleavage, the second polynucleotide after cleavage, the first vector polynucleotide after cleavage and the second vector polynucleotide after cleavage. In some cases, the "double replacement screened vector" can be expressed to form an antigen-binding protein. In other cases, the "double replacement screened vector" cannot be expressed to form an antigen-binding protein. When the expression product of the double replacement screening target vector has the following properties: it can bind to a target that specifically binds to a reference antigen-binding protein, and the binding ability of the target is 30% or more of the binding ability of the reference antigen-binding protein to the target (e.g., 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 99% or more, 1-fold or more, 1.5-fold or more, 2-fold or more, 2.5-fold or more, 3-fold or more), the double replacement screening target vector can be called a double replacement vector. A "double replacement screening target vector library" includes one or more "double replacement screening target vectors".

[0189] In this application, the term "cleavage" generally refers to contacting a restriction endonuclease and a polynucleotide under conditions that allow cleavage. The cleavage generally refers to cleaving the bond between the sugar molecule of a nucleotide and the phosphate adjacent to the nucleotide molecule, for example, the cleavage generally refers to cleavage of a phosphodiester bond between two nucleotides. After cleavage using a restriction endonuclease, the polynucleotide can generate ends of specific sequences.

[0190] As used herein, the term "ligation" generally refers to the ligation (joining) of two or more polynucleotide molecules together. For example, the ligation can be achieved by a ligase (e.g., DNA ligase). For example, the 3' end of one polynucleotide is ligated to the 5' end of another polynucleotide, thereby forming one complete polynucleotide molecule. "Directional ligation" generally refers to the ligation of two or more polynucleotide molecules together in a specific order.

[0191] As used herein, the term "introduction" generally refers to the process of transferring or introducing an exogenous polynucleotide into a cell. The cell may be a host cell. The cell to be introduced includes the primary cell to be entrained and its progeny. The cell may be a prokaryotic cell, e.g., a bacterial cell. The cell may be a eukaryotic cell, e.g., a mammalian cell or a yeast cell.

[0192] In the present application, the term "linker" generally refers to a reagent for ligating two or more polynucleotide molecules or fragments thereof. The linker may be a polynucleotide or a fragment thereof. In the present application, the linker may have different lengths. For example, the length of the linker may be 40 bp or more, 50 bp or more, 60 bp or more, 70 bp or more, 80 bp or more, 90 bp or more, 100 bp or more, 150 bp or more, 200 bp or more. In some cases, the linker includes a signal peptide, and the term "signal peptide pelB" generally refers to a signal peptide of a pectinase degrading enzyme, for example, when the antigen binding protein is a Fab. The signal peptide pelB is generally used in prokaryotic expression systems. For example, the signal peptide pelB may have the GenBank accession number ABS75961.1. In some cases, the linker does not include a signal peptide, for example, when the antigen binding protein is a scFv. In some cases, the first polynucleotide may comprise a nucleic acid molecule encoding a VH, and the second polynucleotide may comprise a nucleic acid molecule encoding a VL; in some cases, the first polynucleotide may comprise a nucleic acid molecule encoding a VL, and the second polynucleotide may comprise a nucleic acid molecule encoding a VH.

[0193] In the present application, the term "light chain or fragment thereof" generally refers to an amino acid fragment having the ability to bind to the heavy chain of the same or similar antibody. In the present application, the light chain or fragment thereof may comprise a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region can be divided into kappa type and lambda type. The light chain further includes a light chain having a lambda variable region (V-λ) ligated to a kappa constant region (C-κ) or a kappa variable region (V-κ) ligated to a lambda constant region (C-λ).

[0194] In the present application, the term "heavy chain or a fragment thereof" generally refers to an amino acid fragment having the ability to bind to the light chain of the same or a similar antibody. In the present application, the heavy chain or a fragment thereof has the ability to bind to the light chain of the same or a similar antibody. In the present application, the heavy chain or the heavy chain fragment may comprise a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region may comprise a CH1 structural region, a hinge region, a CH2 structural region and a CH3 structural region. In the case of IgE, IgM and IgY, the heavy chain constant region may comprise a CH4 structure but does not have a hinge region. In the present application, the "heavy chain constant region" may be a CH1, a hinge region, a CH2, a CH3, a CH4 structural region or any combination thereof.

[0195] In this application, the term "restriction endonuclease" generally refers to an enzyme that cuts double-stranded DNA. The restriction endonuclease can generate protruding sticky ends that allow single-stranded DNA to attach to DNA ligase. recognition and restriction cleavage. For example, the cleavage site of the restriction endonuclease is at a certain distance from its recognition site. For example, the restriction endonuclease may be selected from SfiI, BsmBI and Esp3I.

[0196] As used herein, the term "polynucleotide" generally refers to nucleotides, i.e., at least two nucleotides ligated together. The polynucleotide may be a polymer of any length, including, for example, 200, 300, 500, 1000, 2000, 3000, 5000, 7000, 10,000, 100,000, etc. The polynucleotide may include phosphodiester bonds.

[0197] In this application, the term "comprising" generally refers to the inclusion of the explicitly specified features but does not exclude other elements.

[0198] In this application, the term "about" generally refers to a variation within 0.5%-10% above or below a specified numerical value, for example, a variation within 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below a specified numerical value.

[0199] [ Detailed Description of the Invention ]

[0200] The present application provides a method for selecting functional antigen-binding proteins that combines strand displacement technology and antibody display methods (eg, bacteriophage display and mammalian cell display).

[0201] First substitution

[0202] In the present application, the method uses an antibody heavy chain or fragment thereof (or an antibody light chain or fragment thereof) of a reference antigen-binding protein to construct a library to be screened, comprising a plurality of antibody light chains or fragments thereof (or antibody heavy chains or fragments thereof) and a first vector to be screened. The first vector to be screened may be a bacteriophage display vector or a cell display vector, or any other display vector selected based on practical conditions. Specifically, the method includes: a) providing a first polynucleotide, wherein the first polynucleotide comprises R1-nucleic acid fragment I-R2 in a 5' to 3' direction, and the nucleic acid fragment I can encode an antigen-binding fragment I; b) providing a second polynucleotide, wherein the second polynucleotide comprises R3-reference nucleic acid fragment II-R4 in a 5' to 3' direction, and the reference nucleic acid fragment II can encode a reference antigen-binding fragment II, and the reference antigen-binding fragment II can form a reference antigen-binding protein with the reference antigen-binding fragment I encoded by the reference nucleic acid fragment I; c) providing a first vector polynucleotide, wherein the first vector polynucleotide comprises R2-vector fragment I-R3 in a 5' to 3' direction. d) providing a second vector polynucleotide, wherein the second vector polynucleotide comprises R4-vector fragment II-R1 in the 5' to 3' direction; e) cleaving the first polynucleotide, the second polynucleotide, the first vector polynucleotide and the second vector polynucleotide with a restriction endonuclease to obtain the cleaved first polynucleotide, the cleaved second polynucleotide, the cleaved first vector polynucleotide and the cleaved second vector polynucleotide; f) mixing the cleaved first polynucleotide, the cleaved second polynucleotide, the cleaved first vector polynucleotide and the cleaved second vector polynucleotide, thereby unidirectionally ligating them to form a first screening target vector in a circular form. The structure of the first screening target vector can be seen in Figure 1.The method further comprises expressing the vector to be screened and selecting, by comparison with a reference antigen binding protein (or other positive control antibody), an antigen binding protein having the following properties as a functional antigen binding protein: the reference binding protein is capable of binding to a target it can bind to, and the binding ability of the target is at least 30% of the binding ability of the reference antigen binding protein to the target.

[0203] In the present application, the nucleic acid fragment I may encode an antibody light chain or a fragment thereof, the reference nucleic acid fragment II may encode a reference antigen binding protein heavy chain or a fragment thereof, the vector fragment I may include a linker peptide, and the vector fragment II may be derived from a bacteriophage display vector (e.g., pComb3x vector). In some cases, the nucleic acid fragment I encodes an antibody light chain, and the linker peptide includes a nucleic acid sequence encoding a signal peptide pelB or a fragment thereof. In some cases, the nucleic acid fragment I encodes an antibody light chain variable region. The R1 may include a nucleotide sequence as set forth in SEQ ID NO: 1, the R2 may include a nucleotide sequence as set forth in SEQ ID NO: 2, the R3 may include a nucleotide sequence as set forth in SEQ ID NO: 3, and the R4 may include a nucleotide sequence as set forth in SEQ ID NO: 4.

[0204] In the present application, the nucleic acid fragment I may encode an antibody light chain or a fragment thereof, the reference nucleic acid fragment II may encode a reference antigen binding protein heavy chain or a fragment thereof, and the vector fragment I and / or the vector fragment II may be derived from a mammalian cell expression vector (e.g., pDGB4 vector). The R1 may comprise the nucleotide sequence shown in SEQ ID NO: 7, the R2 may comprise the nucleotide sequence shown in SEQ ID NO: 8, the R3 may comprise the nucleotide sequence shown in SEQ ID NO: 5, and the R4 may comprise the nucleotide sequence shown in SEQ ID NO: 6.

[0205] In the present application, the nucleic acid fragment I can encode an antibody heavy chain or a fragment thereof, the reference nucleic acid fragment II can encode a light chain or a fragment thereof of a reference antigen binding protein, the vector fragment II can include a linker peptide, and the vector fragment I can be derived from a bacteriophage display vector (e.g., pComb3x vector). In some cases, the reference nucleic acid fragment II encodes an antibody light chain, and the linker peptide includes a nucleic acid sequence encoding a signal peptide pelB or a fragment thereof. In some cases, the reference nucleic acid fragment II encodes an antibody light chain variable region. The R1 can include a nucleotide sequence as set forth in SEQ ID NO: 3, the R2 can include a nucleotide sequence as set forth in SEQ ID NO: 4, the R3 can include a nucleotide sequence as set forth in SEQ ID NO: 1, and the R4 can include a nucleotide sequence as set forth in SEQ ID NO: 2.

[0206] In the present application, the nucleic acid fragment I may encode an antibody heavy chain or a fragment thereof, the reference nucleic acid fragment II may encode a reference antigen binding protein light chain or a fragment thereof, and the vector fragment I and / or the vector fragment II may be derived from a mammalian cell expression vector (e.g., pDGB4 vector). The R1 may comprise the nucleotide sequence shown in SEQ ID NO: 5, the R2 may comprise the nucleotide sequence shown in SEQ ID NO: 6, the R3 may comprise the nucleotide sequence shown in SEQ ID NO: 7, and the R4 may comprise the nucleotide sequence shown in SEQ ID NO: 8.

[0207] The first screened vector capable of expressing a functional antigen-binding protein is referred to as the first replacement vector, and correspondingly, nucleic acid fragment I of the first screened vector capable of expressing a functional antigen-binding protein is referred to as nucleic acid fragment I' of the first replacement vector.

[0208] Second substitution

[0209] The method of the present application may further include: obtaining nucleic acid fragment I', and then constructing a double replacement screening target vector with nucleic acid fragment II'. The nucleic acid fragment II' can be obtained by any method in the art, for example, by replacing a reference antigen-binding fragment of a reference antigen-binding protein, as long as it can encode antigen-binding protein II', and the antigen-binding fragment II' can bind to the reference antigen-binding fragment I to form an antigen-binding protein having the following properties: it can bind to a target that the reference antigen can bind to, and the binding ability of the target is 30% or more of the binding ability of the reference antigen-binding protein to the target.

[0210] In some cases, the nucleic acid fragment II' is obtained by constructing a second vector to be screened. The method further includes: a) providing a fifth polynucleotide, wherein the fifth polynucleotide comprises, in a 5' to 3' direction, R9-reference nucleic acid fragment I-R10; b) providing a sixth polynucleotide, wherein the sixth polynucleotide comprises, in a 5' to 3' direction, R11-nucleic acid fragment II-R12; c) providing a fifth vector polynucleotide, wherein the fifth vector polynucleotide comprises, in a 5' to 3' direction, R10-vector fragment V-R11; d) providing a sixth vector polynucleotide, wherein the sixth vector polynucleotide comprises, in a 5' to 3' direction, R12-vector fragment V-R11. e) cleaving the fifth polynucleotide, the sixth polynucleotide, the fifth vector polynucleotide and the sixth vector polynucleotide with a restriction endonuclease to obtain a cleaved fifth polynucleotide, a cleaved sixth polynucleotide, a cleaved fifth vector polynucleotide and a cleaved sixth vector polynucleotide; f) mixing the cleaved fifth polynucleotide, the cleaved sixth polynucleotide, the cleaved fifth vector polynucleotide and the cleaved sixth vector polynucleotide, thereby unidirectionally ligating them to form a circularized second screening target vector. The structure of the second screening target vector is shown in FIG. 3. The method further comprises expressing the screening target vector and selecting an antigen-binding protein having the following properties as a functional antigen-binding protein by comparing with a reference antigen-binding protein (or other positive control antibody): the reference binding protein can bind to a target capable of binding, and the binding ability of the target is 30% or more of the binding ability of the reference antigen-binding protein to the target.

