WO2024016843A1

WO2024016843A1 - Recombination system for expressing fab fragment library

Info

Publication number: WO2024016843A1
Application number: PCT/CN2023/097017
Authority: WO
Inventors: 郝小勃; 刘志刚; 周晓巍; 刘玉兰; 黄维国; 王松猛; 刘子彬
Original assignee: 北京智仁美博生物科技有限公司
Priority date: 2022-07-22
Filing date: 2023-05-30
Publication date: 2024-01-25
Also published as: CN117430696A

Abstract

The present application discloses a plasmid combination for expressing an antibody Fab fragment library, containing a first plasmid and a second plasmid. The first plasmid contains an antibody heavy chain VH-CH1 expression unit and a first recombination site; the second plasmid contains an antibody light chain VL-CL expression unit and a second recombination site; and the first recombination site is different from the second recombination site. The present application provides a recombination system and recombinant cell for expressing an antibody Fab fragment library, and also provides a use of the plasmid combination, the recombination system and the recombinant cell, and a preparation method for an antibody Fab fragment library.

Description

Recombinant systems for expressing Fab fragment libraries

Cross-references to related applications

This application claims priority from Chinese Patent Application No. 202210871809.6 submitted on July 22, 2022, the entire content of which is incorporated herein by reference in its entirety.

Field of invention

This application generally relates to the fields of genetic engineering and antibody drugs; specifically, this application provides plasmid combinations, recombinant systems and recombinant cells for expressing antibody Fab fragment libraries, as well as methods for preparing antibody Fab fragment libraries.

Background of the invention

Large-capacity, high-quality antibody libraries provide an important source of molecules for screening high-affinity antibodies to any antigen, and have high commercial and application value. At present, most of the high-quality antibody libraries reported at home and abroad are constructed through electroconversion technology. However, the size of the antibody library constructed using electroconversion technology is limited by the transformation efficiency. It requires multiple batches of antibody library preparation, which takes a long time and consumes a lot of manpower and material resources.

Respiratory syncytial virus (RSV) is the most important viral pathogen causing acute lower respiratory tract infections (ALRTI) in children under 5 years of age worldwide ¹ . RSV infection is the leading cause of hospitalization for viral respiratory tract infections in infants and young children, seriously endangering children's health, especially premature infants, infants and young children with congenital heart disease or primary immune deficiency. The five countries with the highest incidence of RSV infection are Pakistan, India, Nigeria, China and Indonesia, which contribute nearly half of the global RSV-ALRTI disease ^burden2 .

Respiratory syncytial virus (RSV) belongs to the family Pneumoviridae and the genus Orthopneumovirus. It is a non-segmented single-stranded negative-sense RNA virus ³ and can be divided into two subtypes, A and B, based on differences in surface antigens ⁴ . The RSV genome includes 10 genes encoding 11 proteins. Among them, adhesion protein G and fusion protein F are the main protective antigens on the surface of the virus and can stimulate the body to produce neutralizing antibodies ⁵ . G Proteins vary greatly between subtypes, so antibodies against G protein are mostly subtype-specific antibodies ⁶ , while F protein is highly conserved among subtypes, and antibodies induced by F protein can simultaneously inhibit type A/B RSV. Viral infection.

F protein is a type I transmembrane protein. Its inactive precursor (F0) consists of ⁵⁷⁴ amino acids7. Three F0s form a trimer. When transported through the Golgi apparatus, the host's furin protease binds at the 109th position of F0. Cleavage is performed between amino acids 110 and between amino acids 136 and 137. After cleavage, the short peptide (P27) of the middle 27 amino acids is released, while the remaining two segments, F2 and F1, are connected through disulfide bonds (Cys69–Cys212 and Cys37–Cys439) to form the active F protein ⁸ . There is a highly hydrophobic fusion peptide (FP) at the N-terminus of the F1 protein, which is located in the hydrophobic cavity of the protein and is protected from the influence of the external hydrophilic environment. When F protein appears on the virion surface or cell surface, its structure is not stable, but is in a high-energy metastable pre-fusion conformation structure (Prefusion glyprotein, Pre-F) ⁹ . Subsequently, the N-terminus of the F1 protein undergoes a series of dramatic structural changes. This process induces membrane fusion, thereby achieving the purpose of virus infection of cells. At the same time, this process also causes the F protein to transform from a high-energy metastable Pre-F structure to a stable post-fusion F protein structure (Postfusion glyprotein, Post-F).

There is currently no vaccine to prevent RSV. The U.S. Food and Drug Administration has approved Palivizumab (trade name: Synagis) to prevent RSV infection in high-risk infants and young children. It requires up to 5 injections to cover a typical RSV season. Due to the cost It is expensive and cannot be widely used in infants and young children.

Based on clinical needs, the development of anti-respiratory syncytial virus antibodies is of great medical significance for preventing related diseases caused by RSV infection.

Summary of the invention

In a first aspect, the application provides an antibody against respiratory syncytial virus (RSV), which comprises a heavy chain variable region containing the amino acid sequences of HCDR1, HCDR2 and HCDR3 and a light chain variable region containing the amino acid sequences of LCDR1, LCDR2 and LCDR3. variable area, in which

The amino acid sequence of HCDR1 is shown in SEQ ID NO:32, the amino acid sequence of HCDR2 is shown in SEQ ID NO:33, the amino acid sequence of HCDR3 is shown in SEQ ID NO:34, and the amino acid sequence of LCDR1 The sequence is as shown in SEQ ID NO:35 The amino acid sequence of the LCDR2 is shown in SEQ ID NO:36 and the amino acid sequence of the LCDR3 is shown in SEQ ID NO:37; or

The amino acid sequence of the HCDR1 is shown in SEQ ID NO:38, the amino acid sequence of the HCDR2 is shown in SEQ ID NO:39, the amino acid sequence of the HCDR3 is shown in SEQ ID NO:40, the amino acid sequence of the LCDR1 The sequence is shown in SEQ ID NO:41, the amino acid sequence of the LCDR2 is shown in SEQ ID NO:42 and the amino acid sequence of the LCDR3 is shown in SEQ ID NO:43;

Among them, the HCDR and LCDR amino acid sequences are defined according to Kabat.

In some embodiments of the first aspect, the amino acid sequence of the antibody heavy chain variable region is as shown in SEQ ID NO: 28 or 30.

In some embodiments of the first aspect, the amino acid sequence of the antibody light chain variable region is as shown in SEQ ID NO: 29 or 31.

In some embodiments of the first aspect, the amino acid sequence of the antibody heavy chain variable region is as shown in SEQ ID NO: 28, and the amino acid sequence of the antibody light chain variable region is as shown in SEQ ID NO: 29; or

The amino acid sequence of the variable region of the heavy chain of the antibody is shown in SEQ ID NO: 30, and the amino acid sequence of the variable region of the light chain of the antibody is shown in SEQ ID NO: 31.

In a second aspect, the application provides an antibody against respiratory syncytial virus (RSV), wherein the amino acid sequence of the heavy chain variable region of the antibody has at least 90% identity with SEQ ID NO: 28 or 30, and the The amino acid sequence of the light chain variable region of the antibody has at least 90% identity with SEQ ID NO: 29 or 31.

In some embodiments of either of the first and second aspects, the antibody is a neutralizing antibody.

In some embodiments of either of the first and second aspects, the antibody is capable of binding the F protein of human respiratory syncytial virus (RSV).

In some embodiments of either of the first and second aspects, the antibody is a Fab fragment, a whole antibody, an F(ab') ₂ fragment, or a single chain Fv fragment (scFv).

In some embodiments of either of the first and second aspects, the antibody is a monoclonal antibody.

In some embodiments of any of the first and second aspects, the antibody Comprises a heavy chain constant region selected from the group consisting of IgGl subtype, IgG2 subtype or IgG4 subtype.

In some embodiments of any one of the first and second aspects, the heavy chain constant region comprises the Fc segment sequence of the IgG1 subtype heavy chain constant region and positions 252, 254, and 256 of the Fc segment sequence The amino acid sequences are Y, T and E respectively, wherein the amino acid sequence of the antibody constant region is determined according to EU numbering.

In some embodiments of either of the first and second aspects, the antibody comprises a light chain constant region selected from the kappa subtype or the lambda subtype.

In a third aspect, the present application provides nucleic acid molecules encoding the antibodies described in the first or second aspect.

In a fourth aspect, the present application provides a pharmaceutical composition comprising the antibody described in the first or second aspect and a pharmaceutically acceptable excipient, diluent or carrier.

In the fifth aspect, the application provides the use of the antibody described in the first or second aspect, the nucleic acid molecule described in the third aspect, or the pharmaceutical composition described in the fourth aspect in the preparation of drugs for preventing or treating RSV-related diseases. use.

In a sixth aspect, the present application provides a method for preventing or treating RSV-related diseases, which includes administering the antibody described in the first or second aspect, or the pharmaceutical composition described in the fourth aspect to an individual in need.

In a seventh aspect, the present application provides a plasmid combination for expressing an antibody Fab fragment library, which includes a first plasmid and a second plasmid, wherein

The first plasmid includes an antibody heavy chain VH-CH1 expression unit and a first recombination site;

The second plasmid includes an antibody light chain VL-CL expression unit and a second recombination site; and

The first recombination site is different from the second recombination site.

In some embodiments of the seventh aspect, the plasmid combination comprises a plurality of said first plasmids, and a plurality of said first plasmids carry different said antibody heavy chain VH-CH1 expression units and have the same said First recombination site.

In some embodiments of the seventh aspect, the plasmid combination comprises a plurality of said second plasmids, and a plurality of said second plasmids carry different said antibody light chain VL-CL expression units and have the same said Second recombination site.

In some embodiments of the seventh aspect, the first plasmid and/or the second plasmid for phagemids.

In some embodiments of the seventh aspect, the first plasmid and the second plasmid comprise different origins of replication.

In some embodiments of the seventh aspect, the origin of replication is selected from one or more of the following: pBR ori, CDF ori, origin of replication of filamentous phage M13 (f1 ori), and p15A ori.

In some embodiments of the seventh aspect, the first recombination site and the second recombination site are sites for phage attachment in the respective genomes of the phage and its host bacterium.

In some embodiments of the seventh aspect, the first recombination site is attP and the second recombination site is attB.

In some embodiments of the seventh aspect, the first recombination site and the second recombination site are located at the polyclonal restriction sites of the first plasmid and the second plasmid, respectively.

In some embodiments of the seventh aspect, the antibody light chain constant region CL is a human kappa light chain constant region or a human lambda light chain constant region.

In some embodiments of the seventh aspect, the CH1 segment of the antibody heavy chain constant region is selected from the group consisting of IgG1, IgG2, IgG3 or IgG4 subtypes.

In some embodiments of the seventh aspect, the first plasmid and/or the second plasmid comprise a resistance gene coding region.

In some embodiments of the seventh aspect, the 3' end of the nucleic acid molecule encoding the CH1 segment of the constant region of the antibody heavy chain is fused to the 5' end of the nucleic acid molecule encoding the gIII protein of filamentous phage M13.

In some embodiments of the seventh aspect, the resistance gene is an antibiotic resistance gene.

In some embodiments of the seventh aspect, the resistance gene is selected from the group consisting of: chloramphenicol resistance gene (CmR), ampicillin resistance gene (Ampr), kanamycin resistance gene (KaR), and tetracycline resistance gene. sex gene (TetR).

