WO2024007384A1

WO2024007384A1 - Phage display library panning method for obtaining bispecific antibody

Info

Publication number: WO2024007384A1
Application number: PCT/CN2022/108616
Authority: WO
Inventors: 张军锋; 郭志刚
Original assignee: 浙江蓝盾药业有限公司
Priority date: 2022-07-06
Filing date: 2022-07-28
Publication date: 2024-01-11
Also published as: CN117363606A

Abstract

Provided is a phage display library panning method for obtaining a bispecific antibody. The method comprises: extracting a total RNA of a human PBMC, synthesizing cDNA by reverse transcription with the total RNA serving as a template, performing multiple PCR amplifications by using cDNA as a template, and obtaining a linearized vector fragment and a linearized antibody gene fragment by enzyme cleavage; ligating the fragments to obtain a ligation product and introducing the ligation product into a host cell for phage display to obtain a fully human single-domain antibody phage display antibody library; and carrying out panning and monoclonal ELISA on the library.

Description

A phage display library panning method for obtaining bispecific antibodies

Technical field

The present invention relates to the field of biotechnology, and specifically to a phage display library panning method for obtaining bispecific antibodies.

Background technique

Phage display technology was created by George P. Smith in 1985 by inserting foreign genes into the genome of filamentous phage, so that the polypeptide encoded by the target gene is displayed on the surface of the phage in the form of a fusion protein. Gregory P. Winter established a phage library and used this library to develop a fully human antibody drug - adalimumab (Humira) targeting human tumor necrosis factor α. The drug was first marketed in the United States in 2003 and was the world's first marketed antibody drug derived from phage display technology. In 2018, the Nobel Prize in Chemistry was awarded to George P. Smith and Gregory P. Winter for their contributions to phage display technology.

Since the discovery of phage display technology, the technology has continued to evolve and is widely used in antibody discovery, including antibody affinity maturation. On September 3, 2018, EMA approved Sanofi's nanobody drug Cablivi (Caplacizumab) for the treatment of acquired thrombotic thrombotic purpura (aTTP) in adults. Cablivi became the first single-domain antibody drug to be marketed. In November 2021, Envolimab, a single-domain antibody targeting PD-L1 developed by Alphamab, was approved for marketing in China, becoming the second single-domain antibody drug to be launched in the world and the first in the world. Antibody drugs are injected subcutaneously.

Bispecific antibodies are a new technology that has emerged in recent years. They can exert a synergistic effect from the different mechanisms of action of two antibodies and are therefore more effective than monospecific antibodies. Currently, the discovery of bispecific antibodies takes the route of first screening monospecific antibodies and then constructing bispecific antibodies. The binding properties of individual antibodies in the constructed bispecific antibodies will change. At the same time, there are problems with the instability of bispecific antibody molecules and yield during actual operation. A large number of screenings are required, involving hundreds of configurations, ranging from single Starting from specific antibodies to construct bispecific antibodies is tantamount to re-developing antibodies. This patent constructs a diabody-binding domain phage display library, and directly screening bispecific antibodies from this library is a brand-new idea of bispecific antibody discovery. It not only eliminates the process of re-constructing bispecific antibodies and greatly reduces the workload, but also utilizes the The antibodies screened by the idea have good drug potential because unstable bispecific antibodies with poor expression are screened out during the screening process.

As mentioned above, the current workload of bispecific antibody discovery is huge and it also faces druggability issues. This method can directly eliminate its shortcomings in the early screening process.

Contents of the invention

The purpose of the present invention is to provide a phage display library panning method for obtaining bispecific antibodies.

In a first aspect of the present invention, a method for constructing a phage display library for obtaining bispecific antibodies is provided, and the bispecific antibodies are bispecific fully human single domain antibodies,

The method includes the following steps:

(a) Extract total RNA from human PBMC;

(b) using the total RNA as a template to synthesize cDNA through reverse transcription;

(c) Using the cDNA as a template, use the first primer set to perform the first round of PCR amplification, thereby obtaining the first round of amplification products; wherein the first primer set includes the positive sequences shown in SEQ ID Nos: 1 to 43 Toward primer and reverse primer shown in SEQ ID No:44-47;

(d) Using the first round amplification product as a template, use the second primer set to perform the second round of PCR amplification to obtain the second round amplification product; wherein, the second primer set includes SEQ ID No: 48-90 The first antibody variable region forward primer shown in SEQ ID No:91-94, the first antibody variable region reverse primer shown in SEQ ID No:91-94, the second antibody variable region forward primer shown in SEQ ID No:95～137 Primers, and the second antibody variable region reverse primer shown in SEQ ID No: 138～141;

(e) Using the second round amplification product as a template, use the third primer set to perform the third round of PCR amplification to obtain the third round amplification product; wherein the third primer set includes SEQ ID No: 142~184 The forward primer shown is, the reverse primer shown in SEQ ID No: 185～188;

(f) The vector used to construct the library and the third round amplification product are digested with restriction endonuclease I and restriction endonuclease II to obtain digested linearized vector fragments and linearized Antibody gene fragments;

(g) performing a ligation reaction on the digested linearized vector fragment and the linearized antibody gene fragment to obtain a ligation product;

(h) Introducing the ligation product into a host cell for phage display, thereby obtaining the bispecific fully human single domain antibody phage display antibody library.

In another preferred embodiment, the host cell used for phage display includes Escherichia coli.

In another preferred example, step (a) includes:

(a1) Isolation of human PBMC

Collect anticoagulated blood and isolate PBMC;

(a2) Extraction of total RNA

Take the PBMC and extract total RNA.

In another preferred example, the step (b) includes: cDNA synthesis, that is, using the PBMC total RNA extracted in step (a2) as a template, reverse transcription to synthesize cDNA;

In another preferred example, step (c) further includes: performing nucleic acid electrophoresis detection on the first round of PCR amplification products.

In another preferred example, step (d) further includes: performing nucleic acid electrophoresis detection on the second round of PCR amplification products.

In another preferred example, the vector used to construct the library includes: pCOMB3.

In another preferred embodiment, step (h) also includes measuring diversity: taking positive clones, performing phagemid sequencing and analyzing sequence diversity.

In another preferred example, step (i) also includes:

(i1) Activation of host bacterial TG1;

(i2) Magnetic beads washing and blocking;

(i3) Antigen binding;

(i4) Library closed;

(i5) Phage binding;

(i6) Washing;

(i7) Elution;

(i8) Measure titer;

(i9) Phage amplification;

(i10) Phage precipitation;

(i11) Repeat steps (i2) to (i10) two or three times to obtain phage display antibodies with strong binding capacity.

In another preferred example, the reaction system of the first round of PCR amplification in step (c) is: ddH ₂ O 13.6 μL, 2.5mM dNTPs 1.6 μL, 10× buffer 2 μL, cDNA 0.8 μL, 10 μM upstream primer 1 μL, 1 μL of 10 μM downstream primer, 0.08 μL of Ex Taq, total volume 20 μL.