[0211] Whether the nucleic acid fragment II encodes an antibody light chain or a fragment thereof or an antibody heavy chain or a fragment thereof depends on the type of nucleic acid fragment I. When one of the polypeptide encoded by nucleic acid fragment II and the polypeptide encoded by nucleic acid fragment I is an antibody light chain or a fragment thereof, the other is an antibody heavy chain or a fragment thereof.

[0212] In the present application, the reference nucleic acid fragment I may encode an antibody light chain or a fragment thereof of a reference antigen binding protein, the nucleic acid fragment II may encode an antibody heavy chain or a fragment thereof, the vector fragment V may include a linker peptide, and the vector fragment VI may be derived from a bacteriophage display vector (e.g., a pComb3x vector). In some cases, the reference nucleic acid fragment I encodes an antibody light chain, and the linker peptide includes a nucleic acid sequence encoding a signal peptide pelB or a fragment thereof. In some cases, the reference nucleic acid fragment I encodes an antibody light chain variable region. The R9 may include a nucleotide sequence as set forth in SEQ ID NO: 1, the R10 may include a nucleotide sequence as set forth in SEQ ID NO: 2, the R11 may include a nucleotide sequence as set forth in SEQ ID NO: 3, and the R12 may include a nucleotide sequence as set forth in SEQ ID NO: 4.

[0213] In the present application, the reference nucleic acid fragment I may encode an antibody light chain or a fragment thereof of a reference antigen binding protein, the nucleic acid fragment II may encode a heavy chain or a fragment thereof, and the vector fragment V and / or the vector fragment VI may be derived from a mammalian cell expression vector (e.g., a pDGB4 vector). The R9 may comprise a nucleotide sequence as shown in SEQ ID NO: 7, the R10 may comprise a nucleotide sequence as shown in SEQ ID NO: 8, the R11 may comprise a nucleotide sequence as shown in SEQ ID NO: 5, and the R12 may comprise a nucleotide sequence as shown in SEQ ID NO: 6.

[0214] In the present application, the reference nucleic acid fragment I is an antibody heavy chain or a fragment thereof capable of encoding a reference antigen binding protein, the nucleic acid fragment II is a light chain or a fragment thereof capable of encoding an antigen binding protein, the vector fragment VI may include a linker peptide, and the vector fragment V may be derived from a bacteriophage display vector (e.g., pComb3x vector). In some cases, the nucleic acid fragment II encodes an antibody light chain, and the linker peptide includes a nucleic acid sequence encoding a signal peptide pelB or a fragment thereof. In some cases, the nucleic acid fragment II encodes an antibody light chain variable region. The R9 may include a nucleotide sequence as set forth in SEQ ID NO: 3, the R10 may include a nucleotide sequence as set forth in SEQ ID NO: 4, the R11 may include a nucleotide sequence as set forth in SEQ ID NO: 1, and the R12 may include a nucleotide sequence as set forth in SEQ ID NO: 2.

[0215] In the present application, the reference nucleic acid fragment I may encode an antibody heavy chain or a fragment thereof, the nucleic acid fragment II may encode a reference antigen binding protein light chain or a fragment thereof, and the vector fragment V and / or the vector fragment VI may be derived from a mammalian cell expression vector (e.g., pDGB4 vector). The R9 may comprise the nucleotide sequence shown in SEQ ID NO: 5, the R10 may comprise the nucleotide sequence shown in SEQ ID NO: 6, the R11 may comprise the nucleotide sequence shown in SEQ ID NO: 7, and the R12 may comprise the nucleotide sequence shown in SEQ ID NO: 8.

[0216] double substitution

[0217] The method of the present application further includes: a) obtaining nucleic acid fragment I' and nucleic acid fragment II', wherein one of the polypeptides encoded by nucleic acid fragment I' and nucleic acid fragment II' is an antibody light chain or a fragment thereof and the other is an antibody heavy chain or a fragment thereof; b) providing a third polynucleotide, wherein the third polynucleotide comprises, in a 5' to 3' direction, R5-nucleic acid fragment I'-R6; c) providing a fourth polynucleotide, wherein the fourth polynucleotide comprises, in a 5' to 3' direction, R7-nucleic acid fragment II'-R8, wherein the nucleic acid fragment II' is capable of encoding an antigen binding protein II', and the antigen binding fragment II' is capable of binding to the reference antigen binding fragment I to form an antigen binding protein having the following properties: capable of binding to a target capable of binding to the reference antigen binding, and the binding ability of the target is at least 30% of the binding ability of the reference antigen binding protein to the target. d) providing a third vector polynucleotide, wherein the third vector polynucleotide comprises R6-vector fragment III-R7 in the 5' to 3' direction; e) providing a fourth vector polynucleotide, wherein the fourth vector polynucleotide comprises R8-vector fragment IV-R5 in the 5' to 3' direction; f) cleaving the third polynucleotide, the fourth polynucleotide, the third vector polynucleotide and the fourth vector polynucleotide with a restriction endonuclease to obtain a cleaved third polynucleotide, a cleaved fourth polynucleotide, a cleaved third vector polynucleotide and a cleaved fourth vector polynucleotide; g) mixing the cleaved third polynucleotide, the cleaved fourth polynucleotide, the cleaved third vector polynucleotide and the cleaved fourth vector polynucleotide, thereby ligating them in a certain direction to form a double replacement screening target vector in a circular form. The structure of the double replacement screening target vector can be seen in FIG. 2.The method further comprises expressing the screening vector and selecting, by comparison with a reference antigen binding protein (or other positive control antibody), an antigen binding protein having the following properties as a functional antigen binding protein: the reference binding protein is capable of binding to a target that it can bind to, and the binding ability of the target is 30% or more of the binding ability of the reference antigen binding protein to the target.

[0218] In the present application, the nucleic acid fragment I' can encode an antibody light chain or a fragment thereof of a reference antigen binding protein, the nucleic acid fragment II' can encode an antibody heavy chain or a fragment thereof, the vector fragment III can include a linker peptide, and the vector fragment IV can be derived from a bacteriophage display vector (e.g., pComb3x vector). In some cases, the nucleic acid fragment I' encodes an antibody light chain, and the linker peptide includes a nucleic acid sequence encoding a signal peptide pelB or a fragment thereof. In some cases, the nucleic acid fragment I' encodes an antibody light chain variable region. The R5 can include a nucleotide sequence as set forth in SEQ ID NO: 1, the R6 can include a nucleotide sequence as set forth in SEQ ID NO: 2, the R7 can include a nucleotide sequence as set forth in SEQ ID NO: 3, and the R8 can include a nucleotide sequence as set forth in SEQ ID NO: 4.

[0219] In the present application, the nucleic acid fragment I' can encode the antibody light chain or a fragment thereof of the reference antigen binding protein, the nucleic acid fragment II' can encode the heavy chain or a fragment thereof, and the vector fragment III and / or the vector fragment IV can be derived from a mammalian cell expression vector (e.g., pDGB4 vector). The R5 can comprise the nucleotide sequence shown in SEQ ID NO: 7, the R6 can comprise the nucleotide sequence shown in SEQ ID NO: 8, the R7 can comprise the nucleotide sequence shown in SEQ ID NO: 5, and the R8 can comprise the nucleotide sequence shown in SEQ ID NO: 6.

[0220] In the present application, the nucleic acid fragment I' can encode a reference antigen-binding protein-specific antibody heavy chain or a fragment thereof, the nucleic acid fragment II' can encode an antigen-binding protein-specific light chain or a fragment thereof, the vector fragment IV can include a linker peptide, and the vector fragment III can be derived from a bacteriophage display vector (e.g., pComb3x vector). In some cases, the nucleic acid fragment II' encodes an antibody light chain, and the linker peptide includes a nucleic acid sequence encoding a signal peptide pelB or a fragment thereof. In some cases, the nucleic acid fragment II' encodes an antibody light chain variable region. The R5 can include a nucleotide sequence as set forth in SEQ ID NO: 3, the R6 can include a nucleotide sequence as set forth in SEQ ID NO: 4, the R7 can include a nucleotide sequence as set forth in SEQ ID NO: 1, and the R8 can include a nucleotide sequence as set forth in SEQ ID NO: 2.

[0221] In the present application, the nucleic acid fragment I' can encode an antibody heavy chain or a fragment thereof, the nucleic acid fragment II' can encode a light chain or a fragment thereof of a reference antigen binding protein, the vector fragment III and / or the vector fragment IV can be derived from a mammalian cell expression vector (e.g., pDGB4 vector), the R5 can comprise the nucleotide sequence shown in SEQ ID NO: 5, the R6 can comprise the nucleotide sequence shown in SEQ ID NO: 6, the R7 can comprise the nucleotide sequence shown in SEQ ID NO: 7, and the R8 can comprise the nucleotide sequence shown in SEQ ID NO: 8.

[0222] The method of the present application can be used to simultaneously select functional antigen-binding proteins targeting multiple targets and multiple binding sites for one target. When using the method of the present application and when constructing the first screening target vector and / or the second screening target vector, multiple reference antigen-binding proteins that bind to different epitopes for the same antigen can be used simultaneously to obtain a screening target vector containing multiple reference antigen-binding fragments, which are used to obtain multiple fragments (nucleic acid fragment I' or nucleic acid fragment II') of functional antigen-binding proteins that code for the same antigen but bind to different epitopes. When using the method of the present application, multiple reference antigen-binding proteins for different antigens can also be used simultaneously to obtain a screening target vector containing multiple reference antigen-binding fragments, which are used to obtain fragments (nucleic acid fragment I' or nucleic acid fragment II') of functional antigen-binding proteins that code for different antigens.

[0223] For example, the vector library to be first screened may include vectors to be first screened that include a plurality of reference nucleic acid fragments II, where the reference nucleic acid fragment I encodes fragments of two, three, four or more reference antigen-binding proteins that bind to the same antigen. For example, the vector library to be second screened may include vectors to be second screened that include a plurality of reference nucleic acid fragments I, where the reference nucleic acid fragment I encodes fragments of two, three, four or more reference antigen-binding proteins that bind to the same antigen.

[0224] For example, the first screened vector library may include a first screened vector comprising a plurality of reference nucleic acid fragments II, where the reference nucleic acid fragments II encode fragments of different reference antigen-binding proteins that bind to a plurality of antigens (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more). For example, the second screened vector library may include a second screened vector comprising a plurality of reference nucleic acid fragments I, where the reference nucleic acid fragments I encode fragments of different reference antigen-binding proteins that bind to a plurality of antigens (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more).

[0225] Thus, a double-permutation screened vector library of one or more double-permutation screened vectors may include multiple (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or more) antigen binding proteins against multiple (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) antigens.

[0226] For the construction of the screening vector used for bacteriophage expression in this application, please refer to PCT International Application PCT / CN2020 / 085706.

[0227] In the present application, the linker may include a nucleic acid sequence of a fragment encoding signal peptide pelB, and the remaining nucleic acid sequence of the fragment encoding signal peptide pelB may be further located at the recognition site of the polynucleotide. For example, the 3'-terminal nucleotide of the nucleic acid sequence of the fragment encoding signal peptide pelB may be located at the enzyme cleavage site. For example, when the two cleaved polynucleotides are ligated, the nucleic acid sequence of the fragment encoding signal peptide pelB contained in one polynucleotide may be ligated to the nucleic acid sequence of the fragment encoding signal peptide pelB contained in another polynucleotide, thereby forming a nucleic acid sequence encoding the complete signal peptide pelB.

[0228] In the present application, the length of the linker may be about 50 to about 200 bases. For example, the length of the linker may be about 50 to about 200 bases, about 50 to about 180 bases, about 50 to about 160 bases, about 50 to about 140 bases, about 50 to about 120 bases, about 50 to about 100 bases, about 50 to about 90 bases, about 50 to about 80 bases, about 50 to about 75 bases, about 50 to about 70 bases, or about 50 to about 60 bases.

[0229] In the present application, the ends generated after cleavage of any one of the restriction endonucleases R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11 and R12 may be non-palindromic.