In an eighth aspect, the present application provides a recombinant system for expressing an antibody Fab fragment library, which includes a first plasmid, a second plasmid and a cell expressing phage integrase; wherein

The second plasmid includes an antibody light chain VL-CL expression unit and a second recombination site; with and

The first recombination site is different from the second recombination site.

In some embodiments of the eighth aspect, the recombinant system comprises a plurality of said first plasmids, and a plurality of said first plasmids carry different said antibody heavy chain VH-CH1 expression units and have the same said First recombination site.

In some embodiments of the eighth aspect, the recombinant system comprises a plurality of said second plasmids, and a plurality of said second plasmids carry different said antibody light chain VL-CL expression units and have the same said Second recombination site.

In some embodiments of the eighth aspect, the first plasmid and/or the second plasmid are phagemids.

In some embodiments of the eighth aspect, the first plasmid and the second plasmid comprise different origins of replication.

In some embodiments of the eighth aspect, the origin of replication is selected from one or more of the following: pBR ori, CDF ori, origin of replication of filamentous phage M13 (f1 ori), and p15A ori.

In some embodiments of the eighth aspect, the first recombination site and the second recombination site are sites for phage attachment in the respective genomes of the phage and its host bacterium.

In some embodiments of the eighth aspect, the first recombination site is attP and the second recombination site is attB.

In some embodiments of the eighth aspect, the first recombination site and the second recombination site are located at the polyclonal restriction sites of the first plasmid and the second plasmid, respectively.

In some embodiments of the eighth aspect, the antibody light chain constant region CL is a human kappa light chain constant region or a human lambda light chain constant region.

In some embodiments of the eighth aspect, the CH1 segment of the antibody heavy chain constant region is selected from the group consisting of IgG1, IgG2, IgG3 or IgG4 subtypes.

In some embodiments of the eighth aspect, the first plasmid and/or the second plasmid comprise a resistance gene coding region.

In some embodiments of the eighth aspect, the 3' end of the nucleic acid molecule encoding the CH1 segment of the constant region of the antibody heavy chain is fused to the gIII protein encoding the filamentous phage M13. The 5' end of a nucleic acid molecule.

In some embodiments of the eighth aspect, the resistance gene is an antibiotic resistance gene.

In some embodiments of the eighth aspect, the resistance gene is selected from the group consisting of: chloramphenicol resistance gene (CmR), ampicillin resistance gene (Ampr), kanamycin resistance gene (KaR) and tetracycline resistance gene. sex gene (TetR).

In some embodiments of the eighth aspect, the phage integrase is a tyrosine integrase.

In some embodiments of the eighth aspect, the phage integrase is lambda phage integrase.

In some embodiments of the eighth aspect, the phage integrase is inducibly expressed or constitutively expressed.

In some embodiments of the eighth aspect, the phage integrase is inducibly expressed.

In some embodiments of the eighth aspect, the cell expressing the phage integrase is a prokaryotic cell.

In some embodiments of the eighth aspect, the cell expressing the phage integrase is E. coli.

In some embodiments of the eighth aspect, the cells expressing phage integrase are genetically engineered bacteria.

In some embodiments of the eighth aspect, the first plasmid, the second plasmid and the cell expressing the phage integrase each exist independently, or at least one of the first plasmid and the second plasmid has been introduced into the cell. cells expressing phage integrase.

In a ninth aspect, the present application provides recombinant cells for expressing an antibody Fab fragment library, which comprise a first plasmid, a second plasmid and express phage integrase; wherein

The first recombination site is different from the second recombination site.

In some embodiments of the ninth aspect, the recombinant cell comprises a plurality of said first plasmids, and a plurality of said first plasmids carry different said antibody heavy chain VH-CH1 expression units and have the same said First recombination site.

In some embodiments of the ninth aspect, the recombinant cell comprises a plurality of said second plasmids, and a plurality of said second plasmids carry different said antibody light chain VL-CL expression units and have the same said Second recombination site.

In some embodiments of the ninth aspect, the first plasmid and/or the second plasmid are phagemids.

In some embodiments of the ninth aspect, the first plasmid and the second plasmid comprise different origins of replication.

In some embodiments of the ninth aspect, the origin of replication is selected from one or more of the following: pBR ori, CDF ori, origin of replication of filamentous phage M13 (f1 ori), and p15A ori.

In some embodiments of the ninth aspect, the first recombination site and the second recombination site are sites for phage attachment in the respective genomes of the phage and its host bacterium.

In some embodiments of the ninth aspect, the first recombination site is attP and the second recombination site is attB.

In some embodiments of the ninth aspect, the first recombination site and the second recombination site are located at the polyclonal restriction endonuclease sites of the first plasmid and the second plasmid, respectively.

In some embodiments of the ninth aspect, the antibody light chain constant region CL is a human kappa light chain constant region or a human lambda light chain constant region.

In some embodiments of the ninth aspect, the CH1 segment of the antibody heavy chain constant region is selected from the group consisting of IgG1, IgG2, IgG3 or IgG4 subtypes.

In some embodiments of the ninth aspect, the first plasmid and/or the second plasmid comprise a resistance gene coding region.

In some embodiments of the ninth aspect, the 3' end of the nucleic acid molecule encoding the CH1 segment of the constant region of the antibody heavy chain is fused to the 5' end of the nucleic acid molecule encoding the gIII protein of filamentous phage M13.

In some embodiments of the ninth aspect, the resistance gene is an antibiotic resistance gene.

In some embodiments of the ninth aspect, the resistance gene is selected from the group consisting of: chloramphenicol resistance gene (CmR), ampicillin resistance gene (Ampr), kanamycin resistance gene (KaR), and tetracycline resistance gene. sex gene (TetR).

In some embodiments of the ninth aspect, the phage integrase is a tyrosine integrase.

In some embodiments of the ninth aspect, the phage integrase is lambda phage integrase.

In some embodiments of the ninth aspect, the phage integrase is inducibly expressed or constitutively expressed.

In some embodiments of the ninth aspect, the phage integrase is inducibly expressed.

In some embodiments of the ninth aspect, the recombinant cells are prokaryotic cells.

In some embodiments of the ninth aspect, the recombinant cell is E. coli.

In some embodiments of the ninth aspect, the recombinant cells are genetically engineered bacteria.

In a tenth aspect, the present application provides a method for preparing an antibody Fab fragment library, which includes transducing a first plasmid and a second plasmid into cells expressing phage integrase; wherein

The first recombination site is different from the second recombination site.

In some embodiments of the tenth aspect, a plurality of the first plasmids are transduced into the cell expressing phage integrase, wherein a plurality of the first plasmids carry different VH-CH1 of the antibody heavy chain expression units and have the same first recombination site.

In some embodiments of the tenth aspect, a plurality of said second plasmids are transduced into said cell expressing phage integrase, wherein a plurality of said second plasmids carry different said antibody light chain VL- CL expression unit and have the same second recombination site.

In some embodiments of the tenth aspect, transducing the first plasmid and/or the second plasmid into the cell expressing the phage integrase includes the following steps:

(1) Transfect the first plasmid and/or the second plasmid into competent cells;

(2) Use the helper phage to infect the competent cells obtained in step (1) to construct a phage library; and

(3) Transduce the phage library obtained in step (2) into the cells expressing phage integrase.

In some embodiments of the tenth aspect, the competent cells are prokaryotic cells.

In some embodiments of the tenth aspect, the competent cell does not express phage integrase.

In some embodiments of the tenth aspect, the helper phage is an M13 helper phage.

In some embodiments of the tenth aspect, the first plasmid and/or the second plasmid are phagemids.

In some embodiments of the tenth aspect, the first plasmid and the second plasmid comprise different origins of replication.

In some embodiments of the tenth aspect, the origin of replication is selected from one or more of the following: pBR ori, CDF ori, origin of replication of filamentous phage M13 (f1 ori), and p15A ori.

In some embodiments of the tenth aspect, the first recombination site and the second recombination site are sites for phage attachment in the respective genomes of the phage and its host bacterium.

In some embodiments of the tenth aspect, the first recombination site is attP and the second recombination site is attB.

In some embodiments of the tenth aspect, the first recombination site and the second recombination site are located at the polyclonal restriction enzyme sites of the first plasmid and the second plasmid, respectively.

In some embodiments of the tenth aspect, the antibody light chain constant region CL is a human kappa light chain constant region or a human lambda light chain constant region.

In some embodiments of the tenth aspect, the CH1 segment of the antibody heavy chain constant region is selected from the group consisting of IgG1, IgG2, IgG3 or IgG4 subtypes.

In some embodiments of the tenth aspect, the first plasmid and/or the second plasmid comprise a resistance gene coding region.

In some embodiments of the tenth aspect, the resistance gene is selected from the group consisting of: chloramphenicol resistance gene (CmR), ampicillin resistance gene (Ampr), kanamycin resistance gene (KaR), and tetracycline resistance gene. sex gene (TetR).

In some embodiments of the tenth aspect, the phage integrase is a tyrosine integrase.

In some embodiments of the tenth aspect, the phage integrase is a lambda phage integrase Synthase.

In some embodiments of the tenth aspect, the phage integrase is inducibly expressed or constitutively expressed.

In some embodiments of the tenth aspect, the phage integrase is inducibly expressed.

In some embodiments of the tenth aspect, the cell expressing the phage integrase is a prokaryotic cell.

In some embodiments of the tenth aspect, the cell expressing the phage integrase is E. coli.

In some embodiments of the tenth aspect, the cells expressing phage integrase are genetically engineered bacteria.

In the eleventh aspect, the present application provides the use of the plasmid combination described in the seventh aspect, the recombinant system described in the eighth aspect, or the recombinant cell described in the ninth aspect in preparing an antibody Fab fragment library.

In a twelfth aspect, the present application provides an antibody Fab fragment library prepared by the method described in the tenth aspect.

In a thirteenth aspect, the present application provides the use of the antibody Fab fragment library described in the twelfth aspect in at least one of the following: antibody preparation, prevention, diagnosis and/or treatment of diseases, and vaccine preparation.

Brief description of the drawings

Figure 1 shows a schematic structural diagram of the phagemid vector pHGDisn-attP-new.

Figure 2 shows a schematic structural diagram of the prokaryotic expression vector pHKb-attB-new.

Figure 3 shows ELISA analysis of the binding activity of recombinant anti-RSV F protein monoclonal antibodies (R3B1h1, R22B1 and positive control antibodies) to RSV-DS-Cav1-A (Panel A) and RSV-DS-Cav1-B (Panel B) result.

Figure 4 shows the ELISA analysis of the blocking results of anti-RSV F protein monoclonal antibodies (R3B1h1, R22B1 and Clesrovimab) against RSV F protein phage binding to RSV-DS-Cav1-B; where A-C are R3B1h1, R22B1 and Clesrovimab against RSV respectively. Blocking results of F protein purified phage binding to RSV-DS-Cav1-B.

Figure 5 shows the Rapid Fluorescent Focus Inhibition Test (Rapid Fluorescent Focus Inhibition Test, RFFIT) detects the results of the anti-RSV F protein monoclonal antibody inhibiting the infection of Hep-2 cells by the RSV/A2 strain.

Figure 6 shows the results of RFFIT detection of anti-RSV F protein monoclonal antibodies inhibiting the infection of Hep-2 cells by the RSV/18537 strain.