In another preferred example, the reaction program of the first round of PCR amplification in step (c) is: 94°C for 4 min, 98°C for 10 s, 59°C for 40 s, 72°C for 50 s, 72°C for 10 min, and the amplification cycle is 25 Second-rate.

In another preferred example, the reaction system for the second round of PCR amplification in step (d) is: 2.5 μL of 10× buffer, 1.6 μL of 2.5mM dNTPs, 15 ng of the recovered product of the first round of amplification, and 10 μM upstream primer. 1 μL, 10 μM downstream primer 1 μL, Ex Taq 0.1 μL, ddH ₂ O make up to a total volume of 25 μL.

In another preferred example, the reaction program for the second round of PCR amplification in step (d) is: 94°C for 4 min, 98°C for 10 s, 59°C for 40 s, 72°C for 50 s, 72°C for 10 min, and the amplification cycle is 15 Second-rate.

In another preferred example, the reaction system for the third round of PCR amplification in step (e) is: 2.5 μL of 10× buffer, 1.6 μL of 2.5mM dNTPs, 15 ng of recovered product of the second round of amplification, and 10 μM upstream primer. 1 μL, 10 μM downstream primer 1 μL, Ex Taq 0.1 μL, ddH ₂ O make up to a total volume of 25 μL.

In another preferred example, the reaction program of the third round of PCR amplification in step (e) is: 94°C for 4 min, 98°C for 10 s, 59°C for 40 s, 72°C for 50 s, 72°C for 10 min, and the amplification cycle is 15 Second-rate.

In a second aspect of the present invention, a phage display library of bispecific antibodies is provided, and the phage display library of bispecific antibodies is constructed using the method described in the first aspect of the present invention.

In another preferred embodiment, the positive rate of the library is ≥80%, preferably, ≥90%.

In another preferred embodiment, the antibody sequence diversity of the library is ≥90%, preferably, ≥95%.

In another preferred example, the library capacity is ≥1×10 ⁸ pfu, preferably, ≥5×10 ⁸ pfu.

In a third aspect of the present invention, a method for panning bispecific antibodies is provided, comprising the steps:

(S1) Provide a phage display library of the bispecific antibody according to the second aspect of the present invention;

(S2) Use the antigen A1 and the antigen A2 to pan the antibody library to obtain bispecific antibodies against the antigen A1 and the antigen A2.

In another preferred example, in step (S2), the following sub-steps are included:

(S2a) In the first round of antibody library panning, two antigens (A1 and A2) are added at the same time.

(S2b) In the second round of screening, only a single antigen A1 was used for screening;

(S2c) In the third round of screening, only a single antigen A2 was used for screening;

(That is, the two antigens A1 and A2 are used alternately in these two rounds for screening). Since in the second and third rounds of screening, only a single antigen is used for screening in each round and the two antigens are used alternately, antibodies that only bind to one antigen can be screened out, and all antibodies that bind to both antigens at the same time are retained.

In another preferred embodiment, the method further includes:

(S3) For the bispecific antibody obtained in the previous step, detect its binding performance to antigen A1 and antigen A2 through ELISA.

In a fourth aspect of the present invention, a primer set is provided, the primer set being the primer sequences shown in SEQ ID Nos: 1 to 188.

In another preferred embodiment, the primer set is primers used for the method of claim 1.

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described one by one here.

Description of the drawings

Figure 1 shows part of the total RNA non-denaturing electrophoresis test showing RNA extraction; there are 48 RNAs in two rows of samples, and the three bands in each lane are 28S rRNA, 18S rRNA, and 5S rRNA. Marker is DNA Marker, which is 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp and 100bp from top to bottom.

Figure 2 is an electrophoresis diagram of a partial antibody variable region gene amplification product showing the first round of amplification of the antibody gene; there are 24 samples in two rows. Since the first round of amplification region contains the CH1 region of the antibody gene, the size is approximately 700 bp. (Due to different sizes of antibody genes); DNA Marker from top to bottom is 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp.

Figure 3 shows the electrophoresis diagram of partial antibody variable region gene amplification products showing the second round of amplification of antibody genes; there are 24 samples in two rows, and the target band size is approximately 410bp (different sizes due to different antibody genes); DNA Marker From top to bottom they are 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp.

Figure 4 is an electrophoresis diagram showing the third round of amplification of antibody genes (splicing antibody genes and adding enzyme cutting sites), splicing part of two antibody variable region genes into one gene chain and adding enzyme cutting sites; a total of 24 For each sample, the size of the target band is about 750bp (the size varies depending on the antibody gene); the DNA Marker from top to bottom is 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp and 100bp.

Figure 5 shows the electrophoresis pattern of the pCOMB3 vector; the DNA Markers from top to bottom are 6000bp, 5000bp, 4000bp, 3000bp, 2500bp, 2000bp and 1500bp.

Figure 6 shows the electrophoresis diagram of single clone PCR identification of two antibody variable region gene splicing genes; there are 96 clones in four rows of samples, and the target band size is about 930bp (due to different sizes of different antibody genes); DNA Marker from top to bottom The order is 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp and 100bp.

Figure 7 shows homology analysis of antibody sequences in positive clones.

Figure 8 shows the results of monoclonal phage ELISA.

Detailed ways

After extensive and in-depth research and extensive screening, the inventors constructed a single-domain antibody library based on fully human antibody genes for the first time. Specifically, the inventor used specific primers to amplify the fully human antibody gene and spliced it to form a bispecific single domain antibody construct. After connecting the construct with the vector fragment, it was introduced into the host for phage display. cells, thereby obtaining the bispecific fully human single domain antibody phage display antibody library. The single-domain antibody library based on bispecific fully human antibody genes obtained by the method of the present invention has the advantages of high positive rate, good diversity, large library capacity, etc. On this basis, the present invention was completed.

Phage display library of bispecific antibodies

The present invention provides for the first time a display library of dual-binding domain antibodies (i.e., bispecific antibodies) and a panning method thereof, in which each monospecific antibody element of the dual-binding domain antibody is a human single-domain antibody element. The schematic diagram is as follows:

VHH1

Linker

VHH2

As used herein, the terms "antibody library of the present invention", "phage display library of bispecific antibodies of the present invention", "single domain antibody library of the present invention", etc. are used interchangeably and refer to using the method described in the second aspect of the present invention. Phage display antibody library of dual-binding domain antibodies (i.e., bispecific antibodies). The library of the present invention is prepared by using a specific primer set and through the construction method described in the first aspect of the present invention.

A preferred library of the invention is a phage-based library. Preferably, the library of the present invention is a library based on M13 phage.

The M13 phage coat contains the capsid protein pill. The antibody gene is fused to the pill protein gene. When the phage infects E. coli, during the assembly process of the progeny phage, the antibody is brought to the shell by the pill protein and secreted from the progeny phage. There is an antibody fused to the pill protein on the surface of the progeny phage. , thereby realizing antibody display.