[0230] When selecting an endonuclease recognition site (e.g., in the case of R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11 and R12), an antibody gene library can be selected that prevents destruction (e.g., degradation) during enzyme cleavage by retaining the integrity of the functional region (e.g., light chain variable region or heavy chain variable region) of the antigen-binding fragment that is encoded by the sequence that is not substantially contained in the nucleic acid fragment (e.g., nucleic acid fragment I, nucleic acid fragment II, nucleic acid fragment I', nucleic acid fragment II', reference nucleic acid fragment I, reference nucleic acid fragment II') as much as possible. Furthermore, the end formed after cleavage by the restriction endonuclease may be a non-palindromic sequence, thereby preventing self-ligation and further realizing a reduction in non-target ligation. Furthermore, the selection of the endonuclease recognition site allows unidirectional ligation of multiple fragments, thereby improving ligation efficiency.

[0231] In the present application, two or more of R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11 and R12 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12) are selected from the group consisting of R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11 and R12, and R12 ... recognition and can be cut off.

[0232] In the present application, the restriction endonuclease may be selected from SfiI, BsmBI and Esp3I. In the present application, the BsmBI and Esp3I may be isozymes, which share the same restriction endonuclease recognition site. recognition can.

[0233] For example, SfiI encodes the 13-base sequence (5' to 3') GGCCNNNN / NGGCC (SEQ ID NO:9). recognitionIt can be digested with SfiI to form an overhang at the 3' end (e.g., a single-stranded sequence containing three bases), where N can represent any one of the four bases of GATC. Thus, 64 different sequences can be produced by SfiI. recognition will be done.

[0234] For example, BsmBI and Esp3I recognize the 12-base sequence (5' to 3') CGTCTCN / NNNNN (SEQ ID NO:10). recognition It can be digested with enzymes to form an overhang at the 5' end (e.g., a single-stranded sequence containing 4 bases), where N can represent any one of the four bases of GATC. Thus, 264 different sequences can be generated by BsmBI and Esp3I. recognition will be done.

[0235] In the present application, the bacteriophage display vector or mammalian cell display vector (e.g., pDGB4 and / or pComb3x vector) can be modified / altered to construct the vector of the present application. For example, one or more endonuclease recognition sites can be removed from the vector by site-directed mutagenesis. In some cases, one or more endonuclease recognition sites can be increased in the vector by site-directed mutagenesis.

[0236] For example, the pComb3x vector can be modified to meet the purpose of the present application. For example, the pComb3x vector can include one SfiI recognition site located at the 5' end and one SfiI recognition site located at the 3' end, and the modification can be performed by removing the SfiI recognition site at the 3' end in the pComb3x vector by site-directed mutagenesis. In some cases, the site-directed mutagenesis does not affect the in-frame expression of proteins (e.g., antibody heavy chain fragments and / or antibody light chain fragments) in the vector. For example, the mutation can be a nonsense mutation, e.g., a mutation that only changes the base sequence but does not change the amino acid sequence.

[0237] In some cases, when mammalian cell expression is used for the first screened vector, the second screened vector and / or the screened double replacement vector, the vector fragment (e.g., Vector Fragment I, Vector Fragment II, Vector Fragment III, Vector Fragment IV, Vector Fragment V and Vector Fragment VI) may be derived from a vector capable of expressing any one gene of interest. For example, the vector fragment may be a fragment derived from the display vector pDGB4 (for pDGB4, see Ivan Zhou, et al., "Four-way ligation for construction of a mammalian cell-based full-length antibody display library", Acta Biochim Biophys Sin 2011, 43: 232-238).

[0238] The vector fragments (e.g., Vector Fragment I, Vector Fragment II, Vector Fragment III, Vector Fragment IV, Vector Fragment V, and Vector Fragment VI) of the present application may contain, but are not limited to, a promoter gene, an enhancer, a signal peptide, a screening marker (e.g., an enzyme recognition site, an resistance gene, a reporter gene, a screening gene), and those skilled in the art may adjust the vector fragments based on the desired function (such as inserting / substituting and / or deleting the nucleotide sequence having the specific function). In some cases, the vector fragments may be adjusted under different conditions to obtain different nucleotide sequences.

[0239] In the present application, the vector may be provided with one or more suitable markers (e.g., purification / recognition / A marker for use in screening), which may be, for example, a His tag, a Flag tag, a fluorescent protein, a selective antibiotic, and / or avidin.

[0240] Depending on the length or nature of the antigen-binding fragment to be expressed and the length or nature of the enzyme cleavage site, vector fragments of various lengths or types (e.g., vector fragment I, vector fragment II, vector fragment III, vector fragment IV, vector fragment V and / or vector fragment VI) can be selected.

[0241] In the present application, the nucleic acid fragment I and the nucleic acid fragment II can be obtained by a sample material. In the present application, the sample material may include material derived from a peripheral blood lymphocyte sample. In the present application, the peripheral blood lymphocytes may be human peripheral blood lymphocytes. In the present application, the sample material may be derived from any other tissue and / or cell, and is not limited to the peripheral blood lymphocyte sample.

[0242] For example, the nucleic acid fragment I and the nucleic acid fragment II may be obtained by a library of antibody light chains or fragments thereof already constructed in the prior art, or a library of antibody heavy chains or fragments thereof already constructed in the prior art. The method includes adding the enzyme cleavage site of the present application to both ends of one or more antibody heavy chains or fragments thereof, or one or more antibody light chains or fragments thereof.

[0243] In the present application, a number of methods can be used to detect the binding ability of the expression products of the first screened vector, the second screened vector and / or the double replacement screened vector to the target. The binding ability of the expression products of the screened vectors to a reference antigen-binding protein or other positive control can be compared by quantitative or non-quantitative methods.

[0244] In some aspects, the binding activity of the antigen binding proteins of the present application to a target can be measured by known methods, such as, for example, enzyme-linked immunosorbent assay (ELISA), Western blot (e.g., Western blotting), flow cytometry (e.g., FACS), immunohistochemistry, immunofluorescence, etc. In some cases, the binding affinity can be measured by surface plasmon resonance (SPR), enzyme-linked immunosorbent assay (ELISA), bound antigen precipitation, equilibrium dialysis, biolayer interferometry (BLI). In some cases, the binding affinity and KD value of the PD-1 antigen binding protein to PD-1 can be measured by biolayer interferometry (BLI). For example, it can be measured using an ELISA method. For example, under conditions in which the antigen-binding protein and / or the reference antigen-binding protein can bind to the target, the expression product of the first screened vector, the second screened vector and / or the double-replacement vector to be screened and / or the reference antigen-binding protein are contacted with the target (e.g., an antigen), and whether or not a complex is formed between the expression product of the first screened vector, the second screened vector and / or the double-replacement vector to be screened and the target is detected, and whether or not a complex is formed between the reference antigen-binding protein and the target is detected.

[0245] For the quantitative method, the target binding capacity is determined by the EC 50 The EC value may include a KD value for binding to the target, an OD value of the complex formed upon binding to the target, and / or an absorbance value of the complex formed upon binding to the target. 50 value, KD value, OD value of a complex formed by binding to a target and / or absorbance of a complex formed by binding to a target of the first screening target vector, the second screening target vector and / or the screening target double replacement vector, and EC 50The expression product may be considered to be capable of specifically binding to the target if the EC value, KD value, OD value and / or absorbance of the complex formed upon binding to the target is 30% or more (e.g., 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100%, 110% or 120% or more). 50 The expression product may also be considered to be capable of specifically binding to the target if the absorbance value, KD value, OD value of the complex and / or the absorbance of the complex formed by the binding of the first screened vector, the second screened vector and / or the double replacement vector to be screened to the target to a target is at least twice (e.g., 2.5 times, 3 times, 3.5 times or more) that of a negative control in which no expression product of the first screened vector, the second screened vector, the double replacement vector to be screened, and / or the reference antigen-binding protein is present.

[0246] On the other hand, the present application provides a method for producing said functional antigen binding protein, comprising using said first screened vector, said second screened vector and / or said screened double replacement vector.

[0247] On the other hand, the present application provides a functional antigen-binding protein obtainable by said method of the present application.

[0248] Without being limited by any theory, the following examples are merely illustrative of the methods of the present application and functional antigen-binding proteins obtainable by the methods of the present application, and should not be used to limit the scope of the invention of the present application.

[0249] Working Example

[0250] Example 1: Production of TG1 bacteria carrying 16 reference antibody Fab bacteriophage display vectors

[0251] 1.1 Synthesis of full-length light and heavy chain variable region genes of 16 reference antibodies:

[0252] A literature search obtained the amino acid sequences of the full-length light and heavy chain variable regions of the following 16 reference antibodies: Secukinumab, Selicrelumab, APX-005M, Tafasitamab, Ublituximab, Epratuzumab, Relatlimab, Leramilimab, Coblimab, Tiraglumab, Vibostolimab, Etigilimab, Amatuximab, TRX-518, Bleselumab, Iscalimab.

[0253] For example, a literature search provided the amino acid sequences of the full-length light chain and full-length heavy chain of Selicrelumab (see associated sequence information in WO2018220100), where the amino acid sequence of the full-length light chain of Selicrelumab is set forth in SEQ ID NO. 11, and the amino acid sequence of the full-length heavy chain of Selicrelumab is set forth in SEQ ID NO. 12.

[0254] The amino acid sequences of the full-length light and heavy chain variable regions of selicrelumab were converted into base sequences that could be expressed in E. coli, and base sequences containing appropriate enzyme cleavage sequences and / or signal peptide sequences that match the bacteriophage display vector were added to both ends of the sequence, the gene was synthesized, and inserted into the pUC57 vector (synthesized by GENEWIZ).

[0255] Here, the nucleotide sequence containing the synthesized full-length light chain was shown in SEQ ID NO. 13, and the nucleotide sequence containing the synthesized heavy chain variable region was shown in SEQ ID NO. 14.

[0256] The amino acid sequences of the full-length light and heavy chain variable regions of the 16 reference antibodies were converted into base sequences that could be expressed in Escherichia coli, and base sequences containing appropriate enzyme cleavage sequences and / or signal peptide sequences that match the bacteriophage display vector were added to both ends of the sequences, the genes were synthesized, and the sequences were inserted into the pUC57 vector (GENEWIZ).

[0257] 1.2 Construction of display vectors for 16 reference antibody Fab bacteriophages

[0258] The pUC57 vector containing the 16 full-length light chain nucleotide sequences synthesized was cleaved with SfiI enzyme to obtain 16 full-length light chain fragments, which are the first polynucleotides in FIG.

[0259] The pUC57 vector containing the 16 synthesized heavy chain variable region base sequences was double-digested with SfiI+Esp3I enzymes to obtain 16 heavy chain variable region fragments, which are the second polynucleotides in FIG.

[0260] The obtained 16 full-length light chain fragments (i.e., the first polynucleotide in FIG. 1 of the present application) and 16 heavy chain variable region fragments (i.e., the second polynucleotide in FIG. 1 of the present application) were subjected to 16 4-fragment ligation with the first vector polynucleotide and the second vector polynucleotide shown in FIG. 1 according to the light chain fragment corresponding to the reference antibody, and the base sequence of the clone was sequenced and analyzed to obtain 16 bacteriophage display vectors of positive reference antibody Fab. For the description and preparation of the first vector polynucleotide and the second vector polynucleotide, please refer to the relevant steps of patent application WO2020216191A1.

[0261] For example, the obtained full-length light chain fragment of Selicrelumab (first polynucleotide) was subjected to four-fragment ligation with the heavy chain variable region fragment of Selicrelumab (second polynucleotide) and the first and second vector polynucleotides shown in Figure 1, and the base sequence of the clone was analyzed by sequencing to obtain a bacteriophage display vector of the positive reference antibody Selicrelumab-Fab. For the description and preparation of the first and second vector polynucleotides, please refer to the relevant steps of WO2020216191A1.

[0262] Specifically, the first vector polynucleotide is described in Example 1.6 of WO2020216191A1: the linker component plasmid or the linker-preserving vector in Example 1.4.4 is used as a template, and a 0.8 kb fragment containing the linker is amplified using the linker forward primer (SEQ ID NO: 79 of WO2020216191A1) and the linker reverse primer (SEQ ID NO: 80 of WO2020216191A1), and then the 0.8 kb PCR product is enzymatically cleaved using restriction endonucleases R3 and R4, and purified and recovered by gel electrophoresis (using small fragment gel to recover reagent kit, purchased from Laifeng Biological, Cat# DK402) to obtain a linker fragment of 72 pb.

[0263] The second vector polynucleotide is described in WO2020216191A1, Example 1.8: the display vector DDB-R1R2R5R6 prepared in Example 1.7 was enzymatically digested with restriction endonuclease R7 and restriction endonuclease R8 to obtain a 3.6 kb display vector fragment.