Figure 7 shows the results of anti-RSV F protein monoclonal antibodies inhibiting RSV infection of Hep-2 cells isolated from clinical samples.

Sequence description

SEQ ID NO: 1 shows the nucleotide sequence of the promoter encoding the chloramphenicol resistance gene (Cat promoter) derived from Escherichia coli.

SEQ ID NO: 2 shows the nucleotide sequence encoding the attP attachment site derived from Escherichia phage Lambda.

SEQ ID NO: 3 shows the nucleotide sequence encoding CDF ori derived from E. coli.

SEQ ID NO:4 shows the nucleotide sequence encoding the attB attachment site derived from E. coli.

SEQ ID NO: 5 shows the nucleotide sequence encoding the chloramphenicol resistance gene derived from E. coli.

SEQ ID NO: 6 shows the nucleotide sequence of the complete ampicillin resistance gene expression element derived from E. coli.

SEQ ID NO: 7 shows the nucleotide sequence derived from the lambda phage integrase expression element induced by the lambda phage lactose operon of Escherichia.

SEQ ID NO:8 shows the nucleotide sequence encoding the attL recombination site.

SEQ ID NO:9 shows the nucleotide sequence encoding the attR recombination site.

SEQ ID NO:10 shows the amino acid sequence of the F protein of RSV subtype A strain A2.

SEQ ID NO: 11 shows the amino acid sequence of the F protein of RSV subtype B strain 18537.

SEQ ID NO:12 shows the amino acid sequence of the pre-fusion F protein mutant RSV-DS-Cav1-A of the DS-Cav1 structure.

SEQ ID NO: 13 shows the amino acid sequence of the pre-fusion F protein mutant RSV-DS-Cav1-B of the DS-Cav1 structure.

SEQ ID NO:14 shows the amino acid sequence of the His tag.

SEQ ID NO: 15 shows the amino acid sequence of the heavy chain constant region of human (homo sapiens) IgG1 subtype.

SEQ ID NO: 16 shows the amino acid sequence of the mutant IgG1-YTE of the heavy chain constant region of human (homo sapiens) IgG1 subtype.

SEQ ID NO: 17 shows the amino acid sequence of the heavy chain constant region of mouse (mus musculus) IgG2a subtype.

SEQ ID NO: 18 shows the amino acid sequence of the constant region of the human (homo sapiens) kappa subtype light chain.

SEQ ID NO: 19 shows the amino acid sequence of the constant region of the lambda isoform light chain of humans (homo sapiens).

SEQ ID NO: 20 shows the amino acid sequence of the constant region of the mouse (mus musculus) kappa isoform light chain.

SEQ ID NO:21 shows the amino acid sequence of the mouse (mus musculus) lambda isoform light chain constant region.

SEQ ID NO: 22 shows the amino acid sequence of the heavy chain variable region of the humanized anti-RSV monoclonal antibody palivizumab (Hu1129).

SEQ ID NO: 23 shows the amino acid sequence of the light chain variable region of the humanized anti-RSV monoclonal antibody palivizumab (Hu1129).

SEQ ID NO:24 shows the amino acid sequence of the heavy chain variable region of the fully human anti-RSV monoclonal antibody Nirsevimab (MEDI8897).

SEQ ID NO:25 shows the amino acid sequence of the light chain variable region of the fully human anti-RSV monoclonal antibody Nirsevimab (MEDI8897).

SEQ ID NO:26 shows the amino acid sequence of the heavy chain variable region of the fully human anti-RSV monoclonal antibody Clesrovimab (RB1).

SEQ ID NO:27 shows the amino acid sequence of the light chain variable region of the fully human anti-RSV monoclonal antibody Clesrovimab (RB1).

SEQ ID NO: 28 shows the amino acid sequence of the heavy chain variable region of the Fab antibody R3B1h1 against RSV F protein.

SEQ ID NO:29 shows that the light chain of the Fab antibody R3B1h1 against RSV F protein can Amino acid sequence of the variable region.

SEQ ID NO: 30 shows the amino acid sequence of the heavy chain variable region of the Fab antibody R22B1 against RSV F protein.

SEQ ID NO: 31 shows the amino acid sequence of the light chain variable region of the Fab antibody R22B1 against RSV F protein.

SEQ ID NO:32-34 respectively show the amino acid sequences of the heavy chain variable regions HCDR1, HCDR2 and HCDR3 of the Fab antibody R3B1h1 against RSV F protein.

SEQ ID NO:35-37 respectively show the amino acid sequences of the light chain variable regions LCDR1, LCDR2 and LCDR3 of the Fab antibody R3B1h1 against RSV F protein.

SEQ ID NO:38-40 respectively show the amino acid sequences of the heavy chain variable regions HCDR1, HCDR2 and HCDR3 of the Fab antibody R22B1 against RSV F protein.

SEQ ID NO:41-43 respectively show the amino acid sequences of the light chain variable regions LCDR1, LCDR2 and LCDR3 of the Fab antibody R22B1 against RSV F protein.

SEQ ID NO:44 shows the nucleotide sequence encoding pBR ori derived from E. coli.

SEQ ID NO:45 shows the nucleotide sequence encoding f1 ori derived from f1 phage.

SEQ ID NO: 46 shows the nucleotide sequence encoding the gIII protein of filamentous phage M13 derived from phage M13.

Detailed description of the invention

Using the specific recombination system or recombinant cells of lambda phage integrase (Int) and genetic engineering means, specific recombination, integration and excision of the target nucleotide sequence can be achieved in vivo and in vitro. It has been reported in the literature that the use of heat-induced genetically engineered bacteria expressing Int integrase can achieve specific recombination of antibody light chain expression plasmids and heavy chain expression plasmids in cells to produce complete plasmids expressing functional Fabs ¹⁰ . Thermo Fisher's Gateway homologous recombination technology uses the specific recombination principle of lambda phage integrase, eliminating the need for classic gene cloning steps such as enzyme digestion and ligation. The in vitro recombination efficiency is as high as 95%. Its Gateway kit is used to construct cDNA libraries. The diversity of constructed libraries can reach 1.0E+7.

This application uses the lambda phage integrase recombinant system or recombinant cells to overcome the shortcomings of insufficient DNA conversion efficiency during the construction of the antibody library through efficient phage infection. exploit package The phage equipped with the antibody heavy chain expression unit (such as the heavy chain VH-CH1 expression unit) infects E. coli that can induce the expression of Int integrase and contains the antibody light chain expression unit plasmid, and the antibody heavy chain expression is achieved in vivo through Int integrase. Specific recombination of units (such as heavy chain VH-CH1 expression unit) and light chain expression units, and antibiotic screening, yields a large-capacity Fab antibody library (more than 1.0E+12).

The inventor of the present application used the Fab antibody library constructed above to screen and obtain new Fab antibodies against RSV. In various aspects of the present application, novel antibodies against RSV are provided, nucleic acid molecules encoding the antibodies, vectors comprising the nucleic acid molecules, host cells comprising the nucleic acid molecules or vectors, and cells comprising the antibodies. Compositions, methods of preparing and purifying said antibodies, and medical and biological applications of said antibodies. According to the amino acid sequence of the variable region of the antibody provided in this application, a full-length antibody molecule can be constructed as a drug for preventing or treating RSV-related diseases.

Unless otherwise indicated, the practice of this application employs molecular biology, microbiology, cell biology, biochemistry, and immunology techniques routine in the art.

Unless otherwise specified, the terms used in this application have the meanings commonly understood by those skilled in the art.

definition

The term "recombination site" as used herein refers to a site in a phage genome and a bacterial genome for phage integration. In some embodiments of the present application, the first recombination site and the second recombination site may be sites for phage attachment in the respective genomes of the phage and its host bacteria, including the following situations: the first recombination site The point is a site for phage attachment in the phage genome and the second recombination site is a site for phage attachment in the genome of the host bacterium of the phage; or the second recombination site is a site for phage attachment in the phage genome. The site and the first recombination site are sites used for phage attachment in the genome of the host bacterium of the phage.

As used herein, the term "plasmid" refers to DNA molecules other than chromosomes (or nucleoids) in organisms such as bacteria, yeasts, and actinomycetes that exist in the cytoplasm (except yeast, whose 2 μm plasmids exist in the nucleus), It has the ability to replicate autonomously, allowing it to maintain a constant copy number in daughter cells and express the genetic information it carries. It is a closed circular double-stranded DNA molecule. The term "plasmid" as used herein encompasses bacterial plasmids, phagemids, and the like. "Bacterial plasmid" is a commonly used vector in DNA recombinant technology. It is a tool that can deliver a useful foreign gene into recipient cells for proliferation and expression through genetic engineering means. "Phagemid" refers to a phagemid that is a combination of a plasmid vector and a single-stranded phage vector. It contains single-stranded phage packaging sequences, replicons, plasmid replicons, cloning sites, marker genes, etc. In host cells such as E. coli, it can be replicated as a normal double-stranded plasmid DNA molecule. When a helper phage is present, it replicates according to the rolling circle model to produce single-stranded DNA, which is packaged into phage particles.

As used herein, the term "attP attachment site" refers to a specific sequence on the phage DNA that contains a 15 bp core region that is identical to the host chromosomal sequence.

As used herein, the term "attB attachment site" refers to an attachment site in the bacterial genome that contains a 15 bp core region that is identical to the phage genome sequence.

As used herein, the term "phage integrase" refers to a protein with type I topoisomerase activity that can specifically bind attP and attB attachment sites and is indispensable for integrating phage into the host genome through site-specific recombination. .

The term "origin of replication" as used herein refers to the initiation site of plasmid replication, consisting of the initial replicon (ORI) and its regulatory elements.

The term "antibody" as used herein refers to an immunoglobulin molecule capable of specifically binding to a target via at least one antigen recognition site located in the variable region of the immunoglobulin molecule. Targets include but are not limited to carbohydrates, polynucleotides, lipids, peptides, etc. As used herein, "antibody" includes not only intact (i.e., full-length) antibodies, but also antigen-binding fragments thereof (eg, Fab, Fab', F(ab') ₂ , Fv), variants thereof, including antibody portions Fusion proteins, humanized antibodies, chimeric antibodies, diabodies, linear antibodies, single chain antibodies, multispecific antibodies (such as bispecific antibodies) and any other immunoglobulins containing antigen recognition sites with required specificity Modified configurations of protein molecules, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies.

Typically, intact or full-length antibodies contain two heavy chains and two light chains. Each heavy chain contains a heavy chain variable region (VH) and first, second and third constant regions (CH1, CH2 and CH3). Each light chain contains a light chain variable region (VL) and a constant region (CL). A full-length antibody can be any type of antibody, such as IgD, IgE, IgG, IgA or IgM (or subclasses of the above), but anti- The body does not need to belong to any particular category. Depending on the antibody amino acid sequence of the heavy chain constant domain, immunoglobulins can be assigned to different classes. Generally, there are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy chain constant domains corresponding to the different immunoglobulin classes are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional structures of the different classes of immunoglobulins are well known.

The term "antigen-binding fragment or antigen-binding portion" as used herein refers to a portion or region of an intact antibody molecule responsible for binding to an antigen. The antigen-binding domain may comprise a heavy chain variable region (VH), a light chain variable region (VL), or both. Each of VH and VL typically contains three complementarity determining regions CDR1, CDR2 and CDR3.