The M13 phage infects E. coli in a lysogenic manner and does not lyse E. coli. The single-stranded DNA that its genome can replicate stably exists in E. coli, so its genome can proliferate as E. coli proliferates. This feature-related element on the M13 phage is used to transform the plasmid and construct a phagemid vector for antibody display. Monoclonal antibodies can be easily obtained by monocloning and sequencing.

Build method

The invention also provides methods for constructing the library of the invention.

Typically, the construction method of the invention is as described in the first aspect of the invention.

Includes steps:

(a) Extract total RNA from human PBMC;

panning method

In the present invention, the inventor also developed a novel bispecific antibody screening (or panning) method, which method includes the steps:

First, during the first round of antibody library panning, two antigens (A1 and A2) were added at the same time, so that the phage display antibodies obtained included antibodies that only bind to a single antigen A1, antibodies that only bind to a single antigen A2, and antibodies that bind only to a single antigen A2. Antibodies that bind two antigens (A1 and A2). If only one of the antibodies is used, most of the antibodies that bind to both antigens at the same time will be discarded. The remaining very small amount of antibodies that bind to both antigens at the same time will be difficult to screen, or it will be difficult to screen for those with suitable performance.

Secondly, in the second round of screening, only a single antigen A1 was used for screening; in the third round of screening, only a single antigen A2 was used for screening; (that is, the two antigens A1 and A2 were used alternately in these two rounds. , filter). Since in the second and third rounds of screening, only a single antigen is used for screening in each round and the two antigens are used alternately, antibodies that only bind to one antigen can be screened out, and all antibodies that bind to both antigens at the same time are retained.

The results show that based on the dual-binding domain antibody display library of the present invention and the preferred panning method, a large number of bispecific antibodies (i.e., antibodies that bind two antigens simultaneously) with suitable performance can be efficiently obtained.

Main advantages of the invention

(a) Directly obtaining bispecific antibodies changes the previous method of obtaining monospecific antibodies, then constructing bispecific antibodies, and finally screening bispecific antibodies, which greatly speeds up the experimental process. In addition, once the library is built, it can be easily applied to other targets, saving more work.

(b) In the present invention, the two antibody variable regions are derived from all B cell populations and are rich in variety. However, the traditional method of first screening monospecific antibodies and then constructing bispecific antibodies can only select a limited number of monospecific antibodies. Construct.

(c) The obtained bispecific antibodies have good solubility and/or stability and relatively high expression levels. Antibodies with poor solubility and/or stability and low expression levels will be naturally removed.

The present invention will be further described below in conjunction with specific embodiments. It should be understood that these examples are only used to illustrate the invention and are not intended to limit the scope of the invention. Experimental methods without specifying specific conditions in the following examples usually follow conventional conditions, such as the conditions described in Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to manufacturing Conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

Example 1, PBMC separation

1) Take 2 ml of sample density separation liquid (purchased from Tianjin Haoyang, product number: LTS1077), and add it to the bottom of a 10 ml centrifuge tube.

2) Slowly add 2ml of whole blood to the separation solution, centrifuge at 750g for 20 minutes, and separate the buffy coat layer.

3) Insert the suction pipe into the buffy coat layer, suck the buffy coat layer into a 50ml centrifuge tube, and move the pipette while sucking to ensure that the buffy coat layer is absorbed as much as possible and the separation liquid is absorbed as little as possible.

4) Add PBS to 45ml, invert the centrifuge tube several times, and mix.

5) Centrifuge at 1500g for 7 minutes and aspirate the supernatant. Repeat steps 4) and 5).

6) Pour away the PBS, use the trace residual liquid, and flick the bottom of the tube to fully disperse the PBMC.

7) Add 2ml TRIzol and gently aspirate several times until the cells are completely lysed.

Example 2, RNA extraction

1) Add 0.3mL chloroform, shake well and let stand at room temperature to separate layers.

2) Centrifuge at 12,000 rpm for 10 minutes at 4°C.

3) Transfer the upper water phase to a new 1.5ml centrifuge tube, add 0.8 times the volume of pre-cooled isopropyl alcohol, mix well, and precipitate at 4°C for 30 minutes.

4) Centrifuge at 12,000 rpm for 10 minutes at 4°C.

5) Discard the supernatant, add 1.5 mL of 70% ethanol to wash the precipitate, absorb the washing liquid clean, and dry at room temperature for 5 minutes.

6) Add 20 μL of RNase-free deionized water and pipet to fully dissolve the RNA precipitate.

7) Add the total RNA to the wells of 1.5% argarose gel, and quickly separate it by electrophoresis in 1×TAE electrophoresis buffer.

The electrophoresis results are shown in Figure 1. The three bands are clear and the RNA is intact and not degraded.

Example 3, reverse transcription

1) Add reactants in the following order

Template RNA 3μg

oligo(dT)18 primer 1μL

Nuclease-free deionized water Make up to 12μL

2) Incubate at 70°C for 5 minutes and immediately cool on ice.

3) Add reactants in the following order

4) Incubate at 42°C for 40 minutes, then store at -70°C.

Example 4. First round of amplification of antibody genes (antibody heavy chain variable region gene amplification) and gel recovery

1) Add reactants in the following order

Antibody gene first round amplification primer forward primer (SEQ ID No: 1～43):