[0264] 1.3 Production of TG1 bacteria carrying 16 reference antibody Fab bacteriophage display vectors

[0265] The bacteriophage display vectors of the 16 reference antibody Fabs were introduced into electrocompetent cells (Lucigen, Cat#: 60502) to obtain TG1 bacteria carrying each of the reference antibody Fab bacteriophage display vectors.

[0266] For example, a bacteriophage display vector of the positive reference antibody Selicrelumab-Fab was introduced into a TG1-derived bacterium to obtain TG1 bacteria carrying the reference antibody Selicrelumab-Fab bacteriophage display vector.

[0267] Example 2 Construction of multiple reference antibody light chain replacement gene libraries, bacterial libraries and bacteriophage libraries

[0268] 1. The total human-derived Kappa light chain and Lambda light chain sub-libraries were enzymatically digested with SfiI to prepare Kappa light chain library and Lambda light chain library insert fragments.

[0269] 2. The resulting Kappa light chain library and Lambda light chain library insert fragments (first polynucleotide), 16 reference antibody heavy chain variable region fragments (second polynucleotide), and the first vector polynucleotide and second vector polynucleotide shown in Figure 1 (see the patent for library construction) are ligated to construct a light chain replacement library. The four fragments were mixed in an equal molecular ratio of 1:1:1:1, with a total amount of 2345 ng, and ligated overnight at 20 degrees (15 hours).

[0270] 3. The ligation product was purified to obtain 1062 ng of the ligation product of the light chain replacement library (gene library).

[0271] 4. The three TG1-induced strains were transformed, and the total volume of the bacterial solution was 6000 μl. After incubation at 37°C / 250 rpm for 60 minutes, 10 μl of the bacterial solution was taken to titrate the ligation transformation efficiency. Eight 10-fold dilutions were made, and 5 μl of each dilution was taken on a plate and incubated at 32°C overnight.

[0272] 5. The remaining bacterial solution was diluted with 220 ml of 2YT-Amp-2% glucose culture medium, and cultured at 37°C and 250 rpm for 3 hours to reach OD 600 = 0.6 (bacterial library) was measured.

[0273] 6. 50 ml of the bacterial library solution from step 7 was transferred to a 150 ml shake flask and cultured at 37 degrees and 250 rpm for 4 hours. The bacteria were then collected by centrifugation, suspended in 4 ml of 2TY-Amp-7% DMSO, divided into two tubes, and frozen at minus 80 degrees.

[0274] 7. Add 3 ml of helper bacteriophage (2.9 x 10 13 / ml) was added and incubated at 37°C for 40 minutes, followed by incubation at 37°C, 250 rpm for 40 minutes.

[0275] 8. The bacteria infected with the helper bacteriophage were collected by centrifugation, centrifuged at 3500G for 15 minutes, the supernatant was discarded, and the cultured bacteria were resuspended in 500 ml of 2YT-Amp-Kan and grown overnight at 30°C, 250 rpm.

[0276] 9. The next day, based on the titration and plate spreading results from step 4, the library capacity of the light chain-substituted bacterial library was analyzed and calculated to be 1.2x10 9 The bacteria cultured overnight in step 10 were centrifuged to precipitate, and 500 ml of the supernatant was taken and mixed uniformly with 125 ml of 20% PEG-5M NaCl, incubated in an ice bath for 2 hours, and the light chain-substituted bacteriophage library was then collected by centrifugation.

[0277] The light chain-substituted bacteriophage library precipitated by centrifugation was suspended in 10.5 ml of PBS, and centrifuged at 18,000 g for 5 minutes at 4°C using a tabletop high-speed centrifuge. The supernatant was removed and the OD 280 was measured and the light chain-substituted bacteriophage library concentration was calculated: OD 280 = 13.7, bacteriophage concentration 3.2x10 13 / ml.

[0278] 11. The light chain-substituted bacteriophage library was frozen and stored: 5 ml of bacteriophage library + 2 ml of 80% autoclaved glycerol + 0.5 ml of PBS = 7.5 ml were mixed uniformly and diluted to 0.5 ml / 1x10 13 The solution was dispensed into tubes and frozen at -80°C.

[0279] Example 3 Construction of multiple reference antibody heavy chain replacement gene libraries, bacterial libraries and bacteriophage libraries

[0280] 1. The entire human-derived heavy chain variable region (Vh) sub-library was subjected to double enzyme digestion with SfiI and Esp3I to generate Vh library insert fragments.

[0281] 2. Referring to the steps described in Example 1, the obtained 16 reference antibody light chains were inserted into a fragment (first polynucleotide), and the Vh library was inserted into a fragment (second polynucleotide) to perform 4-fragment ligation with the first vector polynucleotide and the second vector polynucleotide shown in Figure 1 to construct a light chain replacement library. The four fragments were mixed in a ratio of 1:1:1:1 in terms of the number of molecules, etc., with a total amount of 2345 ng, and ligated overnight at 20°C (15 hours).

[0282] 3. The ligation product was purified to obtain 1134 ng of the ligation product of the heavy chain displacement library (gene library).

[0283] 4. The three TG1-induced strains were transformed with a total volume of 6000 μl of the bacterial solution, and then cultured at 37°C / 250 rpm for 60 minutes. 10 μl of the bacterial solution was then taken to titrate the ligation transformation efficiency. 8 10-fold dilutions were made, and 5 μl of each dilution was spread onto a plate and cultured at 32°C overnight.

[0284] 5. The remaining bacterial solution was diluted with 220 ml of 2YT-Amp-2% glucose culture medium, and cultured at 37°C and 250 rpm for 3 hours to reach OD 600 = 0.6 (bacterial library).

[0285] 6. 50 ml of the bacterial library solution from step 7 was transferred to a 150 ml shake flask and cultured at 37°C and 250 rpm for 4 hours. The bacteria were then collected by centrifugation, suspended in 4 ml of 2TY-Amp-7% DMSO, divided into two tubes, and frozen at -80°C.

[0286] 7. Add 3 ml of helper bacteriophage (2.9 x 10 13 / ml) was added, the mixture was left stationary at 37°C for 40 minutes, and then cultured at 37°C and 250 rpm for 40 minutes.

[0287] 8. The bacteria infected with the helper bacteriophage were collected by centrifugation, centrifuged at 3500G for 15 minutes, the supernatant was discarded, and the cultured bacteria were resuspended in 500 ml of 2YT-Amp-Kan and grown overnight at 30°C, 250 rpm.

[0288] 9. The next day, based on the results of the plate titration in step 4, the library capacity of the heavy chain-substituted bacterial library was analyzed and calculated to be 1.2x10 9 The bacteria cultured overnight in step 10 were centrifuged and precipitated, and 500 ml of the supernatant was taken and mixed uniformly with 125 ml of 20% PEG-5M NaCl, incubated in an ice bath for 2 hours, and then centrifuged to collect the heavy chain-substituted bacteriophage library.

[0289] The heavy chain-substituted bacteriophage library precipitated by centrifugation was suspended in 10.5 ml of PBS, and centrifuged at 4°C and 18,000 g for 5 minutes using a tabletop high-speed centrifuge. The supernatant was removed and the OD 280 was measured and the heavy chain-substituted bacteriophage library concentration was calculated: OD 280 = 20.3, bacteriophage concentration 4.7x10 13 / ml.

[0290] 11. The heavy chain replacement bacteriophage library was frozen and stored: 5 ml of the bacteriophage library + 2 ml of 80% autoclaved glycerol + 0.5 ml of PBS = 7.5 ml were mixed uniformly and diluted with 0.5 ml / 1x10 13 The solution was dispensed into tubes and frozen at -80°C.

[0291] Example 4 Preparation of Tg1 with Reference Antibody (Her3 / Patritumab) Fab Bacteriophage Display Vector and Construction of Single Reference Antibody (Patritumab) Light Chain Substituted and Heavy Chain Substituted Gene Libraries, Bacterial Libraries and Bacteriophage Libraries

[0292] 1. Referring to the flow of Example 1, Tg1 bacteria carrying a reference antibody (Patritumab) Fab bacteriophage display vector was prepared. Here, the relevant CDR sequence of Patritumab can be found in CN 102633881B. The light and heavy chain sequences of Patritumab can be found in US 7705130B2.

[0293] 2. Construction of a multiple reference antibody light chain / heavy chain substitution library A light chain substitution library and a heavy chain substitution library of a single reference antibody Patritumab were constructed by referring to the flow chart.

[0294] 3. In the two ligation systems of the light chain replacement library and the mass replacement library, the total amount of ligated fragments was 1173 ng each, the ligation time was 6 hours, and the ligation products were purified to obtain 550 ng each.

[0295] 4. 250ng of each was taken and transformed into TG1-induced bacteria. The volume of the transformed bacteria solution was 2ml each, and 10μl of each was taken and titrated, and the remaining bacteria solution was spread on one large dish and one medium dish each. It was cultured at 32℃ overnight.

[0296] On day 5.2, the library capacity of the bacterial library was calculated based on the titration results, and the library capacity was 2.4 x 10e8 for the heavy chain replacement library and 4.4 x 10e8 for the light chain replacement library. All colonies of each of the two bacterial libraries were collected, and 50 ml of culture medium (2YT-0.2% glucose-Amp) and an appropriate amount of bacterial solution were added to each library, and the OD 600 was measured to be around 0.12.

[0297] 6. Incubate at 37°C and 250 rpm for 50 minutes and measure OD 600 The pH was measured to be around 0.25. 250 μl of helper bacteriophage (OD280=13) was added to each well. After uniform mixing, the mixture was incubated at 37°C for 30 minutes at rest, and then incubated at 37°C for 30 minutes at 250 rpm.

[0298] 7. Collect the bacteria by centrifugation, resuspend the bacteria in 100 ml of each culture medium (2YT-Amp-Kana), and grow them at 30°C, 250 rpm for 6 hours (3 pm-9 pm). Centrifuge to precipitate the bacteria, collect the supernatant, add 25 ml of 5xPEG-NaCl to each, mix uniformly, and then precipitate the bacteriophage overnight at 4°C.

[0299] On day 8.3, the light chain-substituted bacteriophage library and the heavy chain-substituted bacteriophage library were collected by centrifugation. Each bacteriophage library was suspended in 2 ml of PBS, dispensed into four tubes, and frozen at minus 80°C.

[0300] Example 5 Construction of a Single Reference Antibody (CD40 / Selicrelumab) Heavy Chain Replacement Gene Library, Bacterial Library, and Bacteriophage Library

[0301] 1. Construction of a Multiple Reference Antibody Heavy Chain Substitution Library A heavy chain substitution library of a single reference antibody, Selicrelumab, was constructed with reference to the flow chart (Example 3).

[0302] 2. Referring to the flow of Example 1, the obtained Selicrelumab light chain was inserted into a fragment (first polynucleotide), and the all-human Vh library was inserted into a fragment (second polynucleotide), followed by 4-fragment ligation with the first vector polynucleotide and the second vector polynucleotide shown in Figure 1 to construct a heavy chain replacement library. The four fragments were mixed in a ratio of 1:1:1:1 in terms of the number of molecules, etc., with a total amount of 1175 ng, and ligated overnight at 20°C (15 hours).

[0303] 3. The ligation product was purified to obtain 513 ng of the ligation product of the heavy chain displacement library (gene library).

[0304] 4. 300ng was used to transform TG1-induced bacteria, the total volume of the transformed bacteria solution was 2000μl, and after culturing at 37℃ / 250rpm for 60 minutes, 10μl of the culture solution was taken to titrate the ligation transformation efficiency. 8 10-fold dilutions were made, and 5μl of each dilution was placed on a plate and cultured at 32℃ overnight.

[0305] 5. The remaining bacterial liquid was spread onto two large plates and cultured overnight at 32°C.

[0306] 6. The next day, based on the titration results from step 4, the library capacity of the heavy strand displacement library was analyzed and calculated to be 3.2x10 8 In step 5, all the colonies from the two overnight plates were harvested, and the total volume of the culture was 15 ml. The OD of the harvested culture was 600 = 20. 3 ml of the solution was taken, DMSO (final concentration 7%) was added, and the solution was divided into two tubes and frozen at -80°C (bacterial library).

[0307] 7. Take 200 μl of the frozen culture collected in step 6 and add it to 50 ml of 2YT-Amp-0.2% glucose culture medium to obtain the OD 600 = 0.109, and incubated at 37°C, 250 rpm for 60 min. OD 600 =0.247 was measured.