It is well known to those skilled in the art that the complementarity determining region (CDR, usually CDR1, CDR2 and CDR3) is the region in the variable region that has the greatest impact on the affinity and specificity of the antibody. There are two common ways to define the CDR amino acid sequence of VH or VL, namely Chothia definition and Kabat ^{definition11-13} . For the variable region amino acid sequence of a given antibody, the CDR amino acid sequences in the VH and VL amino acid sequences can be determined according to the Chothia definition or the Kabat definition. In embodiments of the present application, Kabat is used to define CDR amino acid sequences.

For the variable region amino acid sequence of a given antibody, the CDR amino acid sequence of the variable region amino acid sequence can be analyzed in a variety of ways, for example, it can be determined using the online software Abysis (http://www.abysis.org/).

Examples of antigen-binding fragments include, but are not limited to: (1) Fab fragments, which may be monovalent fragments having a VL-CL chain and a VH-CH1 chain; (2) F(ab') ₂ fragments, which may be a monovalent fragment having two A bivalent fragment of Fab' fragment, the two Fab' fragments are connected by a disulfide bridge in the hinge region (i.e., a dimer of Fab'); (3) Fv fragment with the VL and VH domains of the single arm of the antibody; ( 4) Single chain Fv (scFv), which may be a single polypeptide chain consisting of a VH domain and a VL domain via a peptide linker; and (5) (scFv) ₂ , which may comprise two peptide linkers. A peptide linker connects a VH domain and two VL domains that are combined with the two VH domains via a disulfide bridge.

In some specific embodiments of the present application, the antibody is a Fab fragment, that is, a monovalent fragment having a VL-CL chain and a VH-CH1 chain.

As used herein, the term "Fd fragment" refers to a fragment composed of the heavy chain variable region VH of an antibody and the heavy chain variable region VH. A fragment consisting of the constant region CH1.

The term "specific binding" as used herein refers to a non-random binding reaction between two molecules, such as the binding of an antibody to an antigenic epitope.

As used herein, the term "neutralizing antibody" refers to an antibody that can bind to the antigen on the surface of a pathogenic microorganism, thereby preventing the pathogenic microorganism from adhering to the target cell receptor, or preventing the fusion of the viral envelope and the cell membrane to prevent invasion of the cell.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, that is, the individual antibodies making up the population are identical except for naturally occurring mutations that may be present in a small number of individuals.

The amino acid sequence of the HCDR1 is shown in SEQ ID NO:32, the amino acid sequence of the HCDR2 is shown in SEQ ID NO:33, the amino acid sequence of the HCDR3 is shown in SEQ ID NO:34, and the amino acid sequence of the LCDR1 The sequence is as shown in SEQ ID NO:35, the amino acid sequence of the LCDR2 is as shown in SEQ ID NO:36 and the amino acid sequence of the LCDR3 is as shown in SEQ ID NO:37; or

In some embodiments of the first aspect, the amino acid sequence of the antibody heavy chain variable region is shown in SEQ ID NO: 28 or 30.

In some embodiments of the first aspect, the amino acid sequence of the antibody heavy chain variable region is shown in SEQ ID NO: 28, and the amino acid sequence of the antibody light chain variable region is shown in SEQ ID NO: 29; or

In some embodiments of the second aspect, the antibody heavy chain variable region has an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96% identical to SEQ ID NO: 28 or 30. %, 97%, 98%, 99% or higher identity.

In some embodiments of the second aspect, the antibody light chain variable region has an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96% identical to SEQ ID NO: 29 or 31 %, 97%, 98%, 99% or higher identity.

In some embodiments of the second aspect, the amino acid sequence of the heavy chain variable region of the antibody differs from the amino acid sequence shown in any one of SEQ ID NO: 28 and 30 by about 1, 2, 3, 4, 5 , substitution, deletion and/or addition of 6, 7, 8, 9 or 10 amino acids.

In some embodiments of the second aspect, the amino acid sequence of the light chain variable region of the antibody differs from the amino acid sequence shown in any one of SEQ ID NO: 29 and 31 by about 1, 2, 3, 4, 5 , substitution, deletion and/or addition of 6, 7, 8, 9 or 10 amino acids.

In some embodiments of the second aspect, the C-terminal or N-terminal region of the amino acid sequence shown in any one of SEQ ID NO: 28 and 30 can also be truncated by about 1, 2, 3, 4, 5, 6 , 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids while still retaining similar functionality to the heavy chain variable region of the antibody.

In some embodiments of the second aspect, 1, 2, 3, 4, 5, 6, 7 can also be added to the C-terminal or N-terminal region of the amino acid sequence shown in any one of SEQ ID NO: 28 and 30 , 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids, and the resulting amino acid sequence still maintains a similar function of the heavy chain variable region of the antibody.

In some embodiments of the second aspect, 1, 2, 3, 4, 5 can also be added or deleted in the region other than the C-terminal or N-terminal of the amino acid sequence shown in any one of SEQ ID NO: 28 and 30 , 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids, as long as the changed amino acid sequence substantially maintains a similar weight of the antibody Function of the chain variable region.

In some embodiments of the second aspect, the C-terminal or N-terminal region of the amino acid sequence shown in any one of SEQ ID NO: 29 and 31 can also be truncated by about 1, 2, 3, 4, 5, 6 , 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids while still retaining similar functionality to the light chain variable region of the antibody.

In some embodiments of the second aspect, 1, 2, 3, 4, 5, 6, 7 can also be added to the C-terminal or N-terminal region of the amino acid sequence shown in any one of SEQ ID NO: 29 and 31 , 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids, and the resulting amino acid sequence still maintains a similar function of the light chain variable region of the antibody.

In some embodiments of the second aspect, 1, 2, 3, 4, 5 can also be added or deleted in the region other than the C-terminal or N-terminal of the amino acid sequence shown in any one of SEQ ID NO: 29 and 31 , 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids, as long as the changed amino acid sequence remains substantially similar to the light chain variable region of the antibody function.

In some specific embodiments of any one of the first and second aspects, the F protein of human respiratory syncytial virus (RSV) is a recombinant human RSV F protein, such as the recombinant human shown in SEQ ID NO: 12 or 13 RSV F protein.

In some specific embodiments of either of the first and second aspects, the antibody is a Fab fragment.

In some embodiments of either of the first and second aspects, the antibody comprises a heavy chain constant region selected from the group consisting of IgGl subtype, IgG2 subtype, or IgG4 subtype.

In some embodiments of any of the first and second aspects, the heavy chain The constant region includes the Fc segment sequence of the heavy chain constant region of the IgG1 subtype, and the amino acid sequences at positions 252, 254, and 256 of the Fc segment sequence are Y, T, and E respectively, wherein the amino acid sequence of the antibody constant region is in accordance with EU numbering to make sure.

In some embodiments of the third aspect, the nucleic acid molecule is operably linked to a regulatory amino acid sequence that is recognized by a host cell transformed with the vector.

In some embodiments of the fourth aspect, the pharmaceutical composition is used to prevent or treat RSV-related diseases.

In some embodiments of the fourth aspect, the RSV-associated disease is respiratory tract infection, such as bronchiolitis and pneumonia.

In some embodiments of the fourth aspect, the pharmaceutical composition may further comprise one or more of the following: lubricants, such as talc, magnesium stearate and mineral oil; wetting agents; emulsifiers; Suspending agent; preservatives, such as benzoic acid, sorbic acid and calcium propionate; sweeteners and/or flavoring agents, etc.

In some embodiments of the fourth aspect, the pharmaceutical composition in the present application can be formulated in the form of tablets, pills, powders, lozenges, elixirs, suspensions, emulsions, solutions, syrups, suppositories or capsules.

In some embodiments of the fourth aspect, the pharmaceutical composition of the present application can be delivered using any physiologically acceptable administration method, including but not limited to: oral administration, parenteral administration, nasal administration. medicine, rectal administration, intraperitoneal administration, intravascular injection, subcutaneous administration, transdermal administration, inhalation administration, etc.

In some embodiments of the fourth aspect, the medicament for therapeutic use may be formulated in the form of a lyophilized formulation or aqueous solution by mixing reagents of desired purity with optional pharmaceutically acceptable carriers, excipients, etc. Composition for storage.

In some embodiments of the fifth aspect, the RSV-associated disease is respiratory tract infection, such as bronchiolitis and pneumonia.

In some embodiments of the sixth aspect, the RSV-related disease is respiratory tract infection, such as bronchiolitis and pneumonia.

In a seventh aspect, the present application provides a plasmid combination for expressing antibody Fab fragments, which includes a first plasmid and a second plasmid, wherein

The first recombination site is different from the second recombination site.

In some embodiments of the eighth aspect, the recombinant system comprises a plurality of said first plasmids, and a plurality of said first plasmids carry different expressions of said antibody heavy chain VH-CH1 units and have the same first recombination site.

In some embodiments of the eighth aspect, the first plasmid, the second plasmid and the cell expressing the phage integrase each exist independently, or at least one of the first plasmid and the second plasmid has been introduced in cells expressing phage integrase.

In some embodiments of the eighth aspect, the second plasmid has been introduced into the cell expressing the phage integrase.

The first recombination site is different from the second recombination site.

In some embodiments of the ninth aspect, the recombinant cell is E. coli.

In some embodiments of the ninth aspect, the recombinant cell is a male E. coli strain with F factor, such as the TG1 strain.

The first recombination site is different from the second recombination site.

In some embodiments of the tenth aspect, a plurality of said first plasmids are transduced into said phage integrase-expressing cell, wherein a plurality of said first plasmids carry different said antibody heavy chain VH-CH1 expression units and have the same first recombination site.

In some embodiments of the tenth aspect, a plurality of said second plasmids are transduced into said cell expressing phage integrase, wherein a plurality of said second plasmids carry different expressions of said antibody light chain VL-CL unit and have the same second recombination site.

(1) Transfect the first plasmid and/or the second plasmid into competent cells;

In some embodiments of the tenth aspect, the competent cell is E. coli.

In some embodiments of the tenth aspect, the competent cells are genetically engineered bacteria.

In some embodiments of the tenth aspect, the competent cell is a male E. coli strain with F factor, such as the TG1 strain.

In some embodiments of any of the seventh to tenth aspects, the first plasmid is a phagemid.

In some embodiments of any of the seventh to tenth aspects, the second plasmid is a phagemid.

In some embodiments of any of the seventh to tenth aspects, the first plasmid and the second plasmid comprise different origins of replication.

In some embodiments of any one of the seventh to tenth aspects, the origin of replication is selected from one or more of the following: pBR ori, CDF ori, origin of replication of filamentous phage M13 (f1 ori) and p15A ori.

In some specific embodiments of any one of the seventh to tenth aspects, the first plasmid comprises pBR ori and f1 ori.

In some specific embodiments of any of the seventh to tenth aspects, the second plasmid comprises CDF ori.

In some embodiments of any one of the seventh to tenth aspects, the first recombination site and the second recombination site are respectively used for phage attachment in the respective genomes of the phage and its host bacterium. site.

In some embodiments of any of the seventh to tenth aspects, the first recombination site is attP.

In some embodiments of any of the seventh to tenth aspects, the second recombination site is attB.