LibF1(SEQ ID No:1)CAGGTGCAGCTGGTGCAG

LibF2(SEQ ID No:2)CAAATGCAGCTGGTGCAGT

LibF3(SEQ ID No:3)GAGGTCCAGCTGGTACAGTCT

LibF4(SEQ ID No:4)CGGGTCACCTTGAGGGAG

LibF5(SEQ ID No:5)GAGGTGCAGCTGGTGAAGT

LibF6(SEQ ID No:6)GAGGTTCAGCTGGTGCAGT

LibF7(SEQ ID No:7)GAGGTGCAGCTGGTGGAG

LibF8(SEQ ID No:8)CAGGTGCAGCTACAGCAGT

LibF9(SEQ ID No:9)CAGGTACAGCTGGTGCAGTC

LibF10(SEQ ID No:10)CAGGTCCAGCTTGTTGCAGT

LibF11(SEQ ID No:11)GAGGTACAACTGGTGGAGTCT

LibF12(SEQ ID No:12)CAGATCACCTTGAAGGAGTC

LibF13(SEQ ID No:13)CAGGTTCAGCTGGTGCAGT

LibF14(SEQ ID No:14)CAGGTACAGCTGCAGGAGTC

LibF15(SEQ ID No:15)CAGGTACAGCTGGTGGAGTCT

LibF16(SEQ ID No:16)CAGGTGCAGCTACAGGAGTC

LibF17(SEQ ID No:17)CAGCTGCAGCTGCAGGAGT

LibF18(SEQ ID No:18)GAGGTGCAGCTGGTGGAG

LibF19(SEQ ID No:19)CAGGTGCAGCTGCAGGAC

LibF20(SEQ ID No:20)CAGGTGCAGCTGTTGGAG

LibF21(SEQ ID No:21)CGGCTGCAGCTGCAGGAGT

LibF22(SEQ ID No:22)GAGGTGCAGCTGGTGCAG

LibF23(SEQ ID No:23)GAGACGCAGCTGGTGGAGT

LibF24(SEQ ID No:24)CAGATGCAGCTGGTGCAG

LibF25(SEQ ID No:25)CAGGTACAGCTGATGCAGTC

LibF26(SEQ ID No:26)CAGGTGCAGCTGGTGCAAT

LibF27(SEQ ID No:27)CAGGTCCAGCTGGTGCAG

LibF28(SEQ ID No:28)GAGGTGCATCTGGTGGAGT

LibF29(SEQ ID No:29)CAGGTGCAGCTACAACAGTG

LibF30(SEQ ID No:30)GAAGGTGCAGCTGGTGCAGT

LibF31(SEQ ID No:31)CAGGTGCAGCTGGTGGAG

LibF32(SEQ ID No:32)GAGGTGCAGCTGGTAGAGTC

LibF33(SEQ ID No:33)CAGGTACAGCTGCAGCAGT

LibF34(SEQ ID No:34)GAGGTGCAGCTGTTGGAGTC

LibF35(SEQ ID No:35)CAGGTCCAGCTGGTACAGTCTG

LibF36(SEQ ID No:36)GAGATGCAGCTGGTGGAGTC

LibF37(SEQ ID No:37)CAGGTCACCTTGAAGGAGTCT

LibF38(SEQ ID No:38)CAGGTCCAGCTGGTGCAA

LibF39(SEQ ID No:39)CAGGTCACCTTGAGGGAGTC

LibF40(SEQ ID No:40)GAAGTGCAGCTGGTGGAG

LibF41(SEQ ID No:41)CAGGTGCAGCTGCAGGAG

LibF42(SEQ ID No:42)CAGGTGCGGCTGCAGGAG

LibF43(SEQ ID No:43)CAGGTGCAGCTGGTGGA

Antibody gene first round amplification primer reverse primer (SEQ ID No: 44～47):

LibR6(SEQ ID No:44)TGGAAGAGGCACGTTCTTTTC

LibR7(SEQ ID No:45)ACTTCCTTGTCCACCTTGGTG

LibR8(SEQ ID No:46)ACTTTCTTGTCCACCTTGGTG

LibR9(SEQ ID No:47)ACTGTTCTTGTCCACCTTGGTG、

2) Run PCR amplification according to the following procedure

94℃, 4min→98℃, 10s→59℃, 40s→72℃, 50s→72℃, 10min amplification cycle 25 times

3) Add the amplification products to the wells of 1% argarose gel and electrophoresis separate them in 1×TAE electrophoresis buffer.

4) Cut out the target antibody heavy chain variable region gene strip, and recover the antibody gene fragment according to the kit instructions (Omega Company EZNA

Gel Extraction Kit, item number: D2500).

The electrophoresis results are shown in Figure 2. All samples were amplified, the 700bp target band was clear, and the mixed bands were weak or absent but there were primer dimers.

Example 5, second round of amplification of antibody genes (two antibody heavy chain variable region genes plus splicing linkers) and gel recovery

1) Add reactants in the following order

Forward primer for the first antibody variable region in the second round of amplification of antibody genes (SEQ ID No: 48～90):

LibF50(SEQ ID No:48)CAGGTGCAGCTGGTGCAG

LibF51(SEQ ID No:49)CAAATGCAGCTGGTGCAGT

LibF52(SEQ ID No:50)GAGGTCCAGCTGGTACAGTCT

LibF53(SEQ ID No:51)CGGGTCACCTTGAGGGAG

LibF54(SEQ ID No:52)GAGGTGCAGCTGGTGAAGT

LibF55(SEQ ID No:53)GAGGTTCAGCTGGTGCAGT

LibF56(SEQ ID No:54)GAGGTGCAGCTGGTGGAG

LibF57(SEQ ID No:55)CAGGTGCAGCTACAGCAGT

LibF58(SEQ ID No:56)CAGGTACAGCTGGTGCAGTC

LibF59(SEQ ID No:57)CAGGTCCAGCTTGTGCAGT

LibF60(SEQ ID No:58)GAGGTACAACTGGTGGAGTCT

LibF61(SEQ ID No:59)CAGATCACCTTGAAGGAGTC

LibF62(SEQ ID No:60)CAGGTTCAGCTGGTGCAGT

LibF63(SEQ ID No:61)CAGGTACAGCTGCAGGAGTC

LibF64(SEQ ID No:62)CAGGTACAGCTGGTGGAGTCT

LibF65(SEQ ID No:63)CAGGTGCAGCTACAGGAGTC

LibF66(SEQ ID No:64)CAGCTGCAGCTGCAGGAGT

LibF67(SEQ ID No:65)GAGGTGCAGCTGGTGGAG

LibF68(SEQ ID No:66)CAGGTGCAGCTGCAGGAC

LibF69(SEQ ID No:67)CAGGTGCAGCTGTTGGAG

LibF70(SEQ ID No:68)CGGCTGCAGCTGCAGGAGT

LibF71(SEQ ID No:69)GAGGTGCAGCTGGTGCAG

LibF72(SEQ ID No:70)GAGACGCAGCTGGTGGAGT

LibF73(SEQ ID No:71)CAGATGCAGCTGGTGCAG

LibF74(SEQ ID No:72)CAGGTACAGCTGATGCAGTC

LibF75(SEQ ID No:73)CAGGTGCAGCTGGTGCAAT

LibF76(SEQ ID No:74)CAGGTCCAGCTGGTGCAG

LibF77(SEQ ID No:75)GAGGTGCATCTGGTGGAGT

LibF78(SEQ ID No:76)CAGGTGCAGCTACAACAGTG

LibF79(SEQ ID No:77)GAAGGTGCAGCTGGTGCAGT

LibF80(SEQ ID No:78)CAGGTGCAGCTGGTGGAG

LibF81(SEQ ID No:79)GAGGTGCAGCTGGTAGAGTC

LibF82(SEQ ID No:80)CAGGTACAGCTGCAGCAGT

LibF83(SEQ ID No:81)GAGGTGCAGCTGTTGGAGTC

LibF84(SEQ ID No:82)CAGGTCCAGCTGGTACAGTCTG

LibF85(SEQ ID No:83)GAGATGCAGCTGGTGGAGTC

LibF86(SEQ ID No:84)CAGGTCACCTTGAAGGAGTCT

LibF87(SEQ ID No:85)CAGGTCCAGCTGGTGCAA

LibF88(SEQ ID No:86)CAGGTCACCTTGAGGGAGTC

LibF89(SEQ ID No:87)GAAGTGCAGCTGGTGGAG

LibF90(SEQ ID No:88)CAGGTGCAGCTGCAGGAG

LibF91(SEQ ID No:89)CAGGTGCGGCTGCAGGAG

LibF92(SEQ ID No:90)CAGGTGCAGCTGGTGGA

Reverse primer for the first antibody variable region in the second round of amplification of antibody genes (SEQ ID No: 91～94):

LibR11(SEQ ID No:91)

LibR12(SEQ ID No:92)

LibR13(SEQ ID No:93)

LibR14(SEQ ID No:94)

Note: Lowercase letters refer to the linker coding area.