[0308] 8. 250 μl of helper bacteriophage (OD280=13) was added, mixed uniformly, and incubated at 37° C. for 30 minutes at rest, and then cultured at 37° C. and 250 rpm for 30 minutes.

[0309] 9. The bacteria infected with the helper bacteriophage were collected by centrifugation, resuspended in 100 ml of 2YT-Amp-Kana broth, and cultured at 30°C and 250 rpm for 8 hours. The bacteria were precipitated by centrifugation, and the supernatant was collected and mixed uniformly with 25 ml of 5 x PEG-NaCL, and the bacteriophage was precipitated overnight at 4°C.

[0310] 10. The next day, the bacteriophage library from step 9 was centrifuged and precipitated, and then suspended in 2 ml of PBS to precipitate the bacteriophage, obtaining the bacteriophage library. OD 280 = 13, and the bacteriophage concentration was 3x10 13 / ml, and 0.2 ml of the heavy chain-substituted bacteriophage library was taken and used for the first screening.

[0311] 11. The heavy chain-substituted bacteriophage library was frozen and stored: 0.6 ml of autoclaved 80% glycerol solution was added until the final glycerol concentration reached 20%, mixed evenly, and dispensed into 4 tubes, 0.6 ml per tube, and frozen at minus 80°C.

[0312] Example 6: Screening of CD40 antigen-specific Selicrelumab light chain replacement positive clones

[0313] Antigen preparation: Biotin-labeled CD40 antigen (AcroBio, Cat#: CD0-H82E8) and non-biotin-labeled CD40 antigen (AcroBio, Cat#: CD0-H5253) were purchased, and the lyophilized products were dissolved according to the instructions. Biotin-labeled antigen was dispensed at 5 μg / tube, and non-biotin-labeled antigen was dispensed at 10 μg / tube, and stored at minus 80°C.

[0314] First screening: 1. One tube of multi-reference antibody light chain replacement bacteriophage library (bacteriophage concentration 1x10) frozen at -80°C 13 / 0.5 ml, Example 2) was taken, thawed in an ice bath, and mixed with 100 μl of MPBST (containing 12% Milk, 0.05% Tween, 1X PBS), the final volume was 0.6 ml, and the final concentration of Milk was 2%.

[0315] 2.60 μl of washed and resuspended magnetic beads (Invitrogen, Cat#: 11206D) were added and rotated at room temperature for 30 minutes to adsorb non-specific bacteriophage that could bind the magnetic beads.

[0316] 3. The magnetic beads were adsorbed to a magnetic frame (Invitrogen Cat#: 12321D) and discarded, and the bacteriophage library solution was transferred to a 2 ml EP tube sealed with fresh 2% MPBST.

[0317] 4.5 μg of biotin-labeled antigen was taken and mixed with the light chain-substituted bacteriophage library (final antigen concentration: 5 μg / 0.6 ml), and the mixture was rotated and shaken at room temperature for 2 hours to allow the bacteriophage expressing antigen-specific Fab to bind to the biotin-labeled antigen.

[0318] 5.60 μl of washed and resuspended magnetic beads were added to the bacteriophage library solution and rotated and shaken at room temperature for 20 minutes to capture antigen-specific bacteriophages by binding of avidin and biotin on the surface of the magnetic beads, forming magnetic bead-avidin-biotin-antigen-Fab antibody fragment cross-linked conjugates.

[0319] 6. The magnetic beads having the cross-linked conjugates with antigen-specific Fab formed in step 5 were collected by a magnetic frame.

[0320] 7. The magnetic beads were washed four times with 1xPBST, and then four times with 1xPBS.

[0321] 8. The magnetic beads were suspended using 300 μl of glycine solution, pH 2.2, and rotated and shaken at room temperature for 10 minutes to elute the bacteriophage exhibiting antigen-specific Fab.

[0322] 9. The magnetic beads were collected by a magnetic frame, and the eluted supernatant not containing the magnetic beads was transferred to a new 1.5 ml EP tube and neutralized to pH 7.0 with 110 μl of pH 8.0 Tris buffer.

[0323] 10. The magnetic beads were resuspended using 100 μl of glycine solution, pH 2.2, and shaken by rotation for 5 minutes to elute the bacteriophage exhibiting antigen-specific Fab.

[0324] 11. The magnetic beads were collected by a magnetic frame, and the eluted supernatant without magnetic beads was transferred to a new 1.5 ml EP tube and neutralized to pH 7.0 with 35 μl of Tris buffer, pH 8.0.

[0325] 12. Approximately 540 μl of the neutralized eluted bacteriophage solutions obtained in step 9 and step 11 were combined and stored on ice for future use.

[0326] 13. 40ml of TG1 bacteria was pre-cultured and monitored to reach an OD600 of 0.2-0.3.

[0327] 14.3 ml of the TG1 bacterial solution was mixed with 400 μl of the eluted neutralized bacteriophage solution, and the mixture was allowed to stand at 37° C. for 30 minutes.

[0328] 15.10 μl of the bacterial solution infected with the bacteriophage library was taken and diluted 10-fold eight times using 2YT culture medium, and 5 μl of each dilution was spread on a 2YT-Amp petri dish and cultured at 32°C overnight to titrate the screening efficiency.

[0329] 16. The remaining bacterial solution was spread onto two large petri dishes (Thermo Cat#: 8-1030-051) and incubated overnight at 32°C.

[0330] 17. The remaining 140 μl eluted neutralized bacteriophage solution was stored at 4° C.

[0331] 18. The next day, the total number of bacteriophages (output) obtained from the first screening of the light chain-substituted bacteriophage library was analyzed and calculated to be 8.8 x 10 8 It was.

[0332] Packaging, amplification, and precipitation of the bacteriophage library obtained from the first round of screening: 1. Collect two colonies from the large plate in the first output (final bacterial liquid volume 10 ml), and take two tubes of 4 ml each for freezing and storing (2 ml / 7% DMSO / tube).

[0333] 2. Take 20 μl of the collected bacterial solution and dilute it 50-fold with 2YT to obtain the OD 600 =0.7, original bacterial solution OD 600=35 was measured.

[0334] 3.60ml 2YT-Amp-2%Glocose culture solution, 200μl OD 600 The bacterial library solution was added and cultured at 37°C and 250 rpm for 1 hour, and the OD 600 was measured to be around 0.35.

[0335] 4. 0.5 ml of helper bacteriophage was added (OD280=13) and mixed with the bacterial library solution, the MOI was between 500-1000. The mixture was left stationary at 37°C for 30 minutes, and then incubated at 37°C, 250 rpm for 30 minutes.

[0336] 5. The bacteria were collected by centrifugation, and the cultured bacteria were resuspended in 100 ml of 2YT-Amp-Kan. The bacteria were cultured overnight at 30°C and 250 rpm, and packaged to amplify the first output bacteriophage.

[0337] 6. The next day, the mixture was centrifuged at 4° C. and 9,000 rpm (Beckman, JA-10) for 15 minutes, and the supernatant was taken and mixed uniformly with 25 ml of 20% PEG-5M NaCl and placed in an ice bath for 2 hours.

[0338] 7. The mixture was centrifuged at 4°C and 9000 rpm (Beckman, JA-10) for 15 minutes, the supernatant was discarded, and the mixture was centrifuged again at 4°C for 5 minutes to concentrate the bacteriophages at the bottom of the centrifuge tube. The remaining supernatant was then aspirated with a pipette tip and discarded.

[0339] The bacteriophage library was suspended in 8.2 ml of PBS, centrifuged at 18,000 g for 5 minutes at 4°C using a tabletop high-speed centrifuge, and the supernatant was collected and analyzed for OD. 280 = 16 and the bacteriophage concentration was calculated to be 3.73 x 10 13 / ml.

[0340] 9. 0.5 ml of the bacteriophage library was taken and a second round of screening was performed.

[0341] 10. The remaining bacteriophage library was added with 1 / 3 of a high-pressure sterilized 80% glycerol solution (final glycerol concentration: 20%) and mixed homogeneously. Each library was then dispensed into three tubes (0.5 ml per tube) and frozen at minus 80°C.

[0342] Screening rounds 2-5: 1. 0.5 ml of the amplified and collected bacteriophage library obtained in the first screening was taken and subjected to a second screening. The screening flow was the same as that of the first screening, but the final antigen concentration during screening was 1 μg / 0.6 ml, and 540 μl of eluted neutralized bacteriophage solution was obtained.

[0343] 2.0.5ml of the second eluted neutralized bacteriophage solution was taken and directly subjected to the third screening without amplification. The screening flow was the same as that of the first screening, and the final antigen concentration during screening was 1μg / 0.6ml. 270μl of the eluted neutralized bacteriophage solution was obtained.

[0344] 3. 200μl of the third eluted neutralized bacteriophage solution was taken and used to infect 1ml of TG1 bacteria at 37℃ for 30min. 10μl was taken for titration and diluted in 8 gradients. 5μl of each dilution was taken and spread on 2YT-Amp petri dishes and cultured overnight at 32℃ for screening efficiency titration. 750μl of the remaining bacterial solution was spread on a large plate and 250μl was spread on a large plate and cultured overnight at 32℃.

[0345] 4. The next day, the total number of light chain-substituted bacteriophages contained in the third 200 μl eluted neutralized bacteriophage solution was calculated: 2.6 x 10 5 .

[0346] 5. The bacteriophage library obtained after packaging, amplified and screening for the third time was precipitated and collected, and the flow was the same as that for the first time.

[0347] 6. 0.5 ml of the amplified and collected bacteriophage library obtained in the third screening was used for the fourth and fifth screenings. The screening flow was the same as the second and third screenings, and the final antigen concentration during screening was 1 μg / 0.6 ml.

[0348] 7. 200 μl of the fifth eluted neutralized bacteriophage solution was taken and used to infect 1 ml of TG1 bacteria at 37°C for 30 min. 10 μl was taken for titration, and 750 μl of the remaining bacterial solution was spread on a large plate and 250 μl was spread on a large plate and incubated at 32°C overnight.

[0349] 8. The following day, the total number of light chain-substituted bacteriophages contained in the fifth 200 μl bacteriophage solution was calculated: 1.7 x 10 6 .

[0350] Deep well plates were inoculated and Fab expression was induced by IPTG: 1. Inoculation culture medium 2YT-Amp-0.2% Glucose was prepared and dispensed into each deep well plate at 40 ml, 400 μl per well.

[0351] 2. The fifth screening colonies were inoculated (deep well plate), the deep well plate was covered with a breathable membrane and incubated at 37°C, 250 rpm for 6 hours.

[0352] 3. IPTG induction culture 2YT-Amp-2mM-IPTG was prepared and each deep well plate was 40 ml. The induction culture (400 μl / well) was added to the inoculated 6-hour cultured deep well plate (IPTG final concentration 1 mM).

[0353] 4. The culture conditions were adjusted to 30°C and 250 rpm, and the strain was induced and cultured overnight.

[0354] 5. Prepare CD40 specific antigen coating solution (9.5ml ddH2O+0.5ml 20x coating solution+10μg non-biotinylated CD40 antigen) and coat one ELISA plate (100μl / well) and leave coated overnight at 4°C.

[0355] ELISA analysis and screening of CD40 antigen-specific light chain replacement positive clones: Prepare 1.3% MPBST (10 ml / ELISA plate), PBST (200 μl / well each time washing the plate).

[0356] The ELISA plates, which had been coated and left overnight at 2.4° C., were removed, the coating solution discarded, and washed 3 times with PBST.

[0357] 3.3% MPBST (100 μl / well) was added, covered with a non-breathable membrane and sealed at 37° C. for 60 minutes.

[0358] 4. The blocking solution was discarded and the cells were washed three times with PBST.

[0359] 5. 100 μl / well of the homogeneously mixed bacterial solution was taken from the deep well plate induced overnight and added to the corresponding wells of the ELISA plate, covered with a non-permeable membrane, and incubated at 37° C. for 60 minutes.

[0360] 6. The bacterial solution was discarded and the plate was washed three times with PBST.

[0361] 7. Prepare secondary antibody solution: Take 5μl Anti-flag-HRP solution (GenScript Cat# A0148-100, dissolved in ddH2O, 0.5μg / μl) and add 5ml of PBS. Add the prepared secondary antibody solution to the ELISA plate, 50μl / well, cover with non-breathable membrane, and incubate at 37℃ for 45 minutes.

[0362] 8. Discard the secondary antibody solution and wash three times with PBST.

[0363] 9. Color development solution (ABTS, Invitrogen, Cat# MD21704) was added, 50 μl / well, and allowed to develop for 30 minutes at room temperature.

[0364] 10. Add stop solution (0.1 M citric acid), 50 μl / well, mix evenly, and measure OD 405 Read the numerical value.