In some specific embodiments of any one of the seventh to tenth aspects, the first recombination site comprises the nucleotide sequence shown in SEQ ID NO: 2 or the nucleic acid sequence shown in SEQ ID NO: 2 The nucleotide sequence is a nucleotide sequence obtained by one or more substitutions, deletions or additions that do not essentially change the function of the first recombination site.

In some specific embodiments of any one of the seventh to tenth aspects, the second recombination site comprises the nucleotide sequence shown in SEQ ID NO: 4 or the nucleic acid sequence shown in SEQ ID NO: 4 The nucleotide sequence is a nucleotide sequence obtained by one or more substitutions, deletions or additions that do not essentially change the function of the second recombination site.

In some embodiments of any one of the seventh to tenth aspects, the one or more substitutions or deletions do not essentially change the function of the first recombination site or the second recombination site. Or the number added is 1-30, preferably 1-20, more preferably 1-10, wherein the obtained nucleotide sequence substantially maintains the unchanged first recombination site or the second Function of the recombination site.

In some embodiments of any of the seventh to tenth aspects, the first plasmid and second plasmid are different.

In some specific embodiments of any one of the seventh to tenth aspects, the The first plasmid is a phagemid, and the second plasmid is a prokaryotic expression vector (eg, a bacterial plasmid).

In some specific embodiments of any one of the seventh to tenth aspects, the first plasmid is pHGDisn-attP-new.

In some specific embodiments of any one of the seventh to tenth aspects, the second plasmid is pHKb-attB-new.

In some embodiments of any one of the seventh to tenth aspects, the first recombination site is located at a polyclonal restriction enzyme cleavage site of the first plasmid.

In some embodiments of any one of the seventh to tenth aspects, the second recombination site is located at the polyclonal restriction site of the second plasmid.

In some specific embodiments of any one of the seventh to tenth aspects, the first recombination site is located between the XbaI and KpnI restriction sites of the pHGDisn-attP-new plasmid.

In some specific embodiments of any one of the seventh to tenth aspects, the second recombination site is located between the HindIII and BamHI restriction sites of the pHKb-attB-new plasmid.

In some embodiments of any one of the seventh to tenth aspects, the antibody light chain constant region CL is a human kappa light chain constant region or a human lambda light chain constant region.

In some embodiments of any one of the seventh to tenth aspects, the CH1 segment of the antibody heavy chain constant region is selected from the group consisting of IgG1, IgG2, IgG3 or IgG4 subtypes.

In some embodiments of any one of the seventh to tenth aspects, the first plasmid and/or the second plasmid comprises a resistance gene coding region.

In some embodiments of any one of the seventh to tenth aspects, the 3' end of the nucleic acid molecule encoding the CH1 segment of the constant region of the antibody heavy chain is fused to the nucleic acid molecule encoding the gIII protein of filamentous phage M13. 5' end.

In some embodiments of any one of the seventh to tenth aspects, the presence of the resistance gene facilitates the screening of recombinant systems or recombinant cells.

In some embodiments of any one of the seventh to tenth aspects, the resistance gene is an antibiotic resistance gene.

In some embodiments of any one of the seventh to tenth aspects, the resistance The gene is selected from: chloramphenicol resistance gene (CmR), ampicillin resistance gene (Ampr), kanamycin resistance gene (KaR) and tetracycline resistance gene (TetR).

In some embodiments of any of the eighth to tenth aspects, the phage integrase is a tyrosine integrase.

In some embodiments of any of the eighth to tenth aspects, the tyrosine integrase is a lambda phage integrase.

In some embodiments of any one of the eighth to tenth aspects, the phage integrase is inducibly expressed or constitutively expressed.

In some embodiments of any one of the eighth to tenth aspects, the phage integrase is inducibly expressed.

In some embodiments of any one of the eighth to tenth aspects, the inducible expression is by using an inducible promoter, such as a lactose promoter (Plac) or an arabinose promoter (Para).

In some embodiments of the eighth or tenth aspect, the cell expressing the phage integrase is a prokaryotic cell.

In some embodiments of the eighth or tenth aspect, the cell expressing the phage integrase is Escherichia coli.

In some embodiments of the eighth or tenth aspect, the cells expressing phage integrase are genetically engineered bacteria.

In some embodiments of the eighth or tenth aspect, the cell expressing the phage integrase is a male E. coli strain with F factor.

In some specific embodiments of the eighth or tenth aspect, the cell expressing the phage integrase is a TG1 strain.

In some embodiments of any one of the seventh to tenth aspects, the first plasmid comprises the antibody heavy chain VH-CH1 expression unit and attP attachment site; and the second plasmid comprises the Antibody light chain VL-CL expression unit and attB attachment site.

In some embodiments of the seventh aspect, the first recombination site and the second recombination site correspond to each other. For example, when the nucleotide sequence of the first recombination site is SEQ ID NO:2, then the nucleotide sequence of the second recombination site is SEQ ID NO:4.

In some embodiments of the eighth or tenth aspect, the first recombination site, The second recombination site, the type of the phage integrase, and the cell type expressing the phage integrase correspond to each other.

In some embodiments of the eighth or tenth aspect, after the first recombination site is determined, the second recombination site, the type of the phage integrase and the type of the phage integrase applicable to the application can be determined. Describe the cell types that express phage integrase.

In some embodiments of the eighth or tenth aspect, after the second recombination site is determined, the first recombination site, the type of the phage integrase and the type of the phage integrase applicable to the application can be determined. Describe the cell types that express phage integrase.

In some embodiments of the eighth or tenth aspect, after the phage integrase type is determined, the cell type expressing the phage integrase suitable for the present application can be determined.

In some embodiments of the eighth or tenth aspect, when the nucleotide sequence of the first recombination site is SEQ ID NO: 2, then the nucleotide sequence of the second recombination site is SEQ ID NO. NO: 4. The phage integrase is lambda phage integrase, and the cell expressing the phage integrase is TG1 Escherichia coli.

In some embodiments of the ninth aspect, the first recombination site, the second recombination site, the type of the phage integrase, and the type of the recombinant cell correspond to each other.

In some embodiments of the ninth aspect, after the first recombination site is determined, the second recombination site, the type of the phage integrase and the recombinant cell that are suitable for the present application can be determined. type.

In some embodiments of the ninth aspect, after the second recombination site is determined, the first recombination site, the type of the phage integrase and the recombinant cell that are suitable for this application can be determined. type.

In some embodiments of the ninth aspect, after the type of the phage integrase is determined, the type of the recombinant cell suitable for the present application can be determined.

In some embodiments of the ninth aspect, when the nucleotide sequence of the first recombination site is SEQ ID NO:2, then the nucleotide sequence of the second recombination site is SEQ ID NO:4, The phage integrase is lambda phage integrase, and the recombinant cell is TG1 Escherichia coli.

In some specific embodiments described in the tenth aspect, the preparation method of the antibody Fab fragment library may include the following steps:

(1) Transfect the first plasmid (for example, multiple first plasmids carrying different antibody heavy chain VH-CH1 expression units and the same attP attachment site, such as multiple phagemids) into competent cells (for example, TG1 competent cells) );

(2) Use helper phage (such as M13 helper phage) to infect the competent cells obtained in step (1) to construct a phage library (such as multiple cells carrying different antibody heavy chain VH-CH1 expression units and the same attP attachment site). phage library of species);

(3) Transfect the second plasmid (for example, multiple second plasmids carrying different antibody light chain VL-CL expression units and the same attB attachment site) into cells expressing phage integrase (for example, TG1 expressing lambda phage integrase competent cells);

(4) The cells obtained in the induction step (3) express phage integrase (such as lambda phage integrase);

(5) using the phage library (for example, a phage library including a variety of phages carrying different antibody heavy chain VH-CH1 expression units and the same attP attachment site) to infect the cells expressing the phage integrase of step (4); and

(6) Use helper phage (such as M13 helper phage) to infect the cells obtained in step (5) to construct the antibody Fab fragment library.

In some embodiments of the thirteenth aspect, the library of antibody Fab fragments can be used for antibody screening.

In some embodiments of the thirteenth aspect, the library of antibody Fab fragments can be used for antibody preparation.

In some embodiments of the thirteenth aspect, the library of antibody Fab fragments can be used for prevention of disease.

In some embodiments of the thirteenth aspect, the library of antibody Fab fragments can be used for diagnosis of disease.

In some embodiments of the thirteenth aspect, the library of antibody Fab fragments can be used for the treatment of disease.

In some embodiments of the thirteenth aspect, the library of antibody Fab fragments can be used for vaccine screening.

In some embodiments of the thirteenth aspect, the library of antibody Fab fragments can be used in vaccine preparation.

In other aspects, the application also provides vectors comprising nucleic acid molecules encoding the antibodies of the invention or their light or heavy chains, host cells comprising the vectors, and methods of producing the antibodies. In some embodiments, the nucleic acid molecule is operably linked to a regulatory sequence that is recognized by a host cell transformed with the vector. In some embodiments, methods of producing antibodies include culturing host cells. In some embodiments, the method of producing an antibody further includes recovering the antibody from the host cell culture medium.

In addition, the antibodies specific for RSV described herein can also be used to detect the presence of RSV in biological samples. Antibody-based detection methods are well known in the art and include, for example, ELISA, immunoblotting, radioimmunoassay, immunofluorescence, immunoprecipitation, and other related techniques.

It should be understood that the above detailed description is only to enable those skilled in the art to understand the contents of the present application more clearly, and is not intended to be limiting in any respect. Those skilled in the art will be able to make various modifications and changes to the described embodiments.

Example

The following examples are for illustrative purposes only and are not intended to limit the scope of the present application.

Example 1: Preparation of fully human recombinant Fab library

The content of this embodiment refers to Chinese Invention Patent No. CN 108251431B, which is incorporated herein by reference.

1.1 Construction of recombinant expression plasmid containing attB and attP attachment sites

Based on the pADG-S plasmid, XbaⅠ and KpnⅠ double enzyme digestion was used to synthesize the chloramphenicol resistance gene promoter (Cat promoter, whose coding sequence is shown in SEQ ID NO: 1) and attP attachment site The recombinant gene fragment of (attP1, whose coding sequence is shown in SEQ ID NO: 2) was cloned into the vector pADG-S, and a phagemid containing the attP attachment site for expressing the antibody heavy chain Fd (VH-CH1) domain was constructed. Vector pHGDisn-attP-new (Figure 1).

Based on the pADK-S plasmid, HindIII and BamHI double enzyme digestion was used to synthesize the CDF origin of replication (CDF ori, whose coding sequence is shown in SEQ ID NO: 3), attB attachment site (attB1, its coding sequence As shown in SEQ ID NO: 4), chloramphenicol resistance gene (CmR, its coding sequence is as shown in SEQ ID NO: 5) and complete ampicillin resistance gene expression element (AmpR, its coding sequence is as SEQ ID NO : The recombinant gene fragment shown in 6) was cloned into the vector pADK-S, and a prokaryotic expression vector pHKb-attB-new containing the attB attachment site for expressing the antibody light chain was constructed (Figure 2).

pHGDisn-attP-new and pHKb-attB-new contain different origins of replication and can replicate and coexist in the same E. coli cell. After lambda phage integrase (λInt) induces specific recombination of attP and attB attachment sites, the new plasmid formed contains a complete chloramphenicol promoter and coding gene, which can express chloramphenicol acetyltransferase and exhibit chloramphenicol expression. protein resistance, which facilitates the screening of recombinant plasmids.