Forward primer for the second antibody variable region in the second round of amplification of antibody genes (SEQ ID No: 95～137):

LibF101(SEQ ID No:95)

LibF102(SEQ ID No:96)

LibF103(SEQ ID No:97)

LibF104(SEQ ID No:98)

LibF105(SEQ ID No:99)

LibF106(SEQ ID No:100)

LibF107(SEQ ID No:101)

LibF108(SEQ ID No:102)

LibF109(SEQ ID No:103)

LibF110(SEQ ID No:104)

LibF111(SEQ ID No:105)

LibF112(SEQ ID No:106)

LibF113(SEQ ID No:107)

LibF114(SEQ ID No:108)

LibF115(SEQ ID No:109)

LibF116(SEQ ID No:110)

LibF117(SEQ ID No:111)

LibF118(SEQ ID No:112)

LibF119(SEQ ID No:113)

LibF120(SEQ ID No:114)

LibF121(SEQ ID No:115)

LibF122(SEQ ID No:116)

LibF123(SEQ ID No:117)

LibF124(SEQ ID No:118)

LibF125(SEQ ID No:119)

LibF126(SEQ ID No:120)

LibF127(SEQ ID No:121)

LibF128(SEQ ID No:122)

LibF129(SEQ ID No:123)

LibF130(SEQ ID No:124)

LibF131(SEQ ID No:125)

LibF132(SEQ ID No:126)

LibF133(SEQ ID No:127)

LibF134(SEQ ID No:128)

LibF135(SEQ ID No:129)

LibF136(SEQ ID No:130)

LibF137(SEQ ID No:131)

LibF138(SEQ ID No:132)

LibF139(SEQ ID No:133)

LibF140(SEQ ID No:134)

LibF141(SEQ ID No:135)

LibF142(SEQ ID No:136)

LibF143(SEQ ID No:137)

Note: Lowercase letters refer to the linker coding area.

Reverse primer for the second antibody variable region in the second round of amplification of antibody genes (SEQ ID No: 138～141):

LibR16(SEQ ID No:138)TGAGGAGACGGTGACCAG

LibR17(SEQ ID No:139)TGAGGAGACAGTGACCAGGG

LibR18(SEQ ID No:140)TGAAGAGACGGTGACCATTGT

LibR19(SEQ ID No:141)TGAGGAGACGGTGACCGT

2) Run PCR amplification according to the following procedure

94℃, 4min→98℃, 10s→59℃, 40s→72℃, 50s→72℃, 10min amplification cycle 15 times

Gel Extraction Kit, item number: D2500).

The electrophoresis results are shown in Figure 3. All samples amplified the target band with different amplification intensities. The target band was clear and the contaminant bands were weak or absent.

Example 6. The third round of amplification of antibody genes (two antibody heavy chain variable region genes are spliced through linker) and gel recovery

1) Add reactants in the following order

Antibody gene third round amplification primer forward primer (SEQ ID No: 142～184):