[0365] 11.OD 405 Clones with an affinity greater than 1.2 were sequenced and analyzed, and 43 Selicrelumab light chain substitution-positive clones were obtained and stored frozen at minus 80°C.

[0366] Example 7: Screening of CD40 antigen-specific Selicrelumab heavy chain replacement positive clones

[0367] First screening: 200 μl (bacteriophage concentration 3x10 13 A single reference antibody heavy chain-substituted bacteriophage library without fresh glycerol was prepared by taking 100 μl of ...

[0368] The heavy chain-replaced bacteriophage was screened using the liquid-phase magnetic bead method, and the screening flow was the same as the first screening flow for the light chain-replaced bacteriophage library. The antigen concentration was 1 μg / 400 μl, and the final eluted neutralized bacteriophage solution was 540 μl. 400 μl of the eluted neutralized solution was infected with 1 ml of TG1 bacteria, 10 μl of the bacterial solution was titrated, 1 ml of the bacterial solution was spread on a large plate, and the remaining bacterial solution was spread on another large plate and cultured at 32°C overnight.

[0369] The total number of bacteriophages (output) obtained in the first screening of the heavy chain replacement bacteriophage library was 8.5 x 10 5 It was.

[0370] The heavy chain-substituted bacteriophage library obtained after packaging, amplified and first round screening was precipitated and collected: The basic flow was the same as that of packaging and amplifying the light chain-substituted bacteriophage library obtained in the first screening, followed by precipitation and collection. The outline is as follows: All colonies (6.3x10e5) spread on a 1ml plate of bacterial liquid were collected, and the total volume was 8ml. Two tubes were frozen and stored. 140μl of the collected bacterial liquid was mixed with 50ml of culture liquid (2YT-AG) to homogenize, and the OD 600 = 0.114, and incubated at 37°C for 60 min to measure OD 600 = 0.234, and 250 μl of M13KO7 helper bacteriophage (OD 280 =13), mixed homogeneously, incubated at 37°C for 30 min, and incubated at 37°C for 30 min at 250 rpm. Bacteria were collected by centrifugation, resuspended in 100 ml of 2YT-Amp-Kana culture medium, and incubated at 30°C for 8 h. Culture supernatant was collected by centrifugation, mixed homogeneously with 25 ml of 5 x PEG-NaCL, and the bacteriophage was precipitated overnight at 4°C. The next day, the bacteriophage library was collected by centrifugation, suspended in 2 ml of PBS, measured for OD280=7, added glycerol to a final concentration of 20%, dispensed into 0.5 ml / tube, and frozen at -80°C.

[0371] Deep well plates were inoculated and Fab expression was induced by IPTG: The heavy chain-replaced bacteriophage library was inoculated for the first screening and spread to colonies in a large dish (deep well plate) of 0.4 ml of bacterial liquid. The preparation of reagents and materials and the induction of Fab flow expression were the same as for the light chain-replaced clones.

[0372] The CD40 antigen-specific heavy chain replacement positive clones were screened by ELISA analysis: The screening flow was the same as that for the analysis and screening of light chain replacement clones.

[0373] Samples were sent and clones with an ELISA reading greater than 0.8 were sequenced and analyzed, and 28 heavy-strand replacement positive clones were obtained and stored frozen at minus 80°C.

[0374] Example 8 Screening of CD40 antigen-specific Selicrelumab light chain heavy chain double replacement positive clones

[0375] Production A light chain fragment of the light chain replacement positive clone (see FIG. 2, third polynucleotide) and a heavy chain variable region fragment of the heavy chain replacement positive clone (see FIG. 2, fourth polynucleotide) were prepared: Frozen stocks of 37 light chain replacement positive clones (Example 6) and 28 heavy chain replacement positive clones (Example 7) were thawed at room temperature, and 5 μl of each was taken and mixed to obtain CD40-LC-ZH-M37 stock and CD40-VH-ZH-M28 stock. 50 μl of each mixed stock + 500 μl of 2YT-Amp was taken, mixed, spread on a large plate, and cultured at 32° C. overnight.

[0376] The next day, all the colonies from both plates were collected, two from each plate were frozen and stored at -80°C.

[0377] Vector DNA of CD40-VH-ZH-M28 (26 μg) and CD40-LC-ZH-M37 (32 μg) was extracted.

[0378] 5 μg CD40-VH-ZH-M28 vector DNA was cleaved with Esp3I and SfiI enzymes and the purified M28-VH fragment (324 ng) (see FIG. 2, fourth polynucleotide) was analyzed by electrophoresis.

[0379] 5 μg CD40-LC-ZH-M37 vector DNA was enzymatically digested with SfiI and the purified M37-LC fragment was analyzed by electrophoresis (432 ng) (see FIG. 2, third polynucleotide).

[0380] Construction of light chain heavy chain double substitution library: The prepared M37-LC was inserted into a fragment (third polynucleotide), and M28-VH was inserted into a fragment (fourth polynucleotide), and the four fragments were ligated with the third and fourth vector polynucleotides shown in Figure 2 to construct a light chain heavy chain double replacement gene. The four fragments were mixed in an equal molecular ratio of 1:1:1:1, with a total amount of 596 ng, and ligated at 20°C for 4 hours.

[0381] The ligation products were purified to obtain 450 ng of a light chain / heavy chain double replacement gene library.

[0382] 50 ng of the purified ligation product was transformed into TG1-induced bacteria, titered, spread onto plates, and grown overnight at 32°C.

[0383] The next day, the library capacity of the light-heavy double-permuted bacterial library was analyzed and calculated to be 2x10 6 It was.

[0384] Colonies were inoculated and induced to express Fab: Two deep well plates were inoculated and induced with IPTG and grown overnight at 30° C. CD40 antigen was coated onto two ELISA plates at 100 ng / well and incubated overnight at 4° C.

[0385] ELISA analysis and screening of CD40 antigen-specific light chain heavy chain double replacement positive clones: The analysis and screening flow was the same as for the single replacement clones.

[0386] Positive clones with ELISA readings greater than 1.5 were sequenced and analyzed, yielding 10 unique new VHs, 19 unique new LCs, constituting 30 double-replacement positive clones, which were stored frozen at minus 80°C.

[0387] Example 9 Analysis of the Function of CD40 Antigen-Specific Selicrelumab Light Chain Replacement, Heavy Chain Replacement, and Light Chain Heavy Chain Double Replacement Positive Clones

[0388] Antibody Expression: Sixteen (#2-17) positive clones (KLC / IgG1) specific for CD40 antigen were selected and confirmed as positive controls, which were expressed and purified by transient transfection of 293 cells, and the reference antibody Selicrelumab (#1) was simultaneously expressed as a positive control.

[0389] 1 positive control: Selicrelumab, #1; Six light chain replacement clones: #2-7; Six heavy chain replacement clones: #8-13; Four light chain heavy chain double replacement clones: #14-17.

[0390] A total of 15 purified antibodies were obtained (clones #8 and #17 were not expressed).

[0391] The details are shown in Table 1: [Table 1]

[0392] Assay of antibody agonist activity: Referring to the reference (Scandinavian Journal of Immunology 65, 479-486), the antibodies were co-cultured with human PBMC for 5 days (antibody concentration 100nM), and the IFN-γ concentration (pg / ml) of the culture supernatant was detected to analyze the agonist activity of 15 antibodies. The results showed that compared with the positive antibody Selicrelumab (#1), all 14 screened antibodies showed different degrees of agonist activity, where the agonist activity of #2, 3, 4, 5, 6, 7 and #16 was equivalent to the positive control antibody.

[0393] The results are shown in FIG.

[0394] Amino acid sequences of the Vh and full-length light chains of six chain-replacement functional antibodies: As shown by comparison of the amino acid sequences of two light chain replacement clones, #2 and #3; two heavy chain replacement clones, #12 and #13; two light chain heavy chain double replacement clones, #14 and #16, the amino acid sequences of the screened light chain (light chain replacement clone), heavy chain (heavy chain replacement clone), light chain and heavy chain (light chain heavy chain double replacement clone), and CDR regions were significantly different from the corresponding light chain or heavy chain amino acid sequences of the positive control antibody Selicrelumab. See Table 2. [Table 2]

[0395] Example 10 Screening of Her3 antigen-specific Patritumab light chain substitution positive clones

[0396] With reference to the screening flow in Example 6, Her3 antigen-specific Patritumab light chain substitution-positive clones and heavy chain substitution-positive clones were screened.

[0397] The first screening was performed using the patritumab light chain-substituted bacteriophage library (Example 4). The total number of light chain-substituted bacteriophages obtained (output) was 1 x 10 5 It was.

[0398] The bacteria were collected, amplified, and the output from the first round was packaged to obtain a light chain-substituted bacteriophage library from the first round of output, which was then screened for the second and third rounds. The total number of light chain-substituted bacteriophages (output) in the third round of screening was 2.2x10 7 It was.

[0399] Light chain-substituted bacteriophage library clones were inoculated into two deep-well plates and grown overnight at 32° C. and 250 rpm, and expression of Fab was induced by IPTG.

[0400] Screening for Her3 antigen-specific light chain substitution positive clones by ELISA analysis, samples were sent to OD 405A total of 51 clones with high IL-1 expression were sequenced, yielding 28 unique light chain replacement positive clones.

[0401] Example 11 Functional analysis of Her3 antigen-specific Patritumab light chain replacement positive clones

[0402] Antibody Expression: Purified Patritumab (#1) expressed by transient transfection in 293 cells and full-length antibodies of 12 screened Her3 antigen-specific light chain substitution positive clones (#2-13, KLC / IgG1) corresponding to the ELISA readings and positive controls were selected and confirmed.

[0403] Assay of endocytic activity of antibodies: Gastric cancer organoids (3-27-T) were harvested and cultured for 7 days; gastric cancer organoids were collected, washed to remove Matrigel, and suspended in phenol red-free D-Hanks suspension to an appropriate volume and mixed evenly; and dispensed into a 96-well plate, with 50 μL per well.

[0404] References (ThermoFisher / Invitrogen,Zenon TM pHrodo TM Referring to iFL IgG Labeling Reagents, Catalog Nos. Z25609, Z25610, Z25611, Z25612, Pub. No. MAN0017436 Rev. B.0, 160 nM antibody solution and 4 x coupling reagent were prepared, mixed in a 1:1 ratio, and incubated at room temperature for 5 minutes.

[0405] The antibody-coupling reagent mixture was added to the 96-well plate containing gastric cancer organoids, 50 μl of which was added to the enzyme well, and incubated at room temperature for 60 minutes.

[0406] The cells were observed under a microscope, the fluorescence field was photographed and recorded, and the endocytosis activity of the antibodies was analyzed.

[0407] The results are shown in Figure 5. As shown in the results, four antibodies, DDBK004-2, DDBK004-3, DDBK004-7, and DDBK004-13, had green fluorescence, indicating that they had endocytosis activity, but weaker than the positive control; the fluorescence intensity of DDBK004-11 was equivalent to the positive control, indicating that the endocytosis activity of this antibody was equivalent to the positive antibody; the fluorescence intensity of DDBK004-9 was the strongest, higher than the positive control, indicating that the endocytosis activity of this antibody was higher than the positive control.

[0408] The CDR and variable region sequences of the antibodies are shown in Table 3: [Table 3]

[0409] Example 12 Screening of TIGIT antigen-specific reference antibody Tiragolumab light chain substitution positive clones, heavy chain substitution positive clones, and double substitution positive clones, and analysis of biological functions of positive clones

[0410] Screening for strand displacement positive clones: Antigens were prepared with reference to Example 6, and TIGIT antigen-specific light chain replacement-positive clones and heavy chain replacement-positive clones were screened from the light chain replacement bacteriophage library and the heavy chain replacement bacteriophage library.

[0411] Nearly 1000 clones were analyzed by ELISA. Based on the results of the ELISA analysis, the OD of the positive control clone was 405 Based on the read values, more than 400 clones with high read values ​​were selected and sent for sequencing to confirm TIGIT antigen-specific light chain replacement positive clones and heavy chain replacement positive clones.

[0412] Referring to Example 8, light chain replacement unique sequence positive clones and heavy chain replacement unique sequence positive clones were selected, unique sequence light chain fragments (see FIG. 2, third polynucleotide) and heavy chain fragments (see FIG. 2, fourth polynucleotide) were produced, and four-fragment ligation was performed with the third vector polynucleotide and the fourth vector polynucleotide shown in FIG. 2 to construct a light and heavy chain double replacement gene library, a bacterial library and a bacteriophage library, and screened to obtain double replacement unique sequence positive clones.