1.2 Preparation of TG1 E. coli edited by λInt gene

The attB attachment site and attP attachment site undergo specific recombination in E. coli cells to generate attL site and attR site. This reaction must be catalyzed by λ phage integrase ¹⁴ . By gene editing means, the gene encoding the lactose operon-induced lambda phage integrase expression element (its nucleotide sequence is shown in SEQ ID NO: 7) was inserted into the TG1 E. coli genome. The TG1-λInt recombinant engineering strain was commissioned by Kings Rui Biotechnology Co., Ltd. completed.

1.3 Construction of fully human heavy chain antibody library

With reference to Chinese Invention Patent No. CN 108251431B, the cryopreserved natural human heavy chain variable region cDNA and the PCR products of introduced mutations VH1, VH3 and VH5 were used as templates. After designing PCR primers to amplify the target fragment, NcoⅠ and PmlⅠ were used. Enzyme digestion and cloning into the vector pHGDisn-attP-new, the ligation product was electrotransformed into TG1 competent cells, and a fully human heavy chain antibody library with 1.73E+9 diversity was constructed. The constructed antibody library was sent for sequencing analysis. The accuracy rate is over 85%.

1.4 Construction of fully human light chain antibody library

With reference to Chinese Invention Patent No. CN 108251431B, the cryopreserved natural human light chain variable region cDNA and the introduced mutated VK1 and VL3 PCR products were used as templates to design PCR primers to amplify the target fragment, and then use NcoⅠ and PmlⅠ double enzyme digestion for cloning. to the vector pHKb-attB-new, the ligated product was electroporated into TG1-λInt competent cells, and a fully human light chain antibody library with 1.95E+8 diversity was constructed. The constructed antibody library was sent for sequencing analysis, and the average accuracy rate exceeded 90%.

1.5 Preparation of fully human recombinant Fab library

The heavy chain library liquid constructed above was inoculated into 200 mL liquid culture medium, cultured to the logarithmic growth phase at 37°C and 220rpm, infected with M13 helper phage, and cultured overnight at 28°C and 220rmp to amplify the phage, and then used The purified heavy chain phage library was prepared by PEG/NaCl precipitation method, and the titer was measured and then frozen and stored in a -80°C refrigerator for later use.

Take the fully human light chain antibody library prepared above and inoculate it into 50 mL liquid culture medium. Add IPTG to a final concentration of 0.1mM. After culturing to the logarithmic growth phase at 37°C and 220 rpm, add the heavy chain phage library for infection. After the infection is completed, the bacterial solution is evenly spread on the bacterial culture dish and cultured overnight in a 32°C constant-temperature incubator.

Count the bacteria growing on the above-mentioned petri dishes and send them to be sequenced to identify the recombination site of the single clone as well as the light chain sequence and heavy chain sequence. The library capacity after recombination was counted by dilution method, and the total number of colonies after recombination was estimated to be 3.80E+12. The single-clone sequencing results showed that the clones growing on the plate were all recombinant plasmids, including the newly generated attL (attL1, whose coding sequence is such as SEQ ID NO: 8) and attR (attR1, whose coding sequence is shown in SEQ ID NO: 9) recombination sites as well as complete light chain genes and heavy chain Fd genes, and the antibody gene accuracy exceeds 76.5%.

Collect the colonies on the above-mentioned bacterial petri dishes, inoculate them into liquid culture medium, culture them at 37°C and 220 rpm to the logarithmic growth phase, and then infect them with M13 helper phage to prepare a purified human Fab phage library.

Example 2: Preparation of RSV F protein and preparation of recombinant antibodies

2.1 Preparation of RSV F protein

The process of preparing RSV F protein monoclonal antibodies requires the use of F recombinant proteins of different subtype viruses, including the F protein of RSV A subtype strain A2 (F-A2, whose amino acid sequence is shown in SEQ ID NO: 10) and the F protein of RSV subtype B strain 18537 (F-18537, whose amino acid sequence is shown in SEQ ID NO: 11). Based on two different subtypes of RSV F proteins, the inventors constructed pre-fusion F protein mutants of DS-Cav1 structure ¹⁵ , respectively named RSV-DS-Cav1-A (their amino acid sequences are shown in SEQ ID NO: 12 ) and RSV-DS-Cav1-B (whose amino acid sequence is shown in SEQ ID NO: 13). Since F protein has post-translational modifications (such as glycosylation and disulfide bonds), the use of mammalian cell expression system will be more conducive to maintaining the structure and function of the recombinant protein. In addition, a His tag (His, whose amino acid sequence is shown in SEQ ID NO: 14) was added to the C-terminus of this recombinant protein, which will be more conducive to the purification of the recombinant protein and the identification of the function of the monoclonal antibody.

Synthesize RSV-DS-Cav1-A and RSV-DS-Cav1-B genes, use conventional molecular biology techniques to clone the synthesized genes into appropriate eukaryotic expression vectors (such as Invitrogen's pcDNA3.1, etc.), and then use Liposomes (such as Invitrogen's 293fectin, etc.) or other cationic transfection reagents (such as PEI, etc.) are used to transfect the prepared recombinant protein expression plasmid into HEK293 cells (such as Invitrogen's HEK293F), and cultured under serum-free suspension culture conditions 3 to 4 days. The culture supernatant is then harvested by centrifugation. Use a metal chelate affinity chromatography column (such as GE's HisTrap FF, etc.) to perform one-step purification of the recombinant protein in the supernatant. Then use a desalting column (such as GE's Hitrap desaulting, etc.) to replace the recombinant protein storage buffer with PBS (pH7.0) or other suitable buffers. If necessary, the samples can be filtered and sterilized, and then stored in aliquots at -20°C.

2.2 Preparation of recombinant antibodies

Using conventional molecular biology methods, the nucleotide sequences encoding the heavy chain variable region and the light chain variable region of the antibody are cloned into eukaryotic expression fusion nucleotide sequences encoding the heavy chain constant region and the light chain constant region. Vectors (such as pcDNA3.1 from Invitrogen Company, etc.) are used to express whole antibodies in combination. The heavy chain constant region of the antibody can be human IgG1 subtype (whose amino acid sequence is shown in SEQ ID NO: 15), human IgG1 subtype mutant IgG1-YTE (whose amino acid sequence is shown in SEQ ID NO: 16), mouse IgG2a subtype (its amino acid sequence is as shown in SEQ ID NO: 17 shown), the light chain constant region can be human kappa isoform (whose amino acid sequence is shown in SEQ ID NO: 18), human lambda isoform (whose amino acid sequence is shown in SEQ ID NO: 19), mouse kappa isoform (whose amino acid sequence is shown in SEQ ID NO: 19) Its amino acid sequence is shown in SEQ ID NO: 20) or mouse lambda subtype (its amino acid sequence is shown in SEQ ID NO: 21).

Use liposomes (such as Invitrogen's 293fectin, etc.) or other transfection reagents (such as PEI, etc.) to transfect the prepared recombinant antibody expression plasmid into HEK293 cells (such as Invitrogen's HEK293F), and culture under serum-free suspension culture conditions 3-5 days. The culture supernatant is then harvested by centrifugation and other methods, and is purified in one step using a ProteinA/G affinity chromatography column (such as GE's Mabselect SURE, etc.). Then use a desalting column (such as GE's Hitrap desaulting, etc.) to replace the recombinant protein storage buffer with PBS (pH 7.0) or other suitable buffers. If necessary, the antibody samples can be filtered and sterilized, then aliquoted and stored at -20°C.

Example 3: Screening of fully human recombinant Fab library using RSV F protein

With reference to the literature (for experimental technical procedures, please refer to Chinese Patent Application No. 201510097117.0, the entire content of the above patent application is incorporated herein by reference), the recombinant RSV F protein (RSV-DS-Cav1-A/ RSV-DS-Cav1-B), through solid-phase screening strategy (for the experimental protocol, please refer to Phage Display: General Experimental Guide/(Clackson, T.), (U.S.) Lowman, H.B. (eds.); Ma Translated by Lan et al. (Chemical Industry Press, 2008.5), the fully human recombinant Fab library constructed in Example 1 was screened, and a total of 4-6 rounds of screening were performed through binding, elution, neutralization, infection, and amplification, and two rounds of screening were finally obtained. Fab antibodies that specifically bind RSV F protein: R3B1h1 and R22B1.

Example 4: Identification of recombinant anti-RSV F protein monoclonal antibodies

Using conventional molecular biology methods, the nucleic acid molecules encoding the light and heavy chains of R3B1h1 and R22B1 were cloned into eukaryotic expression vectors to prepare recombinant human IgG1-κ monoclonal antibodies. At the same time, the human anti-RSV monoclonal antibody palivizumab (Hu1129) was prepared with reference to patent US 7704505B2 (the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 22; the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 23), prepare the human anti-RSV monoclonal antibody Nirsevimab (MEDI8897) with reference to patent US 11186628 B2 (which The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 24; the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 25). The human anti-RSV monoclonal antibody Clesrovimab (RB1) was prepared with reference to patent US 9963500 B2. (The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 26; the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 27) as a positive control antibody.

4.1 Verification of binding activity of recombinant anti-RSV F protein monoclonal antibody

The prepared RSV-DS-Cav1-A and RSV-DS-Cav1-B were coated on a 96-well ELISA plate at 3 μg/mL, 100 μL/well, and coated overnight at 4°C. Use blocking solution PBS-0.1% Tween 20-3% milk (PBST-3% milk) at 37°C for 1 hour, then add each recombinant anti-RSV F protein monoclonal antibody and combine at 37°C for 1 hour. Wash the ELISA plate with PBS-0.1% Tween 20 (PBST) buffer, add HRP-mouse anti-human IgG (Bioss, bsm-0297M-HRP), and bind at 37°C for 1 hour. Wash the ELISA plate with PBST buffer, add OPD substrate chromogenic solution, stop the color development with 1M H ₂ SO ₄ after 5-10 minutes, and measure the optical density value with a microplate reader at 492nm/630nm dual wavelength. ELISA analysis results (Figure 3) show that the recombinant anti-RSV F protein monoclonal antibodies R3B1h1 and R22B1 have comparable activity in binding to the two recombinant proteins RSV-DS-Cav1-A and RSV-DS-Cav1-B.

4.2 Epitope analysis of recombinant anti-RSV virus F protein monoclonal antibodies

Use recombinant protein RSV-DS-Cav1-B to coat on a 96-well ELISA plate (1 μg/mL, 100 μL/well), and coat overnight in a 4°C refrigerator. Use blocking solution PBST-3% milk for 1 hour at 37°C. The recombinant anti-RSV F protein monoclonal antibody (R3B1h1/R22B1/Clesrovimab) was gradient diluted with each anti-RSV F protein purified phage (R3B1h1/R22B1/Clesrovimab/Nirsevimab) at a fixed concentration (1×10 ¹¹ cfu/mL). The starting concentration is 200 μg/mL, 3-fold gradient dilution, 10 concentration gradients, 100 μL/well added to the sealed 96-well ELISA plate, and incubated at 37°C for 1 hour. Wash the ELISA plate with PBST, then add HRP anti-M13 secondary antibody (Beijing Yiqiao Shenzhou Technology Co., Ltd., 11973-MM05T-H), and incubate at 37°C for 1 hour. Use PBST to wash the ELISA plate, add OPD substrate chromogenic solution, stop the color development with 1M H ₂ SO ₄ after 5-10 minutes, and use a microplate reader to measure the 492nm/630nm dual-wavelength optical density value. The ELISA analysis results are shown in Figure 4. The R3B1h1 monoclonal antibody can block the interaction between R22B1/Nirsevimab phage and the recombinant protein RSV-DS- The binding signal of Cav1-B has no effect on the binding of Clesrovimab phage and recombinant protein RSV-DS-Cav1-B (Figure 4A); R22B1 monoclonal antibody can block the binding of R3B1h1/Nirsevimab phage and recombinant protein RSV-DS-Cav1-B The binding signal has no effect on the binding of Clesrovimab phage to recombinant protein RSV-DS-Cav1-B (Figure 4B)); Clesrovimab monoclonal antibody cannot block R3B1h1/R22B1/Nirsevimab phage and recombinant protein RSV-DS-Cav1-B. combination (Figure 4C).