LibF151(SEQ ID No:142)X9-ATGCCCAGGTTCAGCTGGTGCAGT

LibF152(SEQ ID No:143)X9-ATGCCGAAGTGCAGCTGGTGCAGT

LibF153(SEQ ID No:144)X9-ATGCCGAGATGCAGCTGGTGGAG

LibF154(SEQ ID No:145)X9-ATGCCCAGGTCCCAGCTGGTGCAG

LibF155(SEQ ID No:146)X9-ATGCCGAGGTGCAGCTGGTGGA

LibF156(SEQ ID No:147)X9-ATGCCCAGGTCCAGCTGGTACAGTC

LibF157(SEQ ID No:148)X9-ATGCCCAGGTACAGCTGGTGCAGTC

LibF158(SEQ ID No:149)X9-ATGCCCAGATCACCTTGAAGGAGTCTG

LibF159(SEQ ID No:150)X9-ATGCCGAGGTGCAGCTGGTGCAGT

LibF160(SEQ ID No:151)X9-ATGCCGAGGTTCAGCTGGTGCAGTC

LibF161(SEQ ID No:152)X9-ATGCCCAAATGCAGCTGGTGCAGT

LibF162(SEQ ID No:153)X9-ATGCCCAGATGCAGCTGGGTGCAG

LibF163(SEQ ID No:154)X9-ATGCCCAGGTCCAGCTGGTGCAAT

LibF164(SEQ ID No:155)X9-ATGCCCAGGTCCAGCTTGTGCAGT

LibF165(SEQ ID No:156)X9-ATGCCCAGGTACAGCTGATGCAGTCT

LibF166(SEQ ID No:157)X9-ATGCCGAGGTGCAGCTGTTGGAGTC

LibF167(SEQ ID No:158)X9-ATGCCGAGGTCCAGCTGGTACAGTCTG

LibF168(SEQ ID No:159)X9-ATGCCCAGGTGCAGCTGGTGCAAT

LibF169(SEQ ID No:160)X9-ATGCCCAGGTGCAGCTGCAGGAG

LibF170(SEQ ID No:161)X9-ATGCCCAGGTGCAGCTGCAGGAC

LibF171(SEQ ID No:162)X9-ATGCCCAGGTGCAGCTGGTGGAGT

LibF172(SEQ ID No:163)X9-ATGCCGAAGTGCAGCTGGTGGAGTC

LibF173(SEQ ID No:164)X9-ATGCCGAGACGCAGCTGGTGGAGT

LibF174(SEQ ID No:165)X9-ATGCCGAGGTGCAGCTGGTGGAGA

LibF175(SEQ ID No:166)X9-ATGCCCAGGTGCAGCTACAGCAGT

LibF176(SEQ ID No:167)X9-ATGCCCAGGTACAGCTGGTGGAGTC

LibF177(SEQ ID No:168)X9-ATGCCCAGCTGCAGCTGCAGGAGT

LibF178(SEQ ID No:169)X9-ATGCCCAGGTGCGGCTGCAGGAG

LibF179(SEQ ID No:170)X9-CATGCCCAGGTGCAGCTGGTGGA

LibF180(SEQ ID No:171)X9-ATGCCGAGGTACAACTGGTGGAGTCT

LibF181(SEQ ID No:172)X9-ATGCCCAGGTGCAGCTACAACAGTG

LibF182(SEQ ID No:173)X9-ATGCCCAGGTCACCTTGAAGGAGTCTG

LibF183(SEQ ID No:174)X9-ATGCCGAGGTGCAGCTGGTAGAGTCT

LibF184(SEQ ID No:175)X9-ATGCCCGGGTCACCTTGAGGGAGT

LibF185(SEQ ID No:176)X9-ATGCCGAGGTGCATCTGGTGGAGT

LibF186(SEQ ID No:177)X9-ATGCCCAGGTACAGCTGCAGGAG

LibF187(SEQ ID No:178)X9-ATGCCCGGCTGCAGCTGCAGGAGT

LibF188(SEQ ID No:179)X9-ATGCCCAGGTACAGCTGCAGCAGTC

LibF189(SEQ ID No:180)X9-ATGCCCAGGTGCAGCTACAGGAGT

LibF190(SEQ ID No:181)X9-ATGCCCAGGTCACCTTGAGGGAGTC

LibF191(SEQ ID No:182)X9-ATGCCCAGGTGCAGCTGTTGGAG

LibF192(SEQ ID No:183)X9-CATGCCGAGGTGCAGCTGGTGAAG

LibF193(SEQ ID No:184)X9-ATGCCCAGGTGCAGCTGGGTGCAG

Note: X9- represents the 9-nucleotide restriction endonuclease I and its protective bases. Restriction endonuclease I can be AgeI, ApaI, AscI, BamHI, BglII, Bsil, ClaI, CsI, EcoRI, FseI, HindIII, KpnI, MfeI, MluI, NcoI, NheI, NotI, PacI, Pmel, Paul, SacI, One of SauI, SphI, VspI, XbaI, and XhoI.

Reverse primer for the third round of antibody gene amplification (SEQ ID No: 185～188):

LibR21(SEQ ID No:185)Y9-TGAGGAGACGGTGACCAG

LibR22(SEQ ID No:186)Y9-TGAGGAGACAGTGACCAGGG

LibR23(SEQ ID No:187)Y9-TGAAGAGACGGTGACCATTGT

LibR23(SEQ ID No:188)Y9-TGAGGAGACGGTGACCGT

Note: Y9- represents the 9-nucleotide restriction endonuclease II and its protective bases. Restriction endonuclease II can be AgeI, ApaI, AscI, BamHI, BglII, Bsil, ClaI, CspI, EcoRI, FseI, HindIII, KpnI, MfeI, MluI, NcoI, NheI, NotI, PacI, Pmel, Paul, SacI, One of SauI, SphI, VspI, XbaI, and XhoI.

2) Run PCR amplification according to the following procedure

94℃, 4min→98℃, 10s→59℃, 40s→72℃, 60s→72℃, 10min amplification cycle 15 times

Gel Extraction Kit, item number: D2500).

The electrophoresis results are shown in Figure 4. All samples amplified a target band of about 750 bp. The amplification intensity was different, but there were many contaminant bands. These contaminant bands will be removed during the gel cutting, purification and recovery process and will not affect the library. Construct.

Example 7. Digestion and recovery of antibody heavy chain variable region genes and vectors

1) Enzyme digestion

Prepare restriction endonuclease I digestion system according to the following system

Incubate at 37℃ for 1.5h

Add the following ingredients to the above system

10×Tango buffer 42μL

Restriction endonuclease II (Thermo) 15μL

ddH ₂ O 3μL

Mix well and incubate at 37℃ for 1.5h

2) Purification of enzyme digestion products

For the enzyme-digested antibody gene, a centrifugal adsorption column was used to directly purify the PCR product (Omega Company EZNA

Gel Extraction Kit, product number: D2500); for the digested antibody gene, use a centrifugal adsorption column for gel recovery and purification (Omega Company EZNA

Gel Extraction Kit, item number: D2500). Follow the kit instructions.

The electrophoresis results are shown in Figure 5. The vector has been cut, the size is consistent with the expected 5000bp, and the enzyme digestion was successful.

Example 8. Ligation reaction and purification

1) Connection reaction

Mix well and incubate overnight at 16°C

2) Purification

Direct purification of PCR products using centrifugal adsorption columns (Omega Company EZNA

Gel Extraction Kit, product number: D2500), follow the instructions of the kit.

Example 9. Competent state preparation, transformation and plating

1) Preparation of Mini Agar solid medium

Weigh 1.1g of Agar, add 70ml of ultrapure water, sterilize with moist heat at 121°C for 20 minutes, take out and shake well. When the temperature drops to 50-60°C, add disodium hydrogen phosphate to 60mM, potassium dihydrogen phosphate to 17mM, sodium chloride to 8mM, ammonium chloride to 18mM, calcium chloride to 0.1mM, magnesium sulfate to 2mM, and vitamins B1 to 1mM, glucose to 2% (W/V), and the final volume is 100ml. Shake well and pour into a sterile petri dish to solidify.

2) Dip TG1 seed bacteria, streak on Mini Agar solid medium, and culture at 37°C for 2 days until single clones appear.

3) Pick the TG1 single clone from the Mini Agar plate, inoculate it into 10 ml LDM liquid medium, and culture it overnight at 37°C with shaking at 220 rpm. The next day, take 6 ml of bacterial solution and inoculate it into 600 ml of liquid culture medium, and culture it with shaking at 37°C and 220 rpm until OD600=0.7.

4) Transfer the bacterial solution into a 1000ml sterile centrifuge tube, centrifuge at 4000rpm for 10 minutes at 4°C, and discard the supernatant. Use 600 ml of moist heat sterilized ultrapure water pre-cooled on ice and shake thoroughly on ice to resuspend the bacterial pellet. Repeat washing twice. Use 6 ml of moist heat-sterilized 10% glycerol (prepared with ultrapure water) pre-cooled on ice, shake thoroughly on ice to resuspend the bacterial pellet, and dispense 200 μL/tube into 1.5 ml sterile EP tubes on ice.

5) Take 5 μL of the purified ligation product (ie, phagemid), add it to the competent TG1 on ice, transfer it to a 2mm electroshock cup pre-cooled on ice, and keep in ice bath for 10 minutes. Set the electroshock apparatus (Harvard Apparatus) voltage to 2.5kV to transform competent cells. Immediately after electroporation, the cells were transferred to SOC medium and cultured for 1 hour at 37°C with shaking at 180 rpm. Carry out gradient dilution of the bacterial solution and apply it to the 2YT medium plate. Invert the plate and culture it at 37°C overnight to obtain single clones.

6) Pick a single clone, inoculate it into 200 μL of 2YT liquid medium, and culture it overnight at 37°C with shaking at 200 rpm.

7) Take the bacterial liquid and use LD4F and LD3-R for PCR amplification and identification.

LD4F：GTTAGCTCACTCATTAGGCACC(SEQ ID No:189)

LD3-R：GTAAATGAATTTTCTGTATGAGG(SEQ ID No:190)

8) Take positive clones, perform phagemid sequencing and analyze sequence diversity.

The electrophoresis results are shown in Figure 6. There are 4 clones with non-antibody genes. Except for non-specific amplification, the rest are correct positive clones, with a positive rate of 92/96=95.8%.