[0413] Fifteen (#2-16) positive clones specific for TIGIT antigen with full-length antibodies (KLC / IgG1) that were transiently transfected, expressed and purified by 293 cells and had ELISA readings equivalent to or higher than the positive control were selected and confirmed, and the reference antibody Tiragolumab (#1) was simultaneously expressed as a positive control. As shown in Table 4, a total of 15 purified antibodies were obtained (clone #3 was not expressed): [Table 4]

[0414] Here, the amino acid sequence of tiragolumab can be found in WO2009126688A2.

[0415] Antibody tumor cell killing activity analysis: In the experiment, the killing activity of TIGIT antibody and PD-1 antibody (Pembrolizomab) combined in non-small cell lung cancer organs was observed. Based on the results of multiple preliminary experiments, a total of five antibodies, #5, 10, 12, 14, and 15 in Table 4, showed different degrees of killing activity. In order to compare and further confirm the results of the preliminary experiments, the experiment was divided into two groups: TIGIT / PD-1 antibody combination (1-8) and TIGIT antibody alone (9-16), with 8 wells in each group.

[0416] The experimental flow was as follows: 1. Referring to the reference (Tumor organoid-T-cell coculture systems; NATURE PROTOCOLS | VOL 15 JANUARY 2020 15-39), non-small cell lung cancer organoids (4-11-T) were prepared and cultured in culture medium containing IFNγ (200 ng / mL) for 24 hours. The organoids were collected, washed to remove the matrigel, suspended in culture medium to an appropriate volume, and mixed uniformly for use.

[0417] 2. PBMCs (4-11-PBMCs) were taken and counted, their vitality and cell number were calculated, collected, washed, and suspended to an appropriate concentration in T cell culture medium (RPMI1640, 10% FBS, 1% double antibody, 2 mM glutamine, and IL2) and plated in a 96-well U-shaped plate, 5x10 3 PBMC / well.

[0418] 3. An appropriate amount of non-small cell lung cancer organs was added to each well, and the final volume was adjusted to 200 μL / well.

[0419] 4. PD-1 antibody (Pembrolizomab) was added to wells 1-8, the final concentration was 100 nM, then positive control antibody (P), isotype antibody (I), PBS and five preliminary experiment positive clone antibodies were added to wells 1 to 8 in sequence, where PBS was added to one well as a blank control. No PD-1 antibody (Pembrolizomab) was added to wells 9-16, and other reagents were the same as wells 1-8. The results were as shown in Table 5. [Table 5]

[0420] 5. Microscopic observations were continued, and the changes in the organoids and lymphocytes were regularly photographed and recorded, and the killing activity of the antibodies against NSCLC organs was analyzed.

[0421] 6. As shown by the results of using TIGIT antibody alone (Figure 6), compared with the PBS blank control and the isotype antibody, TIGIT antibody alone had no obvious killing activity against non-small cell lung cancer organs.

[0422] 7. The results of combining TIGIT antibody with PD-1 antibody show the following (Figure 7): 1) The “negative” control wells (PD1+I; PD1+PBS) showed no obvious killing activity of PD-1 antibody alone against non-small cell lung cancer organs; 2) The positive control well (PD1+P) showed that on day 11 (D11) the combination of TIGIT antibody and PD-1 antibody had obvious killing activity against non-small cell lung cancer organs; 3) The killing activity of the five experimental wells, TIGIT antibodies #5, #10, #12, #14 and #15 in combination with PD-1 antibodies (PD1+005, PD1+010, PD1+012, PD1+014, PD1+015), against non-small cell lung cancer organs was comparable to that of the positive control (PD1+P), and showed obvious killing activity on day 11 (D11).

[0423] The CDR and variable region sequences of the antibodies are shown in Table 6: [Table 6]

[0424] The above detailed description has been provided for the purposes of explanation and illustration, and is not intended to limit the scope of the appended claims. Variations of the embodiments recited herein to date will be apparent to those skilled in the art and are encompassed within the scope of the claims and their equivalent embodiments.

Claims

1. A method for selecting a functional antigen binding protein, comprising: a) providing a first polynucleotide, wherein the first polynucleotide comprises, in a 5' to 3' direction, R1-nucleic acid fragment I-R2, and wherein the nucleic acid fragment I is capable of encoding an antigen-binding fragment I; b) providing a second polynucleotide, wherein said second polynucleotide comprises in a 5' to 3' direction R3-reference nucleic acid fragment II-R4, said reference nucleic acid fragment II being capable of encoding a reference antigen-binding fragment II, said reference antigen-binding fragment II being capable of forming a reference antigen-binding protein with a reference antigen-binding fragment I encoding the reference nucleic acid fragment I; c) providing a first vector polynucleotide, wherein said first vector polynucleotide comprises, in a 5' to 3' direction, an R2-vector fragment I-R3; d) providing a second vector polynucleotide, wherein said second vector polynucleotide comprises in a 5' to 3' direction an R4-vector fragment II-R1; e) cleaving the first polynucleotide, the second polynucleotide, the first vector polynucleotide, and the second vector polynucleotide with a restriction endonuclease to obtain the cleaved first polynucleotide, the cleaved second polynucleotide, the cleaved first vector polynucleotide, and the cleaved second vector polynucleotide; f) mixing the cleaved first polynucleotide, the cleaved second polynucleotide, the cleaved first vector polynucleotide and the cleaved second vector polynucleotide, thereby unidirectionally ligating them to circularize the first screening target vector; g) expressing the first screening target vector and selecting a first screening target vector that can express an expression product having the following properties as a first replacement vector: the reference binding protein can bind to a target that the reference antigen binding protein can bind to, and the binding ability of the target is 30% or more of the binding ability of the reference antigen binding protein to the target; h) obtaining said functional antigen binding protein from said first replacement vector; wherein R1, R2, R3 and R4 are each independently a restriction endonuclease recognition site.

2. The method of claim 1, comprising: cleaving the first polynucleotide with a restriction endonuclease that specifically recognizes R1 and R2 to obtain a cleaved first polynucleotide; cleaving the second polynucleotide with a restriction endonuclease that specifically recognizes R3 and R4 to obtain the cleaved second polynucleotide; cleaving the first vector polynucleotide with a restriction endonuclease that specifically recognizes R2 and R3 to obtain the cleaved first vector polynucleotide; and / or cleaving the second vector polynucleotide with a restriction endonuclease that specifically recognizes R4 and R1 to obtain the cleaved second vector polynucleotide.

3. The method according to any one of claims 1 to 2, wherein the end generated by specific cleavage of the restriction endonuclease of R1 by specific recognition does not mutually recognize or bind to the end generated by specific cleavage of the restriction endonuclease corresponding to any of R2, R3 and R4; The R2 does not recognize or bind to an end generated by specific cleavage of the restriction endonuclease corresponding to any of the R1, R3, and R4, even when the end generated by specific cleavage of the restriction endonuclease corresponding to the R2 is specifically cleaved by the restriction endonuclease; The end generated by specific cleavage of the restriction endonuclease of R3 by specific recognition does not mutually recognize or bind to the end generated by specific cleavage of the restriction endonuclease corresponding to any of R1, R2 and R4; and / or The method according to the above, wherein the end generated by specific cleavage of R4 with the restriction endonuclease through specific recognition does not mutually recognize or bind to an end generated by specific cleavage with a restriction endonuclease corresponding to any of R1, R2, and R3.

4. The method according to any one of claims 1 to 3, wherein the restriction endonuclease is selected from SfiI and BsmBI.

5. The method according to any one of claims 1 to 4, comprising introducing the first screening target vector into a cell and expressing the first screening target vector.

6. 6. The method according to any one of claims 1 to 5, comprising introducing the first screened vector into a bacterium, producing a bacteriophage library comprising one or more of the first screened vectors, and obtaining the functional antigen-binding protein by means of the bacteriophage library; and / or the display vector is derived from the pComb3x vector; and / or the linker comprises a nucleic acid sequence encoding the signal peptide pelB or a fragment thereof; and / or the linker is about 50 to about 200 bases in length; and / or a) the vector fragment I comprises a linker and the vector fragment II is derived from a display vector; and / or said nucleic acid fragment I encoding an antibody light chain or fragment thereof, said vector fragment I comprising said linker; said reference nucleic acid fragment II encoding an antibody heavy chain or fragment thereof; and said vector fragment II is derived from said display vector; and / or R1 comprises the nucleotide sequence set forth in SEQ ID NO: 1, R2 comprises the nucleotide sequence set forth in SEQ ID NO: 2, R3 comprises the nucleotide sequence set forth in SEQ ID NO: 3, and / or R4 comprises the nucleotide sequence set forth in SEQ ID NO: 4; or b) the vector fragment II comprises a linker and the vector fragment I is derived from a display vector; and / or said nucleic acid fragment I encoding an antibody heavy chain or fragment thereof; said vector fragment I is derived from said display vector; said reference nucleic acid fragment II encoding an antibody light chain or fragment thereof; and said vector fragment II comprises said linker; and / or wherein R1 comprises the nucleotide sequence set forth in SEQ ID NO: 3, R2 comprises the nucleotide sequence set forth in SEQ ID NO: 4, R3 comprises the nucleotide sequence set forth in SEQ ID NO: 1, and / or R4 comprises the nucleotide sequence set forth in SEQ ID NO:

2.

7. 6. A method according to any one of claims 1 to 5, comprising: introducing the vector to be screened first into a bacterium, obtaining DNA of the vector to be screened first from the bacterium, and introducing the DNA of the vector to be screened first into a cell; obtaining the functional antigen-specific binding polypeptide by the cell; and / or the cell is a mammalian cell; and / or said Vector Fragment I and / or said Vector Fragment II are derived from a mammalian cell expression vector; and / or the mammalian cell expression vector is derived from pDGB4; and / or a) said nucleic acid fragment I encodes an antibody light chain or a fragment thereof, and said reference nucleic acid fragment II encodes an antibody heavy chain or a fragment thereof; and / or R1 comprises the nucleotide sequence set forth in SEQ ID NO: 7, R2 comprises the nucleotide sequence set forth in SEQ ID NO: 8, R3 comprises the nucleotide sequence set forth in SEQ ID NO: 5, and / or R4 comprises the nucleotide sequence set forth in SEQ ID NO: 6; or b) said nucleic acid fragment I encodes an antibody heavy chain or a fragment thereof; and said reference nucleic acid fragment II encodes an antibody light chain or a fragment thereof; and / or wherein R1 comprises the nucleotide sequence set forth in SEQ ID NO: 5, R2 comprises the nucleotide sequence set forth in SEQ ID NO: 6, R3 comprises the nucleotide sequence set forth in SEQ ID NO: 7, and / or R4 comprises the nucleotide sequence set forth in SEQ ID NO:

8.

8. The method according to any one of claims 1 to 7, comprising: a) providing a third polynucleotide, wherein said third polynucleotide comprises, in a 5' to 3' direction, R5 through nucleic acid fragment I' through R6; b) providing a fourth polynucleotide, wherein said fourth polynucleotide comprises, in a 5' to 3' direction, R7 to nucleic acid fragment II' to R8, said nucleic acid fragment II' is capable of encoding an antigen-binding protein II', and said antigen-binding fragment II' is capable of binding to said reference antigen-binding fragment I to form an antigen-binding protein having the following properties: the reference antigen-binding protein is capable of binding to a target capable of binding, and the binding ability of said target is at least 30% of the binding ability of said reference antigen-binding protein to said target; c) providing a third vector polynucleotide, wherein said third vector polynucleotide comprises, in a 5' to 3' direction, R6 through vector fragment III through R7; d) providing a fourth vector polynucleotide, wherein said fourth vector polynucleotide comprises, in a 5' to 3' direction, R8 through vector fragment IV through R5; e) cleaving the third polynucleotide, the fourth polynucleotide, the third vector polynucleotide, and the fourth vector polynucleotide with a restriction endonuclease to obtain a cleaved third polynucleotide, a cleaved fourth polynucleotide, a cleaved third vector polynucleotide, and a cleaved fourth vector polynucleotide; f) mixing the cleaved third polynucleotide, the cleaved fourth polynucleotide, the cleaved third vector polynucleotide and the cleaved fourth vector polynucleotide, thereby unidirectionally ligating them to circularize a double replacement screening target vector; g) expressing the double-replacement screening target vector, and selecting a double-replacement screening target vector that can express an expression product having the following properties as a double-replacement vector: it can bind to the target, and the binding ability of the target is 30% or more of the binding ability of the reference antigen-binding protein to the target; h) obtaining said functional antigen binding protein from said double replacement vector; wherein R5, R6, R7 and R8 are each independently a restriction endonuclease recognition site.