4.3 Affinity analysis of recombinant anti-RSV F protein monoclonal antibodies

The affinity of anti-RSV F protein monoclonal antibodies was determined by surface plasmon resonance technology using Biacore T200. Amino coupling kit (BR-1000-50), human antibody capture kit (BR-1008-39), CM5 chip (BR100012) and pH7.4 10×HBS-EP (BR100669) and other related reagents and consumables are Purchased from GE healthcare. According to the instructions in the kit, use 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N -Hydroxysuccinimide (NHS) activates the carboxylated CM5 chip surface, and the anti-human IgG (Fc) antibody (capture antibody) is diluted to 25 μg/mL with 10 mM, pH 5.0 sodium acetate, and then Inject at a flow rate of 10 μL/min to achieve a coupling volume of approximately 10,000 response units (RU). After injection of the capture antibody, 1 M ethanolamine was injected to block unreacted groups. For kinetic measurements, dilute the anti-RSV F protein monoclonal antibody to 0.5-1μg/mL and inject at 10μL/min to ensure that approximately 80RU of the antibody is captured by the anti-human Fc antibody. Then set RSV-DS-Cav1-A or RSV-DS-Cav1-B in a series of concentration gradients (such as 6.17nM, 18.5nM, 55.6nM, 166.7nM, 500nM) at 25°C at 30μL/min from low to low. Inject from the concentration to high concentration, the binding time is 90s, the dissociation time is 3600s, and 3M MgCl ₂ is injected at 10μL/min for 30s to regenerate the chip surface. The association rate (K _on ) and the dissociation rate (K _off ) were calculated by fitting the association and dissociation sensorgrams with a 1:1 binding model using Biacore T200 evaluation software version 3.0. Calculate the dissociation equilibrium constant (K _D ) as the ratio K _off /K _on . The fitting results are shown in Table 1 and Table 2.

Table 1. Affinity constants of recombinant anti-RSV F protein monoclonal antibodies binding to RSV-DS-Cav1-A

Table 2. Affinity constants of recombinant anti-RSV F protein monoclonal antibodies binding to RSV-DS-Cav1-B

4.4 Recombinant anti-RSV F protein monoclonal antibody inhibits RSV infection of Hep-2 cells

4.4.1 Recombinant anti-RSV F protein monoclonal antibody inhibits RSV/A2 infection of Hep-2 cells

Anti-RSV F protein monoclonal antibodies (R3B1h1, R22B1, Nirsevimab, Clesrovimab, and palivizumab) and negative isotype control antibodies were maintained in Hep-2 cell maintenance medium (DMEM+2% FBS+1% P/S+2mM Glu) was diluted with a starting concentration of 66.7 nM, 5-fold gradient dilution, a total of 10 concentration gradients, and 70 μL/well was added to a 96-well sterile fully permeable cell culture plate. Take 1 frozen virus liquid RSV/A2, place it in the P2 laboratory biosafety cabinet and slowly thaw it, dilute it with Hep-2 cell maintenance medium to 1.5×10 ³ PFU/mL, and take 70 μL/mL of the diluted virus liquid. Add the above diluted antibody to the well and mix thoroughly. At the same time, separate cell control wells (Hep-2 cell maintenance medium 140 μL/well) and cells + RSV/A2 wells (Hep-2 cell maintenance medium 70 μL/well + RSV/A2 dilution 70 μL/well) were set. After all samples have been added, cover the culture plate and place it in a cell culture incubator (37±1°C, 5±1% CO ₂ ) for neutralization for 1 hour. Take Hep-2 cells in the logarithmic phase of growth, digest and centrifuge, and use Hep-2 cell maintenance medium to dilute the counted cell suspension to a single cell suspension of 4.3×10 ⁵ cells/mL. Inoculate 70 μL/well in the Place the neutralized 96-well cell culture plate in a cell culture incubator (37±1°C, 5±1% CO ₂ ) and incubate for 60-72 hours. After virus infection, wash twice with PBS, add 80% acetone, and move to a refrigerator at 2 to 8°C to fix for 30 minutes. Discard 80% acetone, wash twice with PBS, add detection antibody palivizumab (100 μL/well) diluted to 10 μg/mL in PBS, and incubate at 37°C in the dark for 60 min. Discard the primary antibody, wash twice with PBS, and add FITC-labeled goat anti-human IgG (Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd., ZF-0308) diluted in PBS (1:400), 100 μL/ wells and incubate at 37°C in the dark for 40 minutes. Discard the secondary antibody, wash twice with PBS, and detect the chemiluminescence value through the Rapid Fluorescent Focus Inhibition Test (RFFIT) using a multifunctional microplate reader (SpectraMax I3). The results showed (Figure 5) that R3B1h1 had the best activity. According to IC ₅₀ value comparison (Table 3), the activity was approximately 5 times higher than Nirsevimab, 12 times higher than Clesrovimab, and 410 times higher than palivizumab.

Table 3. Half-inhibitory concentration of anti-RSV F protein monoclonal antibody in inhibiting RSV/A2 strain infection of Hep-2 cells

4.4.2 Recombinant anti-RSV F protein monoclonal antibody inhibits infection of Hep-2 cells by RSV/18537 strain

Refer to the protocol in 4.4.1 to detect the neutralizing activity of anti-RSV F protein monoclonal antibodies (R3B1h1, R22B1, Nirsevimab, Clesrovimab and palivizumab) against RSV/18537. The results showed (Figure 6) that R3B1h1 had the best activity. According to IC ₅₀ value comparison (Table 4), the activity was approximately 18 times higher than Nirsevimab, approximately 12 times higher than Clesrovimab, and approximately 545 times higher than palivizumab.

Table 4. Half inhibitory concentration of anti-RSV F protein monoclonal antibody in inhibiting infection of Hep-2 cells by RSV/18537 strain

4.4.3 Recombinant anti-RSV F protein monoclonal antibody inhibits clinically isolated RSV infection of Hep-2 cells

Refer to the protocol in 4.4.1 to detect anti-RSV F protein monoclonal antibodies (R3B1h1, R22B1, Neutralizing activity of Nirsevimab, Clesrovimab, and Palivizumab) against clinically isolated RSV.

A total of 11 virus strains isolated from clinical samples during the 2021 RSV epidemic season were obtained from Guangzhou Women and Children's Medical Center. Among them, 9 strains are subtype A (numbered 2033, 2064, 2066, 2406, 2410, 2416, 2425, 2430 and 2438 respectively), and 2 strains are subtype B (numbered 2063 and 2427 respectively). The results showed (Figure 7) that both R3B1h1 and R22B1 could well neutralize RSV clinical isolate samples.

sequence information

references

1.JAMA Pediatr.2016 Mar;170(3):267-87.

2.Lancet Glob Health.2017 Oct;5(10):e984-e991.

3.J Gen Virol.2017 Dec;98(12):2912-2913.

4.J Gen Virol.1985 Oct;66(Pt 10):2111-24.

5.J Virol.2008 Mar;82(5):2040-55.

6.J Virol.1987 Oct;61(10):3163-6.

7. Proc Natl Acad Sci U S A.1984 Dec; 81(24):7683-7.

8.Virus Res.2000 Jun;68(1):25-33.

9.Virology.1993 Nov;197(1):1-11.

10.Gene.1994 Dec 30;151(1-2):109-13.

11. Kabat, "Sequences of Proteins of Immunological Interest", National Institutes of Health, Bethesda, Md. (1991).

12.A1-Lazikani, et al., J. Mol. Biol. 273:927-948 (1997).

13. Martin, et al., Proc. Natl. Acad. Sci. USA 86:9268-9272 (1989).

14.Annu Rev Biochem.1989;58:913-49.

15.Science.2013 Nov 1;342(6158):592-8.

Claims

A plasmid combination for expressing a library of antibody Fab fragments, comprising a first plasmid and a second plasmid, wherein

The first plasmid includes an antibody heavy chain VH-CH1 expression unit and a first recombination site;

The second plasmid includes an antibody light chain VL-CL expression unit and a second recombination site; and

The first recombination site is different from the second recombination site;

Optionally, the plasmid combination comprises a plurality of said first plasmids, and a plurality of said first plasmids carry different said antibody heavy chain VH-CH1 expression units and have the same said first recombination site; and / or

The plasmid combination includes a plurality of the second plasmids, and the plurality of the second plasmids carry different expression units of the antibody light chain VL-CL and have the same second recombination site.
The plasmid combination of claim 1, wherein

The first plasmid and/or the second plasmid are phagemids; and/or

The first plasmid and the second plasmid comprise different origins of replication. Preferably, the origin of replication is selected from one or more of the following: pBR ori, CDF ori, the origin of replication of filamentous phage M13 (f1 ori) and p15A ori; more preferably, the first plasmid contains pBR ori and f1 ori, and/or the second plasmid contains CDF ori; and/or

The first recombination site and the second recombination site are sites for phage attachment in the respective genomes of the phage and its host bacteria; preferably, the first recombination site is attP, and the The second recombination site is attB; more preferably, the first recombination site includes the nucleotide sequence shown in SEQ ID NO:2 or the nucleotide sequence shown in SEQ ID NO:2 through one or A nucleotide sequence obtained by multiple substitutions, deletions or additions that do not essentially change the function of the first recombination site; and/or the second recombination site includes the nucleotides shown in SEQ ID NO:4 The sequence or the nucleotide sequence represented by SEQ ID NO: 4 has been subjected to one or more substitutions, deletions or additions that do not essentially change the function of the second recombination site; and/or

The first recombination site and the second recombination site are respectively located at the polyclonal enzyme cutting sites of the first plasmid and the second plasmid; and/or

The antibody light chain constant region CL is a human kappa light chain constant region or a human lambda light chain constant region; and/or

The CH1 segment of the antibody heavy chain constant region is selected from IgG1, IgG2, IgG3 or IgG4 subtypes; and/or

The first plasmid and/or the second plasmid comprise a resistance gene coding region; and/or

The 3' end of the nucleic acid molecule encoding the CH1 segment of the constant region of the antibody heavy chain is fused to the 5' end of the nucleic acid molecule encoding the gIII protein of filamentous phage M13;

Optionally, the resistance gene is an antibiotic resistance gene; preferably, the resistance gene is selected from: chloramphenicol resistance gene (CmR), ampicillin resistance gene (Ampr), kanamycin resistance gene resistance gene (KaR) and tetracycline resistance gene (TetR).
A recombinant system for expressing an antibody Fab fragment library, which includes a first plasmid, a second plasmid and a cell expressing phage integrase; wherein