The results of the homology analysis of the antibody sequences in the positive clones are shown in Figure 7. From the clones identified as positive, 30 clones were randomly selected for sequencing. Except for one clone that failed to sequence, the sequences of the remaining 29 clones were obtained. The homology of antibody sequences is displayed in a circular phylogenetic tree, in which bold numbers represent different antibody sequences, and the numbers on the branch nodes represent bootstrap values. The greater the homology, the more similar the antibody sequences are, and the shorter the terminal branches on the evolutionary tree. If the terminal branches of the two sequences disappear and only the node vertical line is left, it means that the two antibody sequences are completely identical; on the contrary, the greater the difference in the length of the branch ends, it means that the antibodies on the branches appeared at a farther time due to evolution. In the sequence, the greater the sequence difference is. It can be seen that these sequences are on multiple branches with different levels of homology. No terminal branches disappear and only the node vertical lines are left. All the analyzed sequences are different from each other, so the diversity is 29/29=100 %, showing good antibody sequence diversity in the library.

The following table shows the quality indicators of the constructed antibody library

阳性率Positive rate	多样性Diversity	正确率Correct rate	库容storage space
95.8％95.8%	100％100%	100％100%	1×10 ⁹pfu 1×10 ⁹ pfu

Example 10. Antibody library panning

1) Activation of host bacteria TG1: Prepare a mini agar medium plate, culture TG1 overnight in a 37°C incubator by streaking method.

2) Magnetic bead washing and blocking: absorb 50 μL of magnetic beads (purchased from Invitrogen), place on a magnetic stand, absorb the liquid after adsorption, resuspend in 1 ml of PBS, wash twice, and use 1 ml of 1.5% skimmed milk powder + 1.5% BSA. Block with blocking agent (concentration of blocking agent gradually increases during the second and third rounds of panning) for 1 hour and remove the liquid.

3) Antigen binding: Use 1ml PBS (pH7.2-7.4) to dilute protein A and protein B to a working concentration of 16 μg/ml (both purchased from Acro Biosystem, and the antigen concentration will gradually decrease during each subsequent round of panning). Use this Resuspend the magnetic beads in PBS solution containing protein A and protein B, and rotate and incubate for 1 hour.

4) Library blocking: Synchronize with the binding of the antigen to the magnetic beads, take 10 ¹¹ pfu phage virus particles (from the original antibody library or panning amplification product), and use 1 ml of 1.0% skim milk powder + 1.0% BSA blocking agent to rotate and incubate for 1 Hour.

5) Phage binding: Place the magnetic beads on the magnetic stand and remove the liquid. Add the blocked library to magnetic beads, resuspend and incubate with rotation for 1 hour, and remove the liquid.

6) Washing: Wash with 1ml PBST [0.01M PBS (pH7.4), 0.1% Tween-20 (the second and third rounds of Tween-20 concentration are 0.2% and 0.3% respectively)], and then use 0.01M PBS (pH7) .4) Washing.

7) Elution: Aspirate the liquid, elute with 300μL 0.2M glycine-hydrochloric acid (pH2.2) for 10 minutes, add 20μL neutralizing solution [1M Tris-Cl (pH9.0)], mix well, and temporarily store at 4°C. .

8) Measure the titer: Take 2 μL, 0.2 μL (the original solution is diluted 10 times with 2×YT culture medium and then take 2 μL), 0.02 μL (the original solution is diluted 100 times with 2×YT culture medium and then take 2 μL) eluate, and mix with 0.2 ml of mid-logarithmic (OD600=0.5) TG1 was mixed, incubated at room temperature for 30 minutes, evenly spread on 2×YT-GA100 [containing 2% glucose, 100 μg/ml ampicillin] plate, cultured at 37°C overnight, and counted about 50 The number of clones on the plate, and the titer is calculated based on the dilution factor.

9) Phage amplification: While panning is in progress, pick the TG1 single clone on the mini agar plate, inoculate it into 10ml 2×YT culture medium, and culture it to the mid-log phase (OD600=0.5) at 37°C with shaking at 250rpm. . Add 200 μL of the elution product obtained by panning and incubate at 37°C for 30 minutes. Add helper phage M13KO7, incubate at 37°C for another 30 minutes, and then incubate at 37°C with shaking at 250 rpm for 1 hour. Centrifuge to remove the supernatant, resuspend in 20 ml of 2×YT containing ampicillin at a working concentration of 100 μg/ml and kanamycin at a working concentration of 50 μg/ml, and incubate overnight at 30°C with shaking at 220 rpm.

10) Phage precipitation: Centrifuge at 10,000 rpm for 15 minutes to remove bacterial cells. Add 1/5 volume of 2.5M NaCl/20% PEG8000 to the supernatant and keep in ice bath for 2 hours. Centrifuge at 10,000 rpm for 10 minutes to obtain the phage pellet, remove the residual liquid, add 0.2ml 0.01M PBS (pH7.4) solution to resuspend the pellet, and measure the titer as above.

11) Repeat steps 2)-10) two or three times to obtain phage display antibodies with strong binding capacity. When performing step 3), use 1 ml of PBS solution containing protein A with a working concentration of 10 μg/ml, and rotate and incubate for 1 hour. When repeating 2)-10) next time, step 3) is changed to 10 μg/ml protein B in 1 ml PBS solution, and rotated and incubated for 1 hour.

Example 11. Monoclonal ELISA

1) Coat the enzyme plate with 0.3 μg/ml streptavidin overnight at 4°C, treat with 2% BSA/PBS blocking solution for 2 hours, and wash 3 times with PBS.

2) Add 100 μL of PBS solution with a working concentration of 1 μg/ml protein A to some enzyme plates per well, add 100 μL of PBS solution with a working concentration of 1 μg/ml protein B to some enzyme plates per well, and add 100 μL of PBS solution with a working concentration of 1 μg/ml protein B to each well of some enzyme plates. Add 100 μL of PBS solution. Incubate at 37°C for 1 h, remove the liquid, and wash three times with PBS.

3) Pick a single clone colony from the 2×YT-GA100 plate, culture it with shaking to the mid-log phase, add the helper phage M13KO7, and incubate at 37°C for 30 minutes. Incubate at 37°C with shaking at 220 rpm for 1 hour, and centrifuge at 4000 rpm for 15 minutes. Resuspend in 400 μL of 2×YT containing ampicillin at a working concentration of 100 μg/ml and kanamycin at a working concentration of 50 μg/ml, and incubate overnight at 30°C with shaking at 220 rpm. Centrifuge at 4000rpm for 15 minutes to precipitate the bacteria.

4) Add 50 μL of 4% BSA/PBS to the microplate, and at the same time, take 50 μL of phage supernatant, mix well, and incubate for 1 hour.

5) Remove the liquid, wash 5 times with 0.1% PBST and 3 times with PBS to remove the liquid.