9. The method according to claim 8, further comprising cleaving the third polynucleotide with a restriction endonuclease that specifically recognizes R5 and R6 to obtain the cleaved third polynucleotide; cleaving the fourth polynucleotide with a restriction endonuclease that specifically recognizes R7 and R8 to obtain the cleaved fourth polynucleotide; cleaving the third vector polynucleotide with a restriction endonuclease that specifically recognizes R6 and R7 to obtain the cleaved third vector polynucleotide; and / or cleaving the fourth vector polynucleotide with a restriction endonuclease that specifically recognizes R8 and R5 to obtain the cleaved fourth vector polynucleotide.

10. The method according to any one of claims 8 to 9, wherein the end generated by specific cleavage of the restriction endonuclease of R5 through specific recognition does not mutually recognize or bind to the end generated by specific cleavage of the restriction endonuclease corresponding to any of R6, R7 and R8; The end generated by specific cleavage of the restriction endonuclease of R6 by specific recognition does not mutually recognize or bind to the end generated by specific cleavage of the restriction endonuclease corresponding to any of R5, R7 and R8; The end generated by specific cleavage of the restriction endonuclease of R7 by specific recognition does not mutually recognize or bind to the end generated by specific cleavage of the restriction endonuclease corresponding to any of R5, R6 and R8; and / or The method according to the above, wherein the end generated by specific cleavage of R8 with the restriction endonuclease through specific recognition does not mutually recognize or bind to an end generated by specific cleavage with a restriction endonuclease corresponding to any of R5, R6 and R7.

11. The method according to any one of claims 8 to 10, wherein the restriction endonuclease is selected from SfiI and BsmBI.

12. The method according to any one of claims 8 to 11, comprising introducing the double replacement screening target vector into a cell and expressing the double replacement screening target vector.

13. 13. The method according to any one of claims 8 to 12, comprising introducing said double replacement screened vector into a bacterium, producing a bacteriophage library comprising one or more of said double replacement screened vectors, and obtaining said functional antigen-binding protein by means of said bacteriophage library; and / or the linker comprises a nucleic acid sequence encoding the signal peptide pelB or a fragment thereof; and / or the linker is about 50 to about 200 bases in length; and / or a) the vector fragment III comprises a linker and the vector fragment IV is derived from a display vector; and / or the nucleic acid fragment I' encodes an antibody light chain or fragment thereof; the vector fragment III comprises the linker; the nucleic acid fragment II' encodes an antibody heavy chain or fragment thereof; and the vector fragment IV is derived from the display vector; and / or said R5 comprises the nucleotide sequence set forth in SEQ ID NO: 1, said R6 comprises the nucleotide sequence set forth in SEQ ID NO: 2, said R7 comprises the nucleotide sequence set forth in SEQ ID NO: 3, and / or said R8 comprises the nucleotide sequence set forth in SEQ ID NO: 4; or b) the vector fragment IV comprises a linker and the vector fragment III is derived from a display vector; and / or the nucleic acid fragment I' encodes an antibody heavy chain or fragment thereof; the vector fragment III is derived from the display vector; the nucleic acid fragment II' encodes an antibody light chain or fragment thereof; and the vector fragment IV comprises the linker; and / or the method, wherein R5 comprises the nucleotide sequence set forth in SEQ ID NO: 3, R6 comprises the nucleotide sequence set forth in SEQ ID NO: 4; R7 comprises the nucleotide sequence set forth in SEQ ID NO: 1, and / or R8 comprises the nucleotide sequence set forth in SEQ ID NO:

2.

14. 13. A method according to any one of claims 8 to 12, comprising: introducing the double replacement screened vector into a bacterium, obtaining DNA of the double replacement screened vector from the bacterium, and introducing DNA of the double replacement screened vector into a cell; obtaining the functional antigen-specific binding polypeptide by the cell; and / or the cell is a mammalian cell; and / or said Vector Fragment III and / or said Vector Fragment IV are derived from a mammalian expression vector; and / or the mammalian expression vector is derived from pDGB4; and / or a) said nucleic acid fragment I' encodes an antibody light chain or a fragment thereof; and said nucleic acid fragment II' encodes an antibody heavy chain or a fragment thereof; and / or R5 comprises the nucleotide sequence set forth in SEQ ID NO: 7, R6 comprises the nucleotide sequence set forth in SEQ ID NO: 8, R7 comprises the nucleotide sequence set forth in SEQ ID NO: 5, and / or R8 comprises the nucleotide sequence set forth in SEQ ID NO: 6; or b) said nucleic acid fragment I' encodes an antibody heavy chain or a fragment thereof; and said nucleic acid fragment II' encodes an antibody light chain or a fragment thereof; and / or wherein R5 comprises the nucleotide sequence set forth in SEQ ID NO: 5, R6 comprises the nucleotide sequence set forth in SEQ ID NO: 6, R7 comprises the nucleotide sequence set forth in SEQ ID NO: 7, and / or R8 comprises the nucleotide sequence set forth in SEQ ID NO:

8.

15. The method according to any one of claims 1 to 14, comprising: a) providing a fifth polynucleotide, wherein said fifth polynucleotide comprises, in a 5' to 3' direction, R9 through a reference nucleic acid fragment I through R10; b) providing a sixth polynucleotide, wherein said sixth polynucleotide comprises, in a 5' to 3' direction, R11 through a reference nucleic acid fragment II through R12; c) providing a fifth vector polynucleotide, wherein said fifth vector polynucleotide comprises, in a 5' to 3' direction, R10 through vector fragment V through R11; d) providing a sixth vector polynucleotide, wherein said sixth vector polynucleotide comprises, in a 5' to 3' direction, R12 through vector fragment VI through R9; e) cleaving the fifth polynucleotide, the sixth polynucleotide, the fifth vector polynucleotide, and the sixth vector polynucleotide with a restriction endonuclease to obtain a cleaved fifth polynucleotide, a cleaved sixth polynucleotide, a cleaved fifth vector polynucleotide, and a cleaved sixth vector polynucleotide; f) mixing the cleaved fifth polynucleotide, the cleaved sixth polynucleotide, the cleaved fifth vector polynucleotide and the cleaved sixth vector polynucleotide, thereby unidirectionally ligating them to circularize a second screening target vector; g) expressing the second screening target vector, and selecting as a second replacement vector a second screening target vector capable of expressing an expression product having the following properties: the reference binding protein is capable of binding to a target capable of binding, and the binding ability of the target is 30% or more of the binding ability of the reference antigen-binding protein to the target; h) selecting nucleic acid fragment II of the second replacement vector as nucleic acid fragment II'; wherein R9, R10, R11 and R12 are each independently a restriction endonuclease recognition site.

16. 16. The method according to claim 15, further comprising cleaving the fifth polynucleotide with a restriction endonuclease that specifically recognizes R9 and R10 to obtain the cleaved fifth polynucleotide; cleaving the sixth polynucleotide with a restriction endonuclease that specifically recognizes R11 and R12 to obtain the cleaved sixth polynucleotide; cleaving the fifth vector polynucleotide with a restriction endonuclease that specifically recognizes R10 and R11 to obtain the cleaved fifth vector polynucleotide; and / or cleaving the sixth vector polynucleotide with a restriction endonuclease that specifically recognizes R12 and R9 to obtain a cleaved sixth vector polynucleotide.

17. The method according to any one of claims 15 to 16, wherein the end generated by specific cleavage of the restriction endonuclease of R9 through specific recognition does not mutually recognize or bind to the end generated by specific cleavage of the restriction endonuclease corresponding to any of R10, R11 and R12; The end generated by specific cleavage of the restriction endonuclease of R10 through specific recognition does not mutually recognize or bind to an end generated by specific cleavage of the restriction endonuclease corresponding to any of R11, R12, and R9; The end generated by specific cleavage of the restriction endonuclease of R11 by specific recognition does not mutually recognize or bind to the end generated by specific cleavage of the restriction endonuclease corresponding to any of R9, R10 and R12; and / or The method according to the above, wherein the end generated by specific cleavage of R12 with a restriction endonuclease through specific recognition does not mutually recognize or bind to an end generated by specific cleavage with a restriction endonuclease corresponding to any of R9, R10 and R11.

18. The method according to any one of claims 15 to 17, wherein the restriction endonuclease is selected from SfiI and BsmBI.

19. The method according to any one of claims 15 to 18, comprising introducing the second screening target vector into a cell and expressing the second screening target vector.

20. 20. The method according to any one of claims 15 to 19, comprising introducing the vector to be screened secondly into a bacterium, preparing a bacteriophage library comprising one or more of the vectors to be screened secondly, and obtaining the functional antigen-binding protein by means of the bacteriophage library; and / or the display vector is derived from the pComb3x vector; and / or the linker comprises a nucleic acid sequence encoding the signal peptide pelB or a fragment thereof; and / or the linker is about 50 to about 200 bases in length; and / or a) the vector fragment V comprises a linker and the vector fragment VI is derived from a display vector; and / or said reference nucleic acid fragment I encoding an antibody light chain or fragment thereof; said vector fragment V comprising said linker; said nucleic acid fragment II encoding an antibody heavy chain or fragment thereof; and said vector fragment VI derived from said display vector; and / or R9 comprises the nucleotide sequence set forth in SEQ ID NO: 1, R10 comprises the nucleotide sequence set forth in SEQ ID NO: 2, R11 comprises the nucleotide sequence set forth in SEQ ID NO: 3, and R12 comprises the nucleotide sequence set forth in SEQ ID NO: 4; or b) the vector fragment VI comprises a linker and the vector fragment V is derived from a display vector; and / or the reference nucleic acid fragment I encodes an antibody heavy chain or a fragment thereof; the vector fragment V is derived from the display vector; the nucleic acid fragment II encodes an antibody light chain or a fragment thereof; and the vector fragment VI comprises the linker; and / or The method, wherein R9 comprises the nucleotide sequence set forth in SEQ ID NO: 3, R10 comprises the nucleotide sequence set forth in SEQ ID NO: 4, R11 comprises the nucleotide sequence set forth in SEQ ID NO: 1, and R12 comprises the nucleotide sequence set forth in SEQ ID NO:

2.

21. 20. The method according to any one of claims 15 to 19, comprising: introducing the vector to be screened second into a bacterium; obtaining DNA of the vector to be screened second from the bacterium; introducing DNA of the vector to be screened second into a cell; obtaining the functional antigen-specific binding polypeptide by the cell; and / or the cell is a mammalian cell; and / or said Vector Fragment I and / or said Vector Fragment II are derived from a mammalian expression vector; and / or the mammalian expression vector is derived from pDGB4; and / or a) said reference nucleic acid fragment I encodes an antibody light chain or a fragment thereof; and said reference fragment II encodes an antibody heavy chain or a fragment thereof; and / or said R9 comprises the nucleotide sequence set forth in SEQ ID NO: 7, said R10 comprises the nucleotide sequence set forth in SEQ ID NO: 8, said R11 comprises the nucleotide sequence set forth in SEQ ID NO: 5, and said R12 comprises the nucleotide sequence set forth in SEQ ID NO: 6; or b) said reference nucleic acid fragment I encodes an antibody heavy chain or a fragment thereof; and said nucleic acid fragment II encodes an antibody light chain or a fragment thereof; and / or The method, wherein R9 comprises the nucleotide sequence set forth in SEQ ID NO: 5, R10 comprises the nucleotide sequence set forth in SEQ ID NO: 6, R11 comprises the nucleotide sequence set forth in SEQ ID NO: 7, and R12 comprises the nucleotide sequence set forth in SEQ ID NO:

8.

22. The method of any one of claims 1 to 21, wherein the antigen-binding protein comprises an antibody or an antibody fragment, and / or the antibody fragment is an scFv, Fab, Fab', (Fab) 2 and / or (Fab') 2 The method comprising:

23. 23. The method according to any one of claims 1 to 22, wherein the directional ligation comprises using a ligase and / or the ligase is a DNA ligase.

24. 23. A method for producing an antigen binding protein, the method comprising expressing the first replacement vector of any one of claims 1 to 23, the second replacement vector of any one of claims 15 to 23 and / or the double replacement vector of any one of claims 8 to 23 under conditions allowing expression of the first replacement vector of any one of claims 1 to 23, the second replacement vector of any one of claims 15 to 23 and / or the double replacement vector of any one of claims 8 to 23.

25. A vector which is a first replacement vector prepared by the method of any one of claims 1 to 23, a second replacement vector prepared by the method of any one of claims 15 to 23, or a double replacement vector prepared by the method of any one of claims 15 to 23.

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