The first plasmid includes an antibody heavy chain VH-CH1 expression unit and a first recombination site;

The second plasmid includes an antibody light chain VL-CL expression unit and a second recombination site; and

The first recombination site is different from the second recombination site;

Optionally, the recombinant system includes a plurality of the first plasmids, and a plurality of the first plasmids carry different VH-CH1 expression units of the antibody heavy chain and have the same first recombination site; and / or

The recombinant system includes a plurality of second plasmids, and a plurality of the second plasmids carry different expression units of the antibody light chain VL-CL and have the same second recombination site.
The recombination system of claim 3, wherein

The first plasmid and/or the second plasmid are phagemids; and/or

The first plasmid and the second plasmid comprise different origins of replication. Preferably, the origins of replication are selected from one or more of the following: pBR ori, CDF ori, filamentous phage The origin of replication (f1 ori) and p15A ori of M13; more preferably, the first plasmid contains pBR ori and f1 ori, and/or the second plasmid contains CDF ori; and/or

The first recombination site and the second recombination site are sites for phage attachment in the respective genomes of the phage and its host bacteria; preferably, the first recombination site is attP, and the The second recombination site is attB; more preferably, the first recombination site includes the nucleotide sequence shown in SEQ ID NO:2 or the nucleotide sequence shown in SEQ ID NO:2 through one or A nucleotide sequence obtained by multiple substitutions, deletions or additions that do not essentially change the function of the first recombination site; and/or the second recombination site includes the nucleotides shown in SEQ ID NO:4 The sequence or the nucleotide sequence represented by SEQ ID NO: 4 has been subjected to one or more substitutions, deletions or additions that do not essentially change the function of the second recombination site; and/or

The first recombination site and the second recombination site are respectively located at the polyclonal enzyme cutting sites of the first plasmid and the second plasmid; and/or

The antibody light chain constant region CL is a human kappa light chain constant region or a human lambda light chain constant region; and/or

The CH1 segment of the antibody heavy chain constant region is selected from IgG1, IgG2, IgG3 or IgG4 subtypes; and/or

The first plasmid and/or the second plasmid comprise a resistance gene coding region; and/or

The 3' end of the nucleic acid molecule encoding the CH1 segment of the constant region of the antibody heavy chain is fused to the 5' end of the nucleic acid molecule encoding the gIII protein of filamentous phage M13;

Optionally, the resistance gene is an antibiotic resistance gene; preferably, the resistance gene is selected from: chloramphenicol resistance gene (CmR), ampicillin resistance gene (Ampr), kanamycin resistance gene resistance gene (KaR) and tetracycline resistance gene (TetR).
The recombinant system of claim 3 or 4, wherein

The phage integrase is a tyrosine integrase; preferably, the phage integrase is a lambda phage integrase; and/or

The phage integrase is an inducible expression or a constitutive expression, preferably an inducible expression, and more preferably the inducible expression is through the use of an inducible promoter, such as a lactose promoter (Plac) or an arabinose promoter (Para). );and / or

The cells expressing the phage integrase are prokaryotic cells; preferably, the cells expressing the phage integrase are Escherichia coli; more preferably, the cells expressing the phage integrase are genetically engineered bacteria; most preferably, the cells expressing the phage integrase are genetically engineered bacteria. The cell expressing the phage integrase is a male E. coli strain with F factor, such as the TG1 strain; and/or

The first plasmid, the second plasmid and the cell expressing the phage integrase each exist independently, or at least one of the first plasmid and the second plasmid has been introduced into the cell expressing the phage integrase.
A recombinant cell for expressing an antibody Fab fragment library, which contains a first plasmid, a second plasmid and expresses a phage integrase; wherein

The first plasmid includes an antibody heavy chain VH-CH1 expression unit and a first recombination site;

The second plasmid includes an antibody light chain VL-CL expression unit and a second recombination site; and

The first recombination site is different from the second recombination site;

Optionally, the recombinant cell contains a plurality of the first plasmids, and a plurality of the first plasmids carry different expression units of the antibody heavy chain VH-CH1 and have the same first recombination site; and / or

The recombinant cells comprise a plurality of the second plasmids, and the plurality of second plasmids carry different expression units of the antibody light chain VL-CL and have the same second recombination site.
The recombinant cell of claim 6, wherein

The first plasmid and/or the second plasmid are phagemids; and/or

The first plasmid and the second plasmid comprise different origins of replication. Preferably, the origin of replication is selected from one or more of the following: pBR ori, CDF ori, the origin of replication of filamentous phage M13 (f1 ori) and p15A ori; more preferably, the first plasmid contains pBR ori and f1 ori, and/or the second plasmid contains CDF ori; and/or

The first recombination site and the second recombination site are sites for phage attachment in the respective genomes of the phage and its host bacteria; preferably, the first recombination site is attP, and the The second recombination site is attB; more preferably, the first recombination site Comprising the nucleotide sequence shown in SEQ ID NO: 2 or the nucleotide sequence shown in SEQ ID NO: 2 after one or more substitutions or deletions that do not essentially change the function of the first recombination site Or add the obtained nucleotide sequence; and/or the second recombination site includes the nucleotide sequence shown in SEQ ID NO:4 or the nucleotide sequence shown in SEQ ID NO:4 through one or A nucleotide sequence resulting from multiple substitutions, deletions or additions that do not essentially change the function of the second recombination site; and/or

The first recombination site and the second recombination site are respectively located at the polyclonal enzyme cutting sites of the first plasmid and the second plasmid; and/or

The antibody light chain constant region CL is a human kappa light chain constant region or a human lambda light chain constant region; and/or

The CH1 segment of the antibody heavy chain constant region is selected from IgG1, IgG2, IgG3 or IgG4 subtypes; and/or

The first plasmid and/or the second plasmid comprise a resistance gene coding region; and/or

The 3' end of the nucleic acid molecule encoding the CH1 segment of the constant region of the antibody heavy chain is fused to the 5' end of the nucleic acid molecule encoding the gIII protein of filamentous phage M13;

Optionally, the resistance gene is an antibiotic resistance gene; preferably, the resistance gene is selected from: chloramphenicol resistance gene (CmR), ampicillin resistance gene (Ampr), kanamycin resistance gene sex gene (KaR) and tetracycline resistance gene (TetR); and/or

The phage integrase is a tyrosine integrase; preferably, the phage integrase is a lambda phage integrase; and/or

The phage integrase is an inducible expression or a constitutive expression, preferably an inducible expression, and more preferably the inducible expression is through the use of an inducible promoter, such as a lactose promoter (Plac) or an arabinose promoter (Para). );and / or

The recombinant cells are prokaryotic cells; preferably, the recombinant cells are Escherichia coli; more preferably, the recombinant cells are genetically engineered bacteria; most preferably, the recombinant cells are male Escherichia coli strains with F factor, For example, strain TG1.
A method for preparing an antibody Fab fragment library, which includes transducing a first plasmid and a second plasmid into cells expressing phage integrase; wherein

The first plasmid includes an antibody heavy chain VH-CH1 expression unit and a first recombination site;

The second plasmid includes an antibody light chain VL-CL expression unit and a second recombination site; and

The first recombination site is different from the second recombination site;

Optionally, a plurality of said first plasmids are transduced into said cell expressing phage integrase, wherein a plurality of said first plasmids carry different said antibody heavy chain VH-CH1 expression units and have the same said first recombination site; and/or

A plurality of the second plasmids are transduced into the cells expressing phage integrase, wherein a plurality of the second plasmids carry different expression units of the antibody light chain VL-CL and have the same second recombination site.
The method of claim 8, wherein transducing the first plasmid and/or the second plasmid into the cell expressing phage integrase includes the following steps:

(1) Transfect the first plasmid and/or the second plasmid into competent cells;

(2) Use the helper phage to infect the competent cells obtained in step (1) to construct a phage library; and

(3) transducing the phage library obtained in step (2) into the cells expressing phage integrase;

Optionally, the competent cells are prokaryotic cells; preferably, the competent cells are Escherichia coli; more preferably, the competent cells are genetically engineered bacteria; most preferably, the competent cells are F-factor male E. coli strains, such as the TG1 strain; and/or

The competent cells do not express phage integrase; and/or

The helper phage is M13 helper phage.
The method of claim 8 or 9, wherein

The first plasmid and/or the second plasmid are phagemids; and/or

The first plasmid and the second plasmid comprise different origins of replication. Preferably, the origins of replication are selected from one or more of the following: pBR ori, CDF ori, origin of replication of filamentous phage M13 (f1 ori) and p15A ori; more preferably, the first plasmid contains pBR ori and f1 ori, and/or the second plasmid contains a CDF ori; and/or

The first recombination site and the second recombination site are sites for phage attachment in the respective genomes of the phage and its host bacteria; preferably, the first recombination site is attP, and the The second recombination site is attB; more preferably, the first recombination site includes the nucleotide sequence shown in SEQ ID NO:2 or the nucleotide sequence shown in SEQ ID NO:2 through one or A nucleotide sequence obtained by multiple substitutions, deletions or additions that do not essentially change the function of the first recombination site; and/or the second recombination site includes the nucleotides shown in SEQ ID NO:4 The sequence or the nucleotide sequence represented by SEQ ID NO: 4 has been subjected to one or more substitutions, deletions or additions that do not essentially change the function of the second recombination site; and/or

The first recombination site and the second recombination site are respectively located at the polyclonal enzyme cutting sites of the first plasmid and the second plasmid; and/or

The antibody light chain constant region CL is a human kappa light chain constant region or a human lambda light chain constant region; and/or

The CH1 segment of the antibody heavy chain constant region is selected from IgG1, IgG2, IgG3 or IgG4 subtypes; and/or

The first plasmid and/or the second plasmid comprise a resistance gene coding region; and/or

The 3' end of the nucleic acid molecule encoding the CH1 segment of the constant region of the antibody heavy chain is fused to the 5' end of the nucleic acid molecule encoding the gIII protein of filamentous phage M13;

Optionally, the resistance gene is an antibiotic resistance gene; preferably, the resistance gene is selected from: chloramphenicol resistance gene (CmR), ampicillin resistance gene (Ampr), kanamycin resistance gene sex gene (KaR) and tetracycline resistance gene (TetR); and/or

The phage integrase is a tyrosine integrase; preferably, the phage integrase is a lambda phage integrase; and/or

The phage integrase is an inducible expression or a constitutive expression, preferably an inducible expression, and more preferably the inducible expression is through the use of an inducible promoter, such as a lactose promoter (Plac) or an arabinose promoter (Para). );and / or

The cells expressing the phage integrase are prokaryotic cells; preferably, the cells expressing the phage integrase are Escherichia coli; more preferably, the cells expressing the phage integrase are genetically engineered bacteria; most preferably, the cells expressing the phage integrase are genetically engineered bacteria. The cells expressing the phage integrase are male E. coli strains with F factor, such as the TG1 strain.
The use of the plasmid combination according to claim 1 or 2, the recombinant system according to any one of claims 3-5, or the recombinant cell according to claim 6 or 7 in preparing an antibody Fab fragment library.
A library of antibody Fab fragments prepared by the method of any one of claims 8-10.
The use of the antibody Fab fragment library of claim 12 in at least one of the following: antibody preparation, disease prevention, diagnosis and/or treatment, and vaccine preparation.