6) Dilute the HRP-labeled anti-M13 phage antibody (purchased from Beijing Yiqiao Shenzhou) 3000 times with 2% BSA, add 100 μL to the enzyme plate, incubate for 1 hour, remove the liquid, wash 3 times with 0.1% PBST, and pat dry Residual liquid.

7) Add 100 μL TMB chromogenic solution, incubate at 37°C for 10 minutes or until the blue color is fully developed, add 100 μL 1M sulfuric acid to terminate the reaction, and read the OD450 on a microplate reader.

The results of the monoclonal phage ELISA are shown in Figure 8. The phages obtained by final panning were monocloned and multiplied in large quantities, the host bacteria were removed, and the supernatant was used as a crude phage antibody for ELISA detection. The results show that these monoclones have different binding abilities to protein A or B. Some monoclones bind to protein A stronger than protein B, and some monoclones bind to protein B stronger than protein A. A small number of monoclones are due to experimental errors and combine with both. All weak.

The following table shows the original reading value of OD450 and the ratio of experimental group/PBS

Table 1 OD450 original reading value

Table 2 Experimental group OD450/PBS group

All documents mentioned in this application are incorporated by reference in this application to the same extent as if each individual document was individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

A method for constructing a phage display library for obtaining bispecific antibodies, characterized in that the bispecific antibodies are bispecific fully human single domain antibodies,

The method includes the following steps:

(a) Extract total RNA from human PBMC;

(b) using the total RNA as a template to synthesize cDNA through reverse transcription;

(c) Using the cDNA as a template, use the first primer set to perform the first round of PCR amplification, thereby obtaining the first round of amplification products; wherein the first primer set includes the positive sequences shown in SEQ ID Nos: 1 to 43 Toward primer and reverse primer shown in SEQ ID No:44-47;

(d) Using the first round amplification product as a template, use the second primer set to perform the second round of PCR amplification to obtain the second round amplification product; wherein, the second primer set includes SEQ ID No: 48-90 The first antibody variable region forward primer shown in SEQ ID No:91-94, the first antibody variable region reverse primer shown in SEQ ID No:91-94, the second antibody variable region forward primer shown in SEQ ID No:95～137 Primers, and the second antibody variable region reverse primer shown in SEQ ID No: 138～141;

(e) Using the second round amplification product as a template, use the third primer set to perform the third round of PCR amplification to obtain the third round amplification product; wherein the third primer set includes SEQ ID No: 142~184 The forward primer shown is, the reverse primer shown in SEQ ID No: 185～188;

(f) The vector used to construct the library and the third round amplification product are digested with restriction endonuclease I and restriction endonuclease II to obtain digested linearized vector fragments and linearized Antibody gene fragments;

(g) performing a ligation reaction on the digested linearized vector fragment and the linearized antibody gene fragment to obtain a ligation product;

(h) Introducing the ligation product into a host cell for phage display, thereby obtaining the bispecific fully human single domain antibody phage display antibody library.
The method of claim 1, wherein the reaction system of the first round of PCR amplification in step (c) is: ddH 2 O 13.6 μL, 2.5mM dNTPs 1.6 μL, 10× buffer 2 μL, cDNA 0.8 μL, 1 μL of 10 μM upstream primer, 1 μL of 10 μM downstream primer, Ex Taq 0.08 μL, total volume 20 μL.
The method according to claim 1, characterized in that the reaction program of the first round of PCR amplification in step (c) is: 94°C 4min, 98°C 10s, 59°C 40s, 72°C 50s, 72°C 10 min, amplification cycle 25 times.
The method of claim 1, wherein the reaction system of the second round of PCR amplification in step (d) is: 10×buffer 2.5 μL, 2.5mM dNTPs 1.6 μL, and the first round of amplification is recovered 15ng of product, 1μL of 10μM upstream primer, 1μL of 10μM downstream primer, 0.1μL of Ex Taq, and ddH 2 O to make up the total volume to 25μL.
The method of claim 1, characterized in that the reaction program of the second round of PCR amplification in step (d) is: 94°C 4min, 98°C 10s, 59°C 40s, 72°C 50s, 72°C 10 min, amplification cycle 15 times.
The method of claim 1, wherein the reaction system of the third round of PCR amplification in step (e) is: 10× buffer 2.5 μL, 2.5mM dNTPs 1.6 μL, and the second round of amplification is recovered 15 ng of product, 1 μL of 10 μM upstream primer, 1 μL of 10 μM downstream primer, 0.1 μL of Ex Taq, and ddH 2 O to make up the total volume to 25 μL.
The method according to claim 1, characterized in that the reaction program of the third round of PCR amplification in step (e) is: 94°C 4min, 98°C 10s, 59°C 40s, 72°C 50s, 72°C 10 min, amplification cycle 15 times.
The method of claim 1, wherein step (h) further includes: obtaining positive clones, performing phagemid sequencing and analyzing sequence diversity.
The method of claim 1, wherein step (i) further includes:

(i1) Activation of host bacterial TG1;

(i2) Magnetic beads washing and blocking;

(i3) Antigen binding;

(i4) Library closed;

(i5) Phage binding;

(i6) Washing;

(i7) Elution;

(i8) Measure titer;

(i9) Phage amplification;

(i10) Phage precipitation;

(i11) Repeat steps (i2) to (i10) two or three times to obtain phage display antibodies with strong binding capacity.
A phage display library of bispecific antibodies, characterized in that the phage display library of bispecific antibodies is constructed using the method as described in any one of claims 1 to 9.
The phage display library according to claim 10, wherein the positive rate of the library is ≥80%, preferably, ≥90%.
The phage display library of claim 10, wherein the antibody sequence diversity of the library is ≥90%, preferably, ≥95%.
The phage display library according to claim 10, wherein the library has a library capacity of ≥1×10 8 pfu, preferably ≥5×10 8 pfu.
A method for panning bispecific antibodies, characterized by comprising the steps:

(S1) providing a phage display library of the bispecific antibody according to claim 10;

(S2) Use the antigen A1 and the antigen A2 to pan the antibody library to obtain bispecific antibodies against the antigen A1 and the antigen A2.
The method according to claim 14, characterized in that, in step (S2), it includes the following sub-steps:

(S2a) In the first round of antibody library panning, two antigens (A1 and A2) are added at the same time.

(S2b) In the second round of screening, only a single antigen A1 was used for screening;

(S2c) In the third round of screening, only a single antigen A2 was used for screening;

(That is, the two antigens A1 and A2 are used alternately in these two rounds for screening). Since in the second and third rounds of screening, only a single antigen is used for screening in each round and the two antigens are used alternately, antibodies that only bind to one antigen can be screened out, and all antibodies that bind to both antigens at the same time are retained.
The method of claim 14, further comprising:

(S3) For the bispecific antibody obtained in the previous step, detect its binding performance to antigen A1 and antigen A2 through ELISA.
A primer set, the primer set includes primers with sequences as shown in SEQ ID Nos: 1 to 